Streptomyces 2

I HC THI NGUYN TRNG I HC S PHM ------ ------
BI TH H
NGHIN CU X KHUN THUC CHI STREPTOMYCES SINH CHT KHNG SINH CHNG NM GY BNH TRN CY CH THI NGUYN
LUN VN THC S SINH HC
THI NGUYN - 2008
I HC THI NGUYN TRNG I HC S PHM ------ ------
BI TH H
NGHIN CU X KHUN THUC CHI STREPTOMYCES SINH CHT KHNG SINH CHNG NM GY BNH TRN CY CH THI NGUYN
Chuyn ngnh: Sinh hc thc nghim M s: 60.42.30
LUN VN THC S SINH HC
NGI HNG DN KHOA HC TS. VI TH OAN CHNH
THI NGUYN - 2008
LI CM N
Ti xin by t lng bit n su sc ti c gio TS. Vi Th oan Chnh tn tnh gip ti trong qu trnh thc hin v hon thnh lun vn ny. Xin chn thnh cm n ThS. Nguyn Ph Hng v cc cn b ca b mn Sinh hc thuc Khoa KHTN & XH - HTN nhit tnh gip ti. Xin chn thnh cm n PGS. TS. Ng nh Quang Bnh v cc cn b phng Di truyn vi sinh hc - Vin Cng ngh sinh hc thuc Vin khoa hc v cng ngh Vit Nam. Xin cm n Ban lnh o Trng i hc Y - Dc Thi Nguyn, Khoa Sinh - KTNN, Khoa Sau i hc - Trng i hc S phm - HTN to nhiu iu kin ti hon thnh lun vn ny. Xin cm n Ban ch nhim khoa KHCB, B mn Ha - sinh to mi iu kin cho ti c hc tp v hon thnh lun vn ny. Ti xin cm n cc thy c, bn b ng nghip ng vin, to mi iu kin gip ti trong sut qu trnh lm lun vn. Li cm n su sc nht ti xin dnh cho gia nh v nhng ngi thn yu ca ti.
LI CAM OAN
Ti xin cam oan y l cng trnh nghin cu ca ring ti. Cc s liu, kt qu nghin cu trong lun vn l trung thc v cha tng c cng b trong bt k cng trnh no khc.
Thi nguyn, ngy 15 thng 9 nm 2008 Tc gi
Bi Th H
MC LC
Trang Trang ph ba Li cm n Li cam oan Mc lc Nhng ch vit tt Danh mc cc bng Danh mc cc hnh v, th M U..........................................................................................................1 Chng 1: TNG QUAN TI LIU 1.1. GII THIU V X KHUN..................................................................3 1.1.1. Phn b ca x khun trong t nhin............................................3 1.1.2. c im hnh thi ca x khun..................................................4 1.1.3. S hnh thnh bo t ca x khun...............................................5 1.1.4. Cu to ca x khun....................................................................6 1.2. CC PHNG PHP PHN LOI X KHUN HIN I... ...........8 1.2.1. c im hnh thi v tnh cht nui cy.....................................8 1.2.2. c im ha phn loi (Chemotaxonomy)..................................9 1.2.3. c im sinh l - sinh ha.........................................................10 1.2.4. Phn loi s (Numerical taxonomy)............................................10 1.2.5. Phn loi x khun chi Streptomyces..........................................11 1.3. CHT KHNG SINH T X KHUN.................................................12 1.3.1. Lc s nghin cu cht khng sinh...........................................12 1.3.2. S hnh thnh cht khng sinh x khun..................................15 1.3.3. Cc yu t nh hng n sinh tng hp cht khng sinh........16 1.4. MT S BNH HI CH DO NM GY RA V NG DNG CA CKS TRONG BO V THC VT..................................................18
1.4.1. Mt s bnh hi ch do nm......................................................18 1.4.2. Cc cht khng sinh trong bo v thc vt.................................21 Chng 2 - NGUYN LIU V PHNG PHP NGHIN CU 2.1. Nguyn liu v ha cht.........................................................................24 2.1.1. Nguyn liu................................................................................24 2.1.2. Ha cht, dng c v thit b.................. ...................................24 2.2. Phng php nghin cu..........................................................................27 2.2.1. Phng php phn lp nm gy bnh t cc mu ch...........................27 2.2.2. Phn lp v tuyn chn x khun..........................................................28 2.2.2.1. Phn lp x khun theo Vinogradski........................................28 2.2.2.2. Xc nh hot tnh khng sinh ................................................28 2.2.2.3.Tuyn chn cc chng x khun sinh cht khng sinh............29 2.2.3. Bo qun ging......................................................................................29 2.2.4. Nghin cu cc c im sinh hc v phn loi x khun....................30 2.2.4.1. c im hnh thi...................................................................30 2.2.4.2. c im nui cy....................................................................31 2.2.4.3. c im sinh l - sinh ha......................................................31 2.2.5. Ln men to khng sinh.........................................................................32 2.2.5.1. La chn mi trng ln men thch hp .................................32 2.2.5.2. nh hng ca ngun cacbon..................................................33 2.2.5.3. nh hng ca ngun Nit......................................................33 2.2.6. Cc phng php sinh hc phn t trong phn lp gen 16S - rRNA....33 2.2.6.1. Tch chit DNA ca x khun bng m CTAB.....................33 2.2.62. Khuch i gen 16S - rRNA bng phn ng PCR...................34 2.2.6.3. Phng php in di trn gel agarose......................................35 2.2.7. Phng php x l s liu.....................................................................36
Chng 3 - KT QU V THO LUN

3.1. Phn lp v thun khit cc chng nm gy bnh trn ch......................37
3.2. Phn lp v tuyn chn x khun.............................................................38 3.2.1. Hot tnh khng nm ca cc chng x khun............................38 3.2.2. Tuyn chn cc chng x khun c HTKN cao..........................41 3.3. c im sinh hc v c im phn loi ca 2 chng XK 1 v R2....42 3.3.1. c im hnh thi......................................................................42 3.3.2.c im nui cy........................................................................43 3.3.3. c im sinh l - sinh ha.........................................................45 * Kh nng ng ha cc ngun cacbon...............................................45 * Nhit sinh trng thch hp .........................................................46 * Kh nng chu mui...........................................................................46 * Kh nng sinh enzym ngoi bo........................................................47 3.3.4. Hot tnh khng sinh ca 2 chng R2 v 1..............................48 3.3.5. V tr phn loi ca hai chng x khun R2 v 1....................50 3.4. Kh nng sinh tng hp CKS ca 2 chng x khun la chn...........52 3.4.1. La chn mi trng ln men thch hp.....................................52 3.4.2. nh hng ca ngun cacbon.....................................................54 3.4.3. nh hng ca ngun nit..........................................................56 3.5. Phn loi cc chng x khun theo phng php sinh hc phn t........57 3.5.1. Kt qu tch chit DNA tng s ca cc chng x khun..........57 3.5.2. Kt qu nhn gen 16S - rRNA bng phn ng PCR..................58 Chng 4 - KT LUN V KIN NGH 4.1. Kt lun.........................................................................................60 4. 2. Kin ngh v nhng nghin cu tip theo.....................................61 CNG TRNH CNG B LIN QUAN N LUN VN....................62 TI LIU THAM KHO............................................................................63
NHNG CH VIT TT TRONG LUN VN
XK VSV CKS HSCC HSKS KTKS HTKS HTKN MT KHVQH KHVT DNA RNA ISP TE SDS TAE PCR
: X khun : Vi sinh vt : Cht khng sinh : H si c cht : H si kh sinh : Khun ty kh sinh : Hot tnh khng sinh : Hot tnh khng nm : Mi trng : Knh hin vi quang hc : Knh hin vi in t : Deoxyribonucleic Acid : Ribonucleic Acid : International Streptomyces Project : Tris - Ethylendiamin tetracetic acid : Sodiumdodecyl sulfat : Tris - Acetate - Ethylendiamin tetracetic acid : Polymerase chain reaction (phn ng chui polymerase)
DANH MC CC BNG Trang Bng 3.1. c im mt s mu ch lm ngun phn lp cc chng nm.....37 Bng 3.2. X khun c hot tnh khng nm theo nhm mu................................39 Bng 3. 3.Tnh i khng ca x khun vi 3 chng nm gy bnh trn ch.......40 Bng 3. 4. Hot tnh khng nm ca 2 chng x khun..................................41 Bng 3.5. c im nui cy ca chng R2 v 1.........................................44 Bng 3.6. Kh nng ng ha ngun cacbon ca 2 chng x khun 1 v R2....45 Bng 3.7. Nhit sinh trng thch hp ca 2 chng R2 v 1...................46 Bng 3.8. Kh nng chu mui ca 2 chng R2 v 1...................................47 Bng 3.9. Hot tnh khng sinh ca 2 chng R2 v 1 vi 3 chng nm kim nh.........................................................................................................48 Bng 3.10. So snh c im phn loi ca chng R2 vi S. misawaensis....50 Bng 3.11. So snh c im phn loi ca chng 1 vi A. brunneofungu.52 Bng 3.12: Hot tnh khng sinh ca 2 chng x khun trn cc mi trng ln men khc nhau...........................................................................................53 Bng 3.13. nh hng ca ngun cacbon ln kh nng sinh tng hp CKS ca 2 chng R2 v 1.....................................................................................55 Bng 3.14. nh hng ca ngun nit ln kh nng sinh tng hp CKS ca 2 chng R2 v 1...............................................................................................56
DANH MC CC HNH V, TH Trang Hnh 3.1. Ba chng nm phn lp t cc mu ch b bnh.............................38 Hnh 3. 2. T l cc chng x khun c hot tnh khng nm theo nhm mu.................39 Hnh 3.3. T l cc chng x khun c hot tnh khng nm.........................40 Hnh 3.4. Hot tnh khng nm ca 2 chng x khun la chn.....................42 Hnh 3.5. Cung sinh bo t v b mt bo t chng R2................................42 Hnh 3.6. Cung sinh bo t v b mt bo t chng 1...............................43 Hnh 3.7. Kh nng hnh thnh sc t melanin ca 2 chng...........................44 Hnh 3.8. Hot tnh enzym ca cc chng x khun.......................................47 Hnh 3.9. Hot tnh khng 3 chng nm kim nh ca 2 chng 1 v R2....49 Hnh 3.10: nh hng ca mi trng n kh nng tng hp CKS ............54 Hnh 3.11: nh hng ca ngun cacbon n kh nng tng hp CKS.........55 Hnh 3.12 : nh hng ca ngun nit n kh nng tng hp CKS.............57 Hnh 3.13. nh in di DNA tng s ca 2 chng x khun.........................58 Hnh 3.14. nh in di sn phm PCR ca 2 chng x khun nghin cu.....59
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M U 1. t vn
Vit Nam l nc nng nghip nm trong vng nhit i nng m. Ma l iu kin rt thun li cho VSV pht trin, c bit l cc VSV gy bnh thc vt. V vy vic s dng ho cht trong bo v thc vt t lu ph bin Vit Nam. Song vic s dng ho cht bo v thc vt thng l c hi v khi tn d trong t, nc v nng sn s nh hng nghim trng ti sc kho con ngi, gy nhim mi trng v mt cn bng sinh thi. Chnh v vy, Hi ngh t vn khu vc chu Thi bnh dng ca FAO nm 1992 khng nh u tranh sinh hc l nn tng ca chng trnh IPM (Qun l dch hi tng hp) vi chin lc l s dng tc nhn sinh hc hn ch s pht trin ca cc qun th k sinh. Mt trong nhng hng nghin cu theo xu hng ny l s dng cc tc nhn sinh hc hn ch cc qun th VSV gy bnh. Trong s cc tc nhn sinh hc thng c s dng c ch VSV gy bnh, x khun l nhm c nhiu tim nng nht v t l loi c kh nng sinh CKS cao, trong c nhiu CKS c kh nng chng nm mnh. Thi nguyn l tnh giu tim nng v nng, lm nghip. Trong cy ch l cy loi cy ch o v hng nm cc bnh do nm cng gy ra nhng thit hi nng n, lm gim nng sut v cht lng sn phm. V vy vic tm kim cc chng x khun c kh nng sinh cht khng sinh chng nm gy bnh thc vt c tm quan trng c bit gp phn vo cng tc bo v thc vt v xy dng nn nng nghip an ton v bn vng. Xut pht t nhng l do trn, t xu hng nghin cu trn th gii hin nay cng nh gp phn khai thc ngun vi sinh vt v cng phong ph ca Thi Nguyn. Chng ti thc hin ti: Nghin cu x khun thuc chi Streptomyces sinh cht khng sinh chng nm gy bnh trn cy ch Thi Nguyn.
S ha bi Trung tm Hc liu i hc Thi Nguyn
http://www.lrc-tnu.edu.vn
-22. Mc tiu nghin cu ca ti - Phn lp v tuyn chn cc chng x khun thuc chi Streptomyces c hot tnh khng nm gy bnh trn cy ch. - Nghin cu mt s c im sinh hc v c im phn loi ca mt s chng x khun c hot tnh chng nm mnh, c nhiu trin vng ng dng. 3. i tng v ni dung nghin cu ca ti * i tng nghin cu - Cc chng x khun sinh cht khng sinh chng nm phn lp t t Thi Nguyn. - Cc chng vi nm gy bnh trn cy ch Thi Nguyn. * Ni dung nghin cu 1. Phn lp cc chng nm gy bnh trn cy ch s dng lm VSV kim nh. 2. Phn lp v tuyn chn cc chng x khun c hot tnh khng nm t cc mu t khc nhau. 3. Nghin cu mt s c im sinh hc v c im phn loi ca mt s chng x khun c hot tnh chng nm mnh c la chn. 4. Nghin cu nh hng ca mt s iu kin nui cy n kh nng to thnh CKS ca cc chng la chn.
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Chng 1 TNG QUAN TI LIU

1.1. GII THIU V X KHUN 1.1.1. Phn b ca x khun trong t nhin X khun (Actinobacteria) thuc nhm vi khun tht (Eubacteria) phn b rt rng ri trong t nhin. Chng c trong t, nc, rc, phn chung, bn, thm ch c trong c cht m vi khun v nm mc khng pht trin c. S phn b ca x khun ph thuc vo kh hu, thnh phn t, mc canh tc v thm thc vt. Theo Waksman th trong mt gam t c khong 29.000 - 2.400.000 mm x khun, chim 9 - 45% tng s VSV [43]. S phn b ca x khun cn ph thuc nhiu vo pH mi trng, chng c nhiu trong cc lp t trung tnh v kim yu hoc axit yu 6,8 - 7,5. X khun c rt t trong lp t kim hoc axit v cng him trong cc lp t rt kim, s lng x khun trong t cng thay i theo thi gian trong nm. Mt trong nhng c tnh quan trng nht ca x khun l kh nng hnh thnh cht khng sinh, 60 - 70% x khun c phn lp t t c kh nng sinh cht khng sinh. Cho ti nay khong hn 8000 cht khng sinh hin bit trn th gii th c ti 80% l do x khun sinh ra [6]. Trong s c trn 15% c ngun gc t cc loi x khun him nh Micromonospora Actinomadura, Actinoplanes, Streptoverticillium, Streptosporangium iu ng ch l cc x khun him cung cp nhiu cht khng sinh c gi tr ang dng trong y hc nh gentamixin, tobramixin, vancomixin, rosamixi. Ngoi ra, x khun tham gia tch cc vo cc qu trnh chuyn ho nhiu hp cht trong t, nc. Dng sn xut nhiu enzym nh proteaza,
-4amylaza, xenlulozamt s axit amin v axit hu c. Mt s x khun c th gy bnh cho ngi, ng vt [7]. 1.1.2. c im hnh thi ca x khun * Khun lc c im ni bt ca x khun l c h si pht trin, phn nhnh mnh v khng c vch ngn (ch tr cung bo t khi hnh thnh bo t). H si x khun mnh hn ca nm mc vi ng knh thay i trong khong 0,2 1 m n 2 3 m, chiu di c th t ti mt vi cm [10],[22]. Kch thc v khi lng h si thng khng n nh v ph thuc vo iu kin sinh l v nui cy. Kch thc ca h si x khun l mt trong nhng c im phn bit khun lc ca x khun v khun lc ca nm mc v h si ca nm mc c ng knh rt ln, thay i t 5 quan st bng mt thng. Khun lc ca x khun thng chc, x x c dng da, dng vi, dng nhung t hay dng mng do. Khun lc x khun c mu sc khc nhau: , da cam, vng, nu, xm, trngtu thuc vo loi v iu kin ngoi cnh. Kch thc v hnh dng ca khun lc c th thay i tu loi v tu vo iu kin nui cy nh thnh phn mi trng, nhit , m ng knh mi khun lc ch chng 0,5 lc t ti ng knh 1cm hoc ln hn. Khun lc c 3 lp, lp v ngoi c dng si bn cht, lp trong tng i xp, lp gia c cu trc t ong. Khun ty trong mi lp c chc nng sinh hc khc nhau. Cc sn phm trong qu trnh trao i cht nh: CKS, c t, enzym, vitamin, axit hu cc th c tch lu trong sinh khi ca t bo x khun hay c tit ra trong mi trng. 2 mm nhng cng c khun 50 m, d
-5* Khun ty Trn mi trng c, h si ca x khun pht trin thnh 2 loi: mt loi cm su vo mi trng gi l h si c cht (khun ty c cht substrate mycelium) vi chc nng ch yu l dinh dng. Mt loi pht trin trn b mt thch gi l h si kh sinh (khun ty kh sinh aerial mycelium) vi chc nng ch yu l sinh sn. Nhiu loi ch c h si c cht nhng cng c loi (nh chi Sporichthya) li ch c h si kh sinh. Khi HSKS va lm nhim v sinh sn va lm nhim v dinh dng. di ca khun ty x khun trong giai an pht trin l 11 m/gi [43] v cht nhn ca t bo x khun sp xp u n theo chiu di ca si. Do mt on si (mm x khun) hoc mt bo t x khun gp iu kin thun li s trng ln, sau 1 - 2 gi xut hin qu trnh tng hp ARN, nhn cc gene cn thit t genom v tin hnh tng hp protein, c nh vy si c hnh thnh v pht trin. Mt s x khun c sinh ra nang bo t bn trong cha cc bo t nang [7]. 1.1.3. S hnh thnh bo t ca x khun Bo t x khun c hnh thnh trn cc nhnh phn ha ca khun ty kh sinh - gi l cung sinh bo t. l c quan sinh sn c trng cho x khun. Hnh thi, cung sinh bo t v bo t l cc c im quan trng nht trong phn loi x khun. Cung sinh bo t ca x khun c dng thng hoc ln sng (RF), dng xon l xo (S), chui bo t khng pht trin hoc xon n gin c hnh mc cu (RA). Bo t hnh thnh ng thi trn tt c chiu di ca cung sinh bo t theo 2 cch: kt on hay ct khc v thng c hnh tr, ovan, cu, que vi mp nhn hoc x x, c gai hoc gai pht trin di thnh dng lng [9].
-6Bo t x khun c bao bc bi mng muco polysaccharide giu protein vi dy khong 300 400 A0 chia 3 lp. Cc lp ny trnh cho bo t khi nhng tc ng bt li ca iu kin ngoi cnh nh nhit , pHHnh dng, kch thc chui bo t v cu trc mng bo t l nhng tnh trng tng i n nh v l c im quan trng dng trong phn loi x khun. Tuy nhin nhng tnh trng ny cng c th c nhng thay i nht nh khi nui cy trn mi trng c ngun nit khc nhau [13]. Mun kch thch s hnh thnh bo t trc ht phi kch thch s sinh trng ca khun ty kh sinh. Nu mi trng giu dinh dng qu th qu trnh sinh bo t thng b km hm. Trong nhiu trng hp khi kch thch s hnh thnh bo t, hiu sut sinh tng hp CKS gim i. 1.1.4. Cu to ca x khun X khun c cu trc t bo tng t nh vi khun Gram dng, ton b c th ch l mt t bo bao gm cc thnh phn chnh: thnh t bo, mng sinh cht, nguyn sinh cht, cht nhn v cc th n nhp. Thnh t bo ca x khun c kt cu dng li, dy 10 - 20 nm c tc dng duy tr hnh dng ca khun ty, bo v t bo. Thnh t bo gm 3 lp: Lp ngoi cng dy khong 60 120A0, khi gi c th t ti 150 200A0, lp gia rn chc, dy khong 50A0, lp trong dy khong 50A0. Cc lp ny ch yu cu to t cc lp glucopeptide bao gm cc gc N - axetyl glucozamin lin kt vi N - axetyl muramic bi cc lin kt 1,4 Khi x l bng lyzozym, cc lin kt 1,4 glucozit.
glucozit b ct t, thnh t bo
b ph hu to thnh th sinh cht (protoplast), cu trc si cng b ph hu khi x l t bo vi hn hp este chlorofom v cc dung mi ho tan lipit khc. Nguyn nhn l do lp ngoi cng c cu to ch yu bng lipit (thnh HSKS c nhiu lipit hn so vi HSCC) khc vi nm. Thnh t bo x khun khng cha xenllulose v kitin nhng cha nhiu enzym tham gia vo qu
-7trnh trao i cht v qu trnh vn chuyn vt cht qua mng t bo [25],[30], [32], [35] [36]. Cn c vo thnh phn ho hc, thnh t bo x khun c chia thnh 4 nhm chnh [6], [8]. Nhm I: Thnh phn chnh ca thnh t bo l axit L - 2,6 diaminopimelic (L - ADP) v glyxin. Chi Streptomyces thuc nhm ny. Nhm II: Thnh phn chnh ca thnh t bo l axit meso - 2,6 diaminopimelic (m - ADP) v glyxin. Nhm III: Thnh phn chnh ca thnh t bo l axit meso - 2,6 diaminopimelic. Nhm IV: Thnh phn chnh ca thnh t bo l axit meso - 2,6 diaminopimelic, arabinose v galactose. Di lp thnh t bo l mng sinh cht dy khong 50 nm c cu to ch yu bi 2 thnh phn l photpholipit v protein. Chng c vai tr c bit quan trng trong qu trnh trao i cht v qu trnh hnh thnh bo t ca x khun. Nguyn sinh cht v nhn t bo x khun khng c khc bit ln so vi t bo vi khun. Trong nguyn sinh cht ca x khun cng cha mezoxom v cc th n nhp (cc ht polyphosphate: hnh cu, bt mu vi thuc nhum sudan III v cc ht polysaccharide bt mu vi dung dch lugol). Tuy nhin, im khc bit ca x khun so vi cc sinh vt prokaryote ch chng c t l G + C rt cao trong DNA, thng ln hn 55%, trong khi vi khun t l ny ch l 25 45% [11]. X khun thuc loi vi khun Gram dng nn ngoi yu t di truyn trong nhim sc th cn c cc yu t di truyn ngoi nhim sc th, chng c th t nhn ln m c Lederberg gi l plasmid. Cc plasmid em li cho t bo nhiu c tnh chn lc qu gi nh c thm kh nng phn gii mt s hp
-8cht, chng chu vi nhit bt li, chng chu vi cc khng sinh, chuyn gene, sn xut cc cht khng sinh trong t v mi trng tuyn chn [3]. X khun thuc loi c th d dng, ngun cacbon chng thng dng l ng, tinh bt, ru v nhiu cht hu c khc. Ngun nit hu c l protein, pepton, cao ng, cao nm men. Ngun nit v c l nitrat, mui amnKh nng ng ho cc cht cc loi hay chng x khun khc nhau l khc nhau. 1.2. CC PHNG PHP PHN LOI X KHUN HIN I Cng vi s pht trin mnh ca sinh hc phn t, ha sinh hc, l sinh hc...nn vic nh tn mt chng x khun c tin hnh tng i nhanh chng v chnh xc vi nhiu phng php mi nh phn loi s, nghin cu chng loi pht sinh...Song ngi ta vn ch yu da vo cc c im hnh thi, nui cy c im sinh l, sinh ha, min dch hc v sinh hc phn t. 1.2.1. c im hnh thi v tnh cht nui cy c im hnh thi v tnh cht nui cy l mt trong nhng thng tin quan trng phn loi x khun. lm cho cc chng x khun cn nh loi biu hin y cc c im, ngi ta thng xuyn nui cy chng trn mi trng dinh dng khc nhau trong iu kin nhit v thi gian nht nh. Tin hnh quan st m t chp nh v ghi li nhng c im hnh thi v nui cy ca x khun, c bit l c quan mang bo t, hnh dng v b mt bo t. Theo Pridham v cng s [38], cung sinh bo t chia thnh 3 nhm: RF cho nhng cung sinh bo t thng v ln sng. RA cho nhng cung sinh bo t xon, th s v ngn. S cho nhng cung sinh bo t pht trin mnh v xon. Vic s dng cc c im hnh thi v tnh cht nui cy vn coi l nhng d liu c bn dng trong phn loi x khun. Tuy nhin nh ta bit
-9x khun rt khng bn vng v mt di truyn, thng xuyn xy ra s sp xp li trong phn t DNA. Trong cng mt loi c th biu hin khc nhau v hnh thi hay nhng loi khc nhau c th ging nhau v mt hnh thi. V vy phn loi c chnh xc, ngy nay ngi ta phi dng thm cc ch tiu khc b sung nh cc c im sinh l, sinh ha, min dch hc hay sinh hc phn t [45]. 1.2.2. c im ha phn loi (Chemotaxonomy) c im ha phn loi c s dng rng ri v hiu qu trong vng 20 nm tr li y. y l phng php c bn v c hiu qu thng qua vic nh tnh v nh lng thnh phn ha hc ca t bo VSV. Ha phn loi ch yu da vo cc c im sau: - Typ thnh t bo da trn c s phn tch axit amin trong thnh phn peptit v ng trong thnh t bo hay cc polysaccarit gn vo thnh t bo. - Typ peptidoglycan (PG) da vo cc thng tin v thnh phn v cu trc ca mch tetrapeptit ca PG cu ni peptit v cc lin kt gia cc mt xch ca PG. - Axit mycolic l cc phn t c mch di phn nhnh thuc chi Nocardia, Rhodococus, Mycobacterium v cornebacter. y l c im phn loi c bn cho cc chi . - Axit bo thng c s dng trong phn loi l axit bo bo ha mch thng v khng bo ha vi mch phn nhnh kiu iso v enteiso metyl ha nguyn t cacbon th 10. S c mt ca axit 10 - metylloctade canoit (axit tubereulostearinoic) l c im phn loi n chi [11]. - Photpholipit c 5 typ photpholipit (P I, PII, PIII, PIV, PV) c thnh phn c trng c ngha cho phn loi x khun.
-10Trong phn loi x khun th typ thnh t bo l c im quan trng nht. Khi mun a ra mt loi mi hoc m t mt loi c ngha no , ngi ta khng th no khng xc nh thnh t bo. 1.2.3. c im sinh l - sinh ha phn loi x khun n loi, ngi ta s dng hng lot cc c im sinh l, sinh ha khc nh kh nng ng ha cc ngun cacbon v nit, nhu cu cc cht kch thch sinh trng, kh nng bin i cc cht khc nhau nh h thng enzym. Nhu cu v oxy, gii hn pH, nhit ti u, kh nng chu mui v cc yu t khc ca mi trng, mi quan h vi cht km hm sinh trng v pht trin khc nhau, tnh cht i khng v nhy cm vi cht khng sinh, kh nng to thnh cht khng sinh v cc sn phm trao i cht c trng khc ca x khun. Hopwood (1975) khng nh rng phn ln cc c im sinh l - sinh ha cng c im nui cy d b bin ng v c gi tr thp v mt phn loi. Do tnh khng n nh v bin d cao ca x khun m ngy nay nhng nguyn tc s dng cc c im sinh l - sinh ha phn loi x khun cng phi thay i [29]. 1.2.4. Phn loi s (Numerical taxonomy) pht hin nhng loi mi trn c s s khc nhau v c im sinh l, sinh ha ngi ta cn s dng cc kt qu da trn phn loi s. Phng php ny da trn s nh gi v s lng mc ging nhau gia cc VSV theo mt s ln cc c im ch yu l cc c im hnh thi, sinh l, sinh ha. so snh cc chng x khun vi nhau tng i mt, ngi ta cn c vo h s ging nhau (h s S - Similarity) c 2 cng thc tnh h s S hay c s dng. - Cng thc ca Sokal v Michener (SSM )
-11SSM(AB) NS NS NS NS Nd 100
Trong : SSM : Mc ging nhau gia hai c th A, B (%)

( B) A
NS+ : S cc tnh trng ging nhau Nd : S cc tnh trng khc nhau NS-: S cc tnh trng i lp nhau. - Cng thc ca Jacard (SJ)
S J (AB) NS 100 NS Nd
Trong : S J : mc ging nhau gia hai chng A, B (%)

( B) A
NS: Tng s cc c im dng tnh (ging nhau) ca hai chng so snh. Nd : Tng s cc c im khc nhau (tng s cc c im dng tnh ca chng ny v m tnh ca chng kia). Kt qu cui cng ca phn loi s l v c s phn nhnh (kiu "r cy") ca cc thng s. s ny nhng chng ging nhau nhiu nht c xp vo mt nhm. Bng phn loi s ngi ta chia x khun chi Streptomyces thnh 2 nhm ln, 37 nhm nh v 13 cm vi nhng i din nht nh [44]. 1.2.5. Phn loi x khun chi Streptomyces Chi Streptomyces l mt ging x khun bc cao c Wakman v Henrici t tn nm 1943 [43]. y l chi c s lng loi c m t ln nht. Cc i din chi ny c HSKS v HSCC pht trin phn nhnh. ng knh si x khun khong 1 - 10 m, khun lc thng khng ln c ng knh khong 1 - 5 mm. Khun lc chc, dng da mc m su vo c cht. B
-12mt khun lc thng c ph bi KTKS dng nhung, dy hn c cht, i khi c tnh k nc. X khun chi Streptomyces sinh sn v tnh bng bo t. Trn u si kh sinh hnh thnh cung sinh bo t v chui bo t. Cung sinh bo t c nhng hnh dng khc nhau ty loi: thng, ln sng, xon, c mc, vng ... Bo t c hnh thnh trn cung sinh bo t bng hai phng php phn on v ct khc. Bo t x khun c hnh bu dc, hnh lng tr, hnh cu vi ng knh khong 1,5 m. Mng bo t c th nhn, gai, khi u, np nhn...ty thuc vo loi x khun v mi trng nui cy. Thng trn mi trng c ngun m v c v glucoza, cc bo t biu hin cc c im rt r. Mu sc ca khun lc v h si kh sinh cng rt khc nhau ty theo nhm Streptomyces, mu sc ny cng c th bin i khi nui cy trn mi trng khc nhau. V vy m y ban Quc t v phn loi x khun ISP nu ra cc mi trng chun v phng php chung phn loi nhm VSV ny. Cc loi x khun thuc chi Streptomyces c cu to ging vi khun Gram dng, hiu kh, d dng cc cht hu c. Nhit ti u thng l 25 - 300C, pH ti u 6,5 - 8,0. Mt s loi c th pht trin nhit cao hn hoc thp hn (x khun a nhit v a lnh). X khun chi ny c kh nng to thnh s lng ln cc CKS c ch vi khun, nm si, cc t bo ung th, virus v nguyn sinh ng vt. Cho n nay xc nh thnh phn loi ca chi Streptomyces, cc nh phn loi s dng hng lot cc iu kin v cc kha phn loi khc nhau [48],[49]. 1.3. CHT KHNG SINH T X KHUN 1.3.1. Lc s nghin cu cht khng sinh
-13Theo nh ngha ca Outchinnikov [48]. Cht khng sinh l cht c ngun gc thin nhin v cc sn phm ci bin ca chng bng con ng ha hc c kh nng tc dng chn lc i vi s pht trin ca VSV, t bo ung th ngay nng thp. Ngi u tin t nn mng cho khoa hc nghin cu CKS l Alexander Fleming - Nh sinh vt hc ngi Anh, pht hin ra penixillin vo thng 10 nm 1928. Sau mt thp k, nh s n lc hp tc ca cc nh vi sinh hc v sinh ha hc Anh, M, penixillin c nghin cu, sn sut vi s lng ln v tr thnh "mt loi thuc thn k". Nm 1945, A.Fleming, E. Chain v H.W.Florey c nhn gii thng Nobel v khm ph ra gi tr to ln ca penixillin m ra mt k nguyn mi trong y hc - k nguyn khng sinh. Nhng nm 1940 - 1959 c coi l thi k hong kim ca vic nghin cu CKS vi hng lot CKS mi lin tip c pht hin nh: gramixidin, tiroxidin do Rene' Jules Dubos pht hin nm 1939, streptomycin do Waksman pht hin nm 1941, erythomycin do Gurre pht hin nm 1952... Cng vi vic pht hin ra cc CKS mi, cng ngh ln men sn xut CKS cng ra i v dn c hon thin [31]. Ngay t nhng nm 1950, CKS c nghin cu s dng trong vic phng chng bnh, kch thch s tng trng ca ng vt nui v cy trng. CKS thu ht c s quan tm ca cc nh khoa hc thuc nhiu lnh vc khc nhau trn th gii. Tc tm kim cc CKS trong thi gian gn y vn din ra nhanh chng, nhiu trung tm nghin cu khoa hc v y hc, dc phm v nng nghip ti nhiu nc trn th gii vn lin tc pht hin c hng lot cc CKS mi c gi tr ng dng trong thc tin.
-14Nm 1999 mt khng sinh mi khc c pht hin c tc dng ngn chn hin tng cholesterol, tng sc khng i vi cc cht c ca chut, ngoi ra khng sinh ny cn c hot tnh chng nm gy bnh mnh. l khng sinh loposomal HA - 92, c tch ra t x khun Streptomyces CDRLL - 312. Nm 2003, nhiu nc trn th gii vn tip tc pht hin c hng lot cc CKS mi. Ti Nht Bn, cht khng sinh mi l yatakemycin c tch chit t x khun Streptomyces sp. TP - A0356 bng phng php sc k ct. CKS ny c kh nng km hm s pht trin ca nm Aspergillus fumigalus v Candida albicans. Ngoi ra cht ny cn c kh nng chng li cc t bo ung th vi gi tr Mic l 0,01- 0,3 mg/ml [15]. Nm 2007, ti Hn Quc phn lp c loi x khun Streptomyces sp. C684 sinh CKS laidlomycin, cht ny c th tiu dit c nhng t cu khng methicillin v cc cu khun khng vancomycin [47]. Ngy nay, vi s pht trin mnh m ca sinh hc hin i cng s h tr ca nhiu ngnh khoa hc khc gip cho vic tm kim v ng dng CKS t c nhng thnh tu rc r. sn xut CKS con ngi khng ch tm kim nhng chng VSV sinh CKS t t nhin m cn ci to chng bng nhiu phng php nh dng k thut di truyn v cng ngh gene, gy t bin nh hng, chn dng gene sinh tng hp, to v dung hp t bo trn to ra cc chng c HTKS cao, ng thi nhm mc ch tm kim cc loi khng sinh mi v qu trong thi gian ngn [7],[41]. Nhng thnh tu gn y trong sinh hc phn t nh ti t hp ADN, k thut tch dng gene v biu hin tng hp vi khun E.coli khng nhng em li kt qu to ln trong pht trin chng ging m cn m ra phng hng y trin vng trong sn xut cc CKS.
-151.3.2. S hnh thnh cht khng sinh x khun Mt trong nhng c im quan trng nht ca x khun l kh nng hnh thnh CKS. Trong s 8000 CKS hin bit trn th gii c trn 80% l c ngun gc t x khun [6]. Mt trong nhng tnh cht ca cc CKS c ngun gc t VSV ni chung v t x khun ni ring l c tc dng chn lc. Mi CKS ch c tc dng vi mt nhm VSV nht nh. Hu ht CKS c ngun gc x khun u c ph khng khun rng. Kh nng khng khun ca cc CKS l mt c im quan trng phn loi x khun. C nhiu quan im khc nhau v kh nng hnh thnh CKS. Mt s tc gi cho rng s hnh thnh CKS l do c ch gip cho VSV tn ti trong mi trng t nhin. S khc cho rng, s hnh thnh CKS l do s cnh tranh trong mi trng dinh dng. Hu ht cc tc gi cho rng khng sinh l sn phm chuyn ha th cp c hnh thnh vo cui pha tch ly tha, u pha cn bng ca chu k sinh trng [5]. Mc d CKS c cu trc khc nhau v VSV sinh ra chng cng a dng, nhng qu trnh sinh tng hp chng ch theo mt s con ng nht nh. - CKS c tng hp t mt cht chuyn ha s cp, thng qua mt chui phn ng enzym. - CKS c hnh thnh t hai hoc ba cht chuyn ha s cp khc nhau. - CKS c hnh thnh bng con ng polyme ha cc cht chuyn ha s cp, sau tip tc bin i qua cc phn ng enzym khc. Nhiu chng x khun c kh nng tng hp ng thi hai hay nhiu CKS c cu trc ha hc v c tc dng tng t nhau. Qu trnh sinh tng hp CKS ph thuc vo c ch iu khin a gene, ngoi cc gene chu trch nhim tng hp CKS, cn c c cc gene chu trch nhim tng hp cc tin cht, enzym v cofactor.
-161.3.3. Cc yu t nh hng n sinh tng hp cht khng sinh 1.3.3.1. nh hng ca iu kin nui cy * Nhit Nhit c nh hng rt ln n sinh trng v kh nng tng hp CKS ca x khun. a s cc x khun pht trin tt nhit 28 - 300C, nhng nhit ti u cho sinh trng tng hp CKS thng ch nm trong khong 18 - 280C [20]. * pH mi trng Sinh tng hp cht khng sinh ph thuc rt nhiu vo pH mi trng. pH tc ng trc tip n tnh cht h keo ca t bo, n hot lc ca cc enzym v tc ng gin tip qua mi trng. pH thch hp cho sinh tng hp CKS thng l trung tnh, pH kim hay axit u c ch qu trnh tng hp CKS [24],[46]. * thng kh X khun l loi VSV c nhu cu thng kh cao hn so vi cc VSV khc, nht l giai on nhn ging (khong t gi th 6 n gi th 12 ca qu trnh nui cy)[21]. Do vy m bo thng kh tt, ngi ta thng b sung vo mi trng ln men benzilthioxyanat... lm tng kh nng ha tan oxy. Nng oxy thch hp cho sinh tng hp CKS l 2 - 8ml O2/ 100ml mi trng ln men [8]. * Tui ging Vic tng hp CKS khng ch ph thuc vo iu kin ln men, m cn ph thuc vo cht lng ca bo t v ging sinh dng. Tui ging cy truyn vo mi trng ln men cho hiu sut CKS cao nht thng l 24 gi tui. Lng ging cy truyn khong t 2 - 10% [14].
-171.3.3.2. nh hng ca thnh phn mi trng ln men CKS l sn phm th cp nn qu trnh sinh tng hp CKS ph thuc cht ch vo thnh phn mi trng dinh dng. Trc ht l ngun cacbon, nit, t l C/N v cc cht khong...[5],[26],[29]. * Ngun cacbon Cc hp cht cacbon c ngha hng u trong s sinh trng v hnh thnh CKS. i vi nhiu chng x khun, ngun cacbon thch hp l tinh bt. Tuy nhin, ty tng chng khc nhau m kh nng s dng cc loi ng l khc nhau, c chng s dng tt cc loi ng n nh glucoza, mannoza, fructoza...c chng s dng tt loi ng i nh sacaroza, maltoza...Ngoi ra mt s chng cn c th s dng cc loi acid hu c v cht bo lm ngun thc n cacbon trong ln men sinh CKS. * Ngun nit Hu ht cc chng x khun sinh CKS u i hi c hai ngun nit hu c v v c. Ngun nit hu c thch hp nht thng l cc hp cht t thc vt nh bt u tng, cao ng. Cao ng l ngun b sung c nit v protein, tuy nhin lng pht pht v c trong cao ng cao s c ch sinh tng hp CKS[27]. Ngun nit v c thng s dng l mui amon. Mui nitrat khng thch hp cho s sinh tng hp CKS ca nhiu chng x khun. * Ngun photphat v c Photphat v c ng vai tr nh l tc nhn iu chnh sinh tng hp CKS. Nng photphat thch hp cho sinh tng hp CKS thng khng vt qu 10 mg/ml. Nng photphat ban u cao s lm tng lng axit nucleic dn n ko di pha sinh trng, rt ngn pha tng hp, lm tng ATP trong t bo, dn n gim hoc ngng hn sinh tng hp CKS. * Cc yu t vi lng
-18y l thnh phn khng th thiu trong mi trng ln men. Nu mi trng ln men c ngun dinh dng t nhin th hu ht cc nguyn t vi lng c sn. Vic b sung cc cht giu nguyn t vi lng vo mi trng s lm thay i ng k kh nng tng hp CKS ca nhiu chng x khun. * Hnh thc ln men Trong tng hp CKS, phng php nui cy cng l mt trong nhng yu t quyt nh. Khi nui cy b mt, c im hai pha thng khng quan st thy v CKS c to thnh trong sut pha sinh trng. Qu trnh sn xut CKS thng c tin hnh theo phng php nui cy chm trong ni ln men c cch khuy o v sc kh. 1.4. MT S BNH HI CH DO NM GY RA V NG DNG CA CKS TRONG BO V THC VT 1.4.1. Mt s bnh hi ch do nm 1.4.1.1. Bnh phng l ch Bnh c pht hin nm 1868 n , nhng n nm 1895 Masse mi nghin cu pht hin nguyn nhn gy bnh. Bnh do nm Esobasidium vexans Mase gy ra [1],[4]. Bnh pht sinh l non, cnh non, vt bnh phn ln mp l. u tin trn l xut hin nhng chm nh hnh git du mu vng nht. Sau vt bnh ln dn, mu nht dn. Pha di vt bnh (mt di l) phng ln v mt trn lm xung, pha li c ht phn mu trng, c gii hn r rt vi phn l kho. Cnh b nm hi s b cht [50]. m bo t (Basidiospore) ca nm bnh c hnh gy, pha nh phn nhnh, mi nhnh nh mt bo t c hnh bu dc hoc hnh thn khng mu. Lc u bo t l n bo, v sau gia c vch ngn to thnh hai bo t. Bo t rt d rng.
-19Di iu kin m cao, nhit thp bnh pht sinh mnh. Cc thi im bnh thng pht sinh mnh l thng 3 n thng 5 v thng 9 n thng 10. Nhit thch hp l 15 1.4.1.2. Bnh m nu Bnh m nu (cn gi l kh l ch hnh bnh xe) l bnh hi l thng thy cc nng ch Vit Nam. Bnh pht sinh vo thng 5, 6 ma nhiu v bnh pht sinh mnh nht vo thng 8, 9. Bnh nng c th lm kh l v rng sm. Tc nhn gy bnh l do nm Colletotrichum camelliae Masse [1],[4]. Bnh m nu ch yu hi l gi, cnh v qu. Trn l vt bnh bt u t mp l, mu nu, khng c hnh dng nht nh hoc hnh bn nguyt. Trn vt bnh c cc ng trn ng tm, gia vt bnh l b kh, mu xm tro en lan dn theo hnh gn sng bnh xe. Trn cnh cng c triu trng nh vy, b phn b bnh c th b rch (v) ra [50]. Bo t nm tn ti trn vt bnh v l bnh, thm ch c khi l ri xung t. Nm sau, khi nhit tng ln, bo t pht tn nh gi ma truyn n cc l ch v sau ly nhim 5 15 ngy bnh pht trin rt nng. vng t thp c mc nc ngm cao, thot nc khng tt, phn bn khng u to iu kin cho bnh pht sinh. Trong qu trnh chm sc ch b xy xt nhiu, nh sng qu mnh hoc khi gp ma, bnh pht sinh cng nng, ging ch l to bnh d pht sinh mnh. 18 ngy th xut hin vt bnh. Bnh a nng m nn thng pht sinh vo thng 7, 8. Sau ma lin tc 10 200C.
-201.4.1.3. Bnh m trng Vit Nam, bnh m trng c mi vng ch, pht sinh trn vn ch mi trng, gy hi l non v cnh non. Ma ma bnh pht sinh mnh nht. Tc nhn gy bnh l do nm Phyllosticta theafolia Hara [4]. Lc u vt bnh cn nh, hnh trn, mu nu phn gia lm xung c mu trng sng, xung quanh c mu lng sm. ng knh vt bnh 0,5 1,5 mm. Thi k sau, nhiu vt nh lin kt vi nhau to thnh vt ln khng c hnh dng nht nh, cung l b bnh d lm cho l rng. Bnh c th pht sinh tt c cnh non. Mt trn vt bnh c nhng vt nh mu en. Bnh m trng c nhng nt mc v c nhng vt nh hnh mi kim. Si nm hoc bo t tn ti trong l b rng hoc cnh cy qua ng. Ma xun bo t pht tn v xm nhp vo cc cnh non v l bnh t. Nhit t 18 250C rt thch hp cho s pht sinh ca bnh. Ma xun v ma thu, ma nhiu l iu kin cho s pht sinh mnh. Trn cc ging ch l nh (nh i bch tr, Gruzia) bnh cng pht sinh mnh. 1.4.1.4. Bnh m xm Bnh m xm l bnh ph bin cc vng trng ch. Bnh pht sinh vo ma ma, nhit 27 300C. Bnh nng lm l ch kh rng, cy ch ci cc. Tc nhn gy bnh l do nm Pestalossia theae Sawada [1],[4]. Vt bnh trn l c mu nu sm, lc u ch c chm nh mu en sau lan ra khp l. Bnh thng bt u t mp l v lm cho l rng. Vt bnh c hnh gn sng, trn vt bnh c cc hnh vn en. L thng b rng khi bnh lan khp l hoc 1/2 l.
-21Nhng im nh trn l l bo t phn sinh nh trn cung ngn v nh bo t. Bo t c 3 ngn, u nh c cung, u ln c 3 lng, bo t mu nu xm. 1.4.1.5. Bnh thi bp ch Bnh thi bp ch thng thy cc nc trng ch ca vng Chu . Bnh ny c pht hin Ph H t nm 1961 - 1962 trn nhng nng ch tng sn v ly hom ging. Tc nhn gy bnh l do nm Colletotrichum theae Petch [4]. Bnh thng xut hin l non, cung l v cnh non. Vt bnh lc u bng u kim c mu en, sau lan dn ra ht c bp v cnh ch. Sau 8 10 ngy vt bnh c th di ti 15 20 mm. Khi thi tit nng m l d b rng. Trong vn m thng hay b nng hn nng ch hi bp. T thng 7 n thng 9 thng c ma ko di, bnh d gy hi nng. Nhit 270C v m > 90% l iu kin thun li cho bnh pht sinh. Bo t nm lan truyn nh ma gi. Ch cnh v vn m bn nhiu phn m v trn nn thm canh cao, thng b bnh nng hn. 1.4.2. Cc cht khng sinh trong bo v thc vt * X khun chng nm gy bnh thc vt Cc nh bnh hc thc vt trn ton th gii iu tra nghin cu tnh hnh s dng CKS trong vic ngn chn cc bnh thc vt. Tuy cn mc thp nhng thu c nhng thnh tu nht nh trong nn nng nghip hin i. S i khng gia cc VSV trong t l c s ca bin php sinh hc phng chng bnh cy. S c mt ca x khun i khng trong t lm gim
-22r rt t l mc bnh ca cy. Thng thng, mt loi x khun i khng c th c ch mt vi loi nm gy bnh nhng c nhng loi hot ph rng c th c ch nhiu tc nhn gy bnh c trong t. Khng phi tt c cc x khun c hot tnh khng nm invitro u th hin trong t (khong 4 - 5%) nhng chng c vai tr quan trng trong vic c ch nm gy bnh v ngn nga kh nng nhim bnh cho cy. y l quy lut cn bng sinh hc trong t nhin. Nu s cn bng mt i, lp tc s ny sinh ra bnh khi trong t c mm gy bnh. X khun chng nm ngoi vic tit ra cc CKS, cn tc ng ln khu h VSV thng qua cc enzym phn gii. Ngoi ra, nhiu XK cn tit ra cc cht kch thch sinh trng thc vt cng nh kch thch cc khu h VSV c li trong vng r [14]. * Cc cht khng sinh c ngun gc x khun trong phng tr nm g y bnh thc vt trnh dch bnh trong nng nghip, ngi ta c th s dng mt s bin php k thut, nh thay i c cu cy trng, ma v. Tuy nhin bin php ny gy xo trn h sinh thi ng rung, to iu kin pht sinh mt s bnh m trc y t gp. Vic tuyn chn cc dng cy khng bnh cng ch c vi nm, sau cc tc nhn gy bnh li khng li. Vic s dng CKS trong trng trt nhm cc mc ch nh chng bnh do nm gy ra trn rau qu v cy trng, chng bnh do vi khun gy ra, dit cn trng v c di... kim ch cc bnh thc vt sinh ra t t. So vi thuc ha hc, dng cc CKS trong bo v thc vt va c tc dng nhanh, d phn hy, c tc dng chn lc cao, c thp khng gy nhim mi trng, cn c kh nng c ch cc VSV khng thuc ha hc. CKS v dch ln men cc chng sinh CKS cn dng x l ht ging vi mc ch tiu dit ngun bnh bn ngoi v trong ht, dit bnh c cc b phn nm trn t ca cy v kh trng t [15].
-23Ngy nay, vic s dng cc CKS trong bo v thc vt c ph bin rng ri trn th gii nht l cc nc Nga, Nht, Trung Quc, n ... Trung Quc tuyn chn c nhiu chng x khun t t v nghin cu sn xut nhiu CKS phng chng bnh cy c hiu qu cao nh policin chng bnh o n, jangamicin chng bnh kh vn. Nm 2002, n phn lp c chng Streptomyces sp. 201 c kh nng sinh CKS mi l z - methylheptyl iso - nicotinate, cht khng sinh ny c kh nng khng c nhiu loi nm gy bnh nh Fusarium oxysporum, F. solani...[15]. Vit Nam cng s dng nhiu ch phm khng sinh trong bo v thc vt nhp t Trung Quc hay Nht Bn v phn lp c mt s chng x khun c kh nng chng Pyricularia oryzae gy bnh o n v F. oxysporum gy bnh thi r thc vt [2]. Tuy nhin vic s dng CKS trong lnh vc bo v thc vt nc ta cn mc thp bi tp qun canh tc ch quen dng mt s ha cht bo v thc vt nht nh. Ngoi ra, gi thnh ca cc ch phm sinh hc cha ph hp vi iu kin sn xut ca ngi nng dn. Do cn c s phi hp thng nht trong vic nghin cu, sn xut cc ch phm phng tr sinh hc vi vic truyn thng, xy dng phng php canh tc mi nhm thu c hiu qu to ln trong phng chng dch bnh, nng cao nng sut cy trng v hiu qu kinh t ng thi bo v mi trng v sc khe con ngi [14].
-24-
Chng 2 NGUYN LIU V PHNG PHP NGHIN CU

2.1 NGUYN LIU V HA CHT 2.1.1. Nguyn liu 2.1.1.1 Mu cy Mu nghin cu l cc l ch, bp ch b bnh thu thp t cc a im khc nhau ca Thi Nguyn, c bo qun trong t lnh khng qu 24 gi. 2.1.1.2. Mu t Cc mu t c ly cc su 5 - 10 cm ti cc a im khc nhau ca tnh Thi Nguyn. 2.1.1.3. Vi sinh vt kim nh Ngoi cc chng nm phn lp c t cc l ch b bnh, 3 chng nm gy bnh thc vt c s dng lm VSV kim nh l: - Rhizoctonia solani VCM 3047 - Fusarium oxysporum VCM 3028 - Fusarium moniliforme VCM 3027 Cc chng nm ny nhn c t phng Di truyn VSV - Vin Cng ngh Sinh hc thuc Vin khoa hc v cng ngh Vit Nam. 2.1.2 Ha cht, dng c v thit b 2.1.2.1. Ha cht - Cc loi ng chun: glucose, saccarose, lactose, fructose, mantose... ca hng Merck (c), Trung Quc sn xut - Cc loi mui: K2HPO4, KH2PO4, KI, MgSO4. 7H2O, KNO3, NaCl, FeSO4. 7H2O, (NH4)2SO4, CaCO3, MnCl2, Na2CO3, ZnCl2, ZnSO4.... nhp t Trung Quc, Anh sn xut
-25- Cc loi cao: Cao tht, cao nm men, cao malt, peptone... ca hng Merck (c). - Cc loi ha cht khc: thch, tinh bt tan, casein, CMC (Carboxyl Methyl Cellulose)... ca Nht, Trung Quc, Vit Nam sn xut. - Cc dung mi: etanol, iso-propanol, metanol, aceton.. ca Trung Quc. 2.1.2.2. Dng c v thit b - KHVQH Olympus (Nht) - KHVT Joel (Nht) - My lc (Hn Quc) - Box cy v trng Nuaire (M) - My ly tm Hettich (c) 2.1.2.3. Cc cng thc mi trng Mi trng phn lp v gi ging nm *Mi trng CZ (Czapek - Dox - Agar) (g/l) NaNO3 - 2; KH2PO4 - 1; MgSO4. 7H2O - 0,5; KCl - 0,5; FeSO4 - 0,01; Thch - 20; H2O - 1lt. *Mi trng khoai ty (PDA - Potato Dextrose Agar) (g/l) Khoai ty ming - 200; Thch - 18; ng - 20, H2O 1lt; pH 7 - 1,4. Mi trng phn lp, gi ging v nghin cu x khun * Mi trng Gause - I (g/l) Tinh bt tan - 20; K2HPO4 - 0,5; MgSO4. 7H2O - 0,5; NaCl - 0,5; (NH4)2SO4 2; KNO3 - 0,5; FeSO4 - 0,01; Thch - 18 g; H2O - 1lit; pH 7 - 7,4. * Mi trng Gause - II (g/l) Nc chit tht - 30ml; pepton - 5; NaCl - 5; glucoza - 10; Thch - 18g; H2O 1lt; pH 7 - 7,4. * Mi trng ISP - 1 (g/l) - T m, t sy Memmert (c) - Cn phn tch, cn k thut - Ni kh trng (i Loan) - My o pH 151 Martini - My ct nc Hamilton.
- Cc dng c thy tinh ca Trung Quc, c, Vit Nam...
-26Trypton - 5, cao nm men - 3; thch - 18; H2O - 1lt; pH 7 - 7,2. * Mi trng ISP -2 (g/l) Cao nm men - 3; Cao malt - 10; Dextrose - 4; Thch - 20; Nc ct - 1lit; pH 7 - 7,2. * Mi trng ISP - 3 (g/l) Bt kiu mch - 20; Mui A - 1ml; Thch - 20; Nc ct - 1lit; pH - 7,2. * Mi trng ISP - 4 (g/l) Tinh bt tan - 10; K2HPO4 - 1,0; MgSO4. 7H2O - 1,0; NaCl - 1,0; (NH4)2SO4 2; CaCO3 - 2; dung dch khong - 1ml; thch - 18g; H2O - 1lt; pH 7 - 7,2. * Mi trng ISP - 5 (g/l) Asparagin - 1; Glixerin - 10; K2HPO4 - 1; Mui A - 1ml; Thch - 20; Nc ct - 1lit; pH 7,0 - 7,4. * Mi trng ISP - 6 (g/l) Peptone - 10; cao nm men - 1; xitrat st - 0,5; thch - 18; H2O - 1lt; pH 7 - 7,2. * Mi trng ISP - 7 (g/l) Glycerol - 15; L - tyrosine - 0,5; L - asparagine - 1; FeSO4.7H2O - 0,01; K2 HPO4 0,5; MgSO4. 7H2O - 0,5; Nacl - 0,5; Mui A - 1ml; Thch - 20; Nc ct 1lit; pH 7,2 - 7,4. * Mi trng ISP - 9 (g/l) (NH4)2SO4 - 2,64; KH2PO4 - 2,38; K2HPO4 - 5,65; MgSO4 - 1; dung dch mui B - 1ml; ngun cacbon - 10; thch - 20; H2O - 1lt; pH 6,8 - 7. - Dung dch mui A (%): FeSO4 - 0,1; ZnSO4 - 0,15; MnCl2 - 0,1; Nc ct - 100ml. - Dung dch mui B (%): CuSO 4 - 0,64; FeSO4 - 0,11; MgCl2 - 0,79; nc ct - 100ml. * Mi trng 79 (g/l)
-27Glucoza -10; peptone -10; cazein thy phn - 2; cao nm men - 2; NaCl - 6, K2HPO4 - 0,2; thch - 18; H2O - 1lt; pH 7 - 7,4. * Mi trng A4 - H (g/l) Glucoza - 15; bt u tng - 15; NaCl - 5; CaCO3 - 1; H2O - 1lt; pH 7. * Mi trng A - 4 (g/l) Glucoza - 10; bt u tng - 10; NaCl - 5; CaCO3 - 1; H2O - 1lt; pH 7. * Mi trng Tinh bt (g/l) Tinh bt - 20; K2HPO4 - 0,5; MgSO4 - 0,5; KNO3 - 1; NaCl - 0,5; FeSO4 0,01; Agar - 30,0; Nc - 1 lt; pH 7,2 - 7,4. * Mi trng Czapek Glycerin (g/l) Glycerin - 30,0; NaNO3 - 2,0; K2HPO4 - 1.0; MgSO4 - 0,5; KCl - 0,5; FeSO4 0,01; Agar - 20,0; Nc - 1 lt. * Mi trng Czapek Gluco (g/l) Gluco - 30,0; NaNO3 - 2,0; K2HPO4 - 1.0; MgSO4 - 0,5; KCl - 0,5; FeSO4 0,01; Agar - 20,0; Nc - 1 lt. 2.2 Phng php nghin cu 2.2.1. Phng php phn lp nm gy bnh t cc mu ch Chn cc mu ch c triu chng in hnh, mi. Ra mu bnh sch t ct di vi nc,ct chn mnh m bnh thch hp. Kh trng b mt cc mnh m trn bng cn (etanol 70%) trong vng 3 pht. Ra li bng nc ct v trng, thm kh mnh m bng giy thm v trng. Dng dao m v trng ct cc mnh m trn thnh cc mnh nh 1 - 3mm (cha c phn m bnh v m khe). Dng panh v trng t cc mnh m nh trn vo mi trng Czapek trn a petri. Ghi ch cn thn bng bt vit knh: ngy, cy, bnh.. a mi trng t mu trong t m 300C. Theo di s pht trin ca si nm mc ra t m bnh. Khi nm pht trin t m bnh ra mi trng, ly phn nh si nm chuyn sang mi trng thch hp: PDA hoc
-28CZ. Thu nhn ging thun khit trong ng thch nghing v bo qun trong t lnh 40C [14]. 2.2.2. Phn lp v tuyn chn x khun 2.2.2.1. Phn lp x khun theo Vinogradski Cn 1g t cho vo bnh nn 50 ml cha 9ml nc ct v trng, lc trn my lc 30 pht. Dng pipet v trng ht 0,5 ml dch t sang ng nghim c cha 4,5 ml nc v trng v tip tc pha long n 10-2, 10-3...10-6. T mi nng pha long cc nng 10-3 n 10-6, nh 0,1ml sang a Petri cha mi trng Gause - I. Dng que gt v trng chang u, sau nui nhit phng t 4 - 7 ngy. Trn mi mu t, khun lc ca x khun c cy 3 pha sang a Petri cha mi trng Gause - I lm sch. Sau nui nhit phng 4 - 7 ngy, t cc khun lc c lm sch cy truyn sang ng nghim cha mi trng thch nghing Gause - I, tip tc nui iu kin trn trong 10 - 14 ngy [12]. Theo ISP mu sc khun ty kh sinh ca cc chng x khun c chia thnh 8 nhm mu: White (W) nhm trng, Gray (Gy) nhm xm, Red (R) nhm , Yellow (Y) nhm vng, Green (Gn) nhm xanh, Blue (B) nhm xanh da tri, Violet (V) nhm tm, v nhm mu khng xc nh (X) [16],[19], [42]. 2.2.2.2. Xc nh hot tnh khng sinh Phng php thi thch (dng s tuyn X khun) X khun c nui cy trn mi trng ISP - 4 hoc Gause - I trong a petri sau 7 ngy dng khoan nt chai khoan cc thi thch t vo a petri cy VSV kim nh. Sau vo t lnh 4 - 5h cho CKS khuch tn vo mi trng thch sau vo t m. VSV kim nh nui 28 300C, c kt qu sau vi ngy [7].
-29HTKS c xc nh da vo kch thc vng v khun (D - d, mm), D l kch thc vng v khun v d l ng knh thi thch. Phng php c l (Xc nh hot tnh khng sinh trong dch th) Dng khoan nt chai c cc l trn mi trng thch cy VSV kim nh trong a petri. Nh vo cc l khoan dung dch cn th (cc bc tip theo tin hnh nh phng php thi thch). Phng php khoanh giy lc (Xc nh hot tnh khng sinh trong dung mi) Khoanh giy lc F8 c ng knh 6mm c tm mt lng dch CKS (1ml/10 khoanh giy lc). Sau sy kh 400C, t khoanh giy lc tm CKS vo a petri cy VSV kim nh (cc bc tip theo ging phng php thi thch). 2.2.2.3. Tuyn chn cc chng x khun sinh cht khng sinh T ng thch nghing ging hot ha c cy truyn sang cc bnh nn dung tch 250 ml cha 100 ml mi trng Gause - I v nui lc 220 vng/ pht trong 48 gi nhit phng. Ging pht trin c cy truyn vi t l 10% sang bnh nn dung tch 250 ml cha 80 ml mi trng Gause - I dch th. Qu trnh ln men ko di 96 gi nhit phng trn my lc 220 vng /pht. Sau th HTKS trong dch lc [7]. 2.2.3. Bo qun ging Cc chng x khun la chn c cy trn mi trng thch nghing Gause - I nhit phng. Sau 10 - 14 ngy khi x khun mc tt, cc ng ging c bo qun trong t lnh 4 - 60C. Sau 2 - 3 thng cy truyn li mt ln. bo qun lu ging c gi trong ng ct, trong parafin lng v trng gi lnh su trong glycerin hoc ng kh, bo qun c 6 thng n 2 nm.
-302.2.4. Nghin cu cc c im sinh hc v phn loi x khun 2.2.4.1. c im hnh thi Quan st mu sc ca h si kh sinh Da theo ti liu ca ISP, Gause v cng s (1983), xc nh mu sc ca HSKS da vo bng mu ca Tresner v Bakus. Cn c vo mu sc HSKS ca cc chng mi phn lp phn thnh cc nhm mu theo Gause v cng s: White (W) nhm trng, Gray (Gy) nhm xm, Red (R) nhm , Yellow (Y) nhm vng, Green (Gn) nhm xanh... Quan st mu sc ca h si c cht Mu sc ca HSCC c xc nh qua quan st trc tip trn mi trng thch a hoc thch nghing v m t theo thang mu chun ca Bondarsev (1953), Tresner v cng s (1961). Quan st cung sinh bo t X khun c nui cy trn mi trng Gause - I c gm lamen nghing 450 trn b mt mi trng. Sau 7 - 9 ngy nui nhit phng ly ra quan st hnh dng chui sinh bo t trn lamen di knh hin vi quang hc. Chui sinh bo t c cc dng thng hay hi ln sng k hiu l RF (Rectiflexibiles), hnh mc cu hay hnh xon khng hon ton k hiu l RA (Retinaculiaperti) v xon hon ton k hiu l S (Spirales) [33], [35], [41]. Quan st b mt bo t Dng mng cacbon t trc tip ln b mt khun ty kh sinh c bo t, sau quan st v chp nh di knh hin vi in t qut. Bo t c cc hnh dng: trn, ovan, elp, hnh que [33]. B mt bo t x khun c cc dng: Nhn k hiu l Sm (Smooth), dng mn cc k hiu l Wa (Warty), dng gai k hiu l Sp (Spiny) v dng tc k hiu l Ha (Hairy).
-312.2.4.2. c im nui cy Mu sc khun ty kh sinh, khun ty c cht v sc t tan X khun c nui cy trn mi trng: Gause - I, Gause - II, ISP - 1, ISP - 2, ISP - 3, ISP - 4, ISP - 5, ISP - 6, ISP - 7 nhit phng. Sau 7, 14, 21 ngy ly ra quan st mu sc KTKS, KTCC v sc t tit ra mi trng [33], [37]. S hnh thnh sc t melanin X khun c nui cy trn mi trng ISP - 6 nhit phng. Bt u quan st mu ca mi trng sau 24 gi cho n ngy th 14. Nu sinh melanin, mu ca mi trng s chuyn t mu vng nht sang mu nu m cho n mu en [33]. 2.2.4.3. c im sinh l - sinh ha Kh nng sinh enzym ngoi bo - Chit dch enzym Thu enzym t mi trng lng: x khun sau khi c nui trn mi trng Gause II dch th s c lc b sinh khi hoc ly tm 5000 vng/ pht trong 15 pht, chit dch trong th thu c enzym th. - Xc nh hot tnh cc enzym ngoi bo bng phng php khuch tn trn thch vi mt trong cc ngun c cht sau: - Tinh bt tan (xc nh hot tnh amylaza) - Cazein (xc nh hot tnh proteaza) - CMC (xc nh hot tnh endoglucanaza). Chun b mi trng c cht - thch gm: 20g thch + 2 - 10g c cht, pha mi trng trong 1000 ml nc. Thanh trng 1 atm trong 30 pht. mi trng vo cc a Petri sao cho b dy lp thch khong 3 mm. Dng khoan nt chai c l (d = 10 mm) trn lp thch cch nhau 40 mm. Nh 0,2 ml dung dch enzym cn th v 0,2 ml nc lm i chng, t lnh 40C khong 4 gi, sau t vo t m 300C trong 24 gi [23].
-32Hot tnh enzym c xc nh bng gi tr D - d (mm). Sau khi nhum mu Cng g (c cht CMC), axit tricloaxetic (c cht casein) v thuc th Lugol (c cht tinh bt). Vng c cht b phn gii khng bt mu xung quanh l thch. Kh nng chu mui Cy x khun trn mi trng thch nghing ISP - 1 c b sung thm NaCl vi cc nng 0,5; 3; 7; 9; 11; 12 (%). Sau 7 - 10 ngy ly ra quan st s sinh trng. Kh nng s dng cc ngun cacbon X khun c nui cy trn mi trng ISP - 9 c b sung 1% cc ngun ng: Glucoza, maltoza, fructoza, lactoza, sacaroza.... Cch lm: Cn 1g ng, thanh trng bng n UV ri b sung vo 100ml mi trng ISP - 9 cn nng. Lc cho tan ng ri vo a Petri v nui cy x khun nhit 28 -300C, sau 14 ngy quan st s sinh trng ca cc chng v so snh vi i chng [33]. Trong mi trng c glucoza c coi l i chng dng (+) v mi trng khng c ng c coi l i chng m (-). - Nu x khun sinh trng mnh bng hoc km hn i chng dng mt t: c kh nng s dng loi ng . k hiu l (+). - Nu x khun sinh trng mnh hn i chng dng, k hiu l (++). - Nu x khun sinh trng bng i chng m hoc khng mc: khng c kh nng s dng loi ng , k hiu l (-). - Nu x khun sinh trng tt hn i chng m mt t nhng km hn i chng dng nhiu, k hiu l (). 2.2.5. Ln men to khng sinh 2.2.5.1. La chn mi trng ln men thch hp X khun c ln men trn cc mi trng c bn (theo Porter) l:
-33A - 4, A - 4H, A - 9, ISP - 4, Gause - I v Gause - II. Sau 96 gi nui cy trn my lc 220 vng/ pht nhit phng. Xc nh HTKS trong dch ln men (phng php c l) v sinh khi (phng php khoanh giy lc) chn ra mi trng c bn ph hp cho nhng nghin cu tip theo [34]. 2.2.5.2. nh hng ca ngun cacbon B sung ngun cacbon (%): Tinh bt tan 1 v 2; glucoza - 1,5; lactoza 1,5; sacaroza - 1,5; r ng - 1,5; vo hn hp dung dch mui (dung dch mui A) b sung 0,2 % (NH4)2SO4. B sung ging v ln men trn my lc trn vi tc 220 vng/ pht. Sau 96 gi ln men xc nh HTKS trong dch ln men bng phng php c l thch. 2.2.5.3. nh hng ca ngun Nit B sung ngun Nit (%): Cao nm men, bt u tng, (NH4)2SO4 v KNO3 vo hn hp dung dch mui b sung thm 1,5% glucoza. Sau b sung ging ri ln men v th HTKS bng phng php c l. 2.2.6. Cc phng php sinh hc phn t trong phn lp gen 16S - rRNA 2.2.6.1. Tch chit DNA ca x khun bng m CTAB Nguyn tc: Ph v thnh t bo ca x khun nh lyzozym. Loi b protein khi DNA nh cc enzym protease K. Phn tch cc thnh phn ca t bo sau khi ph v thnh hai pha nh dung dch m chlorofom: isoamyl alcohol (24:1): pha trn cha cn t bo, pha di cha axit nucleic. Sau cng, DNA c ta bng ethanol tuyt i ( c lm lnh - 22), c lm kh v ha tan trong m TE [39]. Tin hnh: Ly 1,5ml dch nui cy t bo em ly tm tc 3000 vng/ pht trong 30 pht thu cn t bo.
-34Ha tan cn t bo trong 0,8 ml TE 25S, lc u bng vontex. B sung 40 l lysozym, 370C trong 90 pht (nng cui cng ca lysozym l 2mg/ ml). B sung 10 l protease K, mix u, thm 60 l 10% SDS, o nh, 1gi nhit 550C (nng cui cng ca protease K l 0,18mg/ml, SDS l 0,5%). B sung 150 l NaCl 5M, mix u. Sau b sung 130 l CTAB, mix u, 10 pht 550C. t nhit phng 5 pht, chia u ra 2 ng eppendorf mi. Sau b sung vo mi ng 700 l chlorofom/ isoamyl alcohol, mix u 15 pht. Ly tm 9000 vng/pht trong 20 pht tch pha. Chuyn dch pha trn sang cc eppendorf mi v b sung 0,6V isopropanol, o nh. t trong t - 200C trong 30 - 40 pht. Ly tm 9000 vng/pht, ra ta bng etanol 70%. Ha tan DNA trong 100 l TE. 2.2.6.2. Khuch i gen rRNA - 16S bng phn ng PCR Nguyn tc: PCR l phn ng trng hp, cho php khuch i mt on DNA c th ln hng triu ln nh s xc tc ca DNA polymerase chu nhit. Enzym ny c kh nng hot ng nhit cao. S hot ng ca enzym c kim sot ca mt chu k nhit xc nh. on DNA c tng hp c gii hn v mt kch thc bi mt cp mi c hiu. Cp mi c s dng trong nghin cu c trnh t l: Trnh t mi: Mi xui 27 Mi ngc 1525 Tin hnh: 5' - agagtttgatcctggctcag - 3' 5' - aaaggaggtgatccagcc - 3' 27- 47 1525 -1507
-35Thnh phn phn ng H2O Buffer (10X) dNTP (2,5mM) Primer 1 (10pml) Primer 2 (10pml) ADN khun Enzyme Taq Tng 17,1l 940C 2,5l 940C 2l 64 C 1l 720C 1l 1l 0,4l 25l 4C
0 0
Chu k nhit 3 pht 1 pht 1 pht 1 pht 30 giy
2.2.5.3. Phng php in di trn gel agarose Phng php in di trn gel agrose c s dng kim tra DNA plasmid, kim tra cc phn on DNA c nhn bng k thut PCR v kim tra kt qu ct gii hn. Phng php ny c chia lm mt s bc nh sau: - Chun b gel agarose: cn mt lng agarose tng ng (ty theo nng gel yu cu) vo trong dung dch 100ml TAE 1X. un si bng l vi sng, ngui xung khong 500C ri dung dch vo bn gel ci sn lc. Sau khong 30 pht, t bn gel vo b in di v dung dch in di TAE ngp bn gel. - Tra mu: ly 1 - 2 g DNA pha long trong 16 l m TE, sau b sung 4 l dye mu. Mt lng DNA maker tng ng cng c tra vo bn gel trc khi in di. - in di: in di ti hiu in th khng i 100V trong khong 20 - 30 pht. Quan st s di ng ca DNA t cc m sang cc dng thng qua dye mu.
-36- Nhum bn gel: Bn gel sau in di c ngm vo dung dch thuc nhum EtBr nng 2 g/ml trong khong 10 pht, lc nh trn my lc, sau soi gel trn n UV v chp nh bng my chp gel ca hng Bio - Rad. 2.2.7. Phng php x l s liu Kt qu nghin cu c chng ti x l theo phng php thng k sinh hc trn phn mm Excel.
-37-
Chng 3 KT QU V THO LUN

3.1. Phn lp v thun khit cc chng nm gy bnh trn ch T cc mu ch b bnh thu thp cc khu vc khc nhau ca tnh Thi Nguyn, chng ti phn lp v thun khit c 3 chng nm c k hiu l CT - 1A, CT - 2E v CT - 5X. Cc chng nm phn lp c u c c im chung l: cc si nm u khng c mu sc, si nm c vch ngn (hnh 3.1). Mi chng nm c phn lp t mt mu ch c biu hin bnh khc nhau. Kt qu c trnh by trn bng 3.1. Bng 3.1. c im mt s mu ch lm ngun phn lp cc chng nm K hiu chng nm CT - 1A Ngun phn lp L ch non m nu Vt bnh c mu nu sm, lc u CT - 2E L ch non ch c chm nh mu en, sau lan ra khp l. CT - 5X L ch non Cc l b xon, trn mt l c th b nhn. Biu hin bnh Trn b mt l v mp l c nhiu
Sau khi c tinh sch, 3 chng nm trn c gi trong ng thch nghing trn mi trng Czapek v c s dng lm VSV kim nh tuyn chn x khun sau ny.
-38-
Chng CT - 1A
Chng CT - 2E
Chng CT - 5X Hnh 3.1. Ba chng nm phn lp t cc mu ch b bnh 3.2. Phn lp v tuyn chn x khun 3.2.1. Hot tnh khng nm ca cc chng x khun T cc mu t ly su 5 - 10 cm ti cc a im khc nhau thuc tnh Thi Nguyn, chng ti phn lp v thun khit c 80 chng x khun thuc chi Streptomyces. Sau khi th HTKS theo phng php khuch tn trn thch vi 3 chng nm CT - 1A, CT - 2E, CT - 5X, chng ti tuyn chn s b c 30 chng trong tng s 80 chng x khun c hot tnh khng nm cc mc khc nhau, chim t l 37,5%. So snh vi t l chng x khun khng nm gy bnh o n cng b trc y ca L Gia Hy v cng s [18], t l chng x khun c hot tnh khng nm gy bnh trn ch ca chng ti c phn cao hn.
-39Bng 3.2. X khun c hot tnh khng nm theo nhm mu Nhm mu x khun Trng (Allbus) Xm (Griseus) Xanh (Azeureus) Hng (Roseus) Nu (Chromogenes) Lc (Viridis) Tng cng Cc chng c HTKN S lng T l(%) 11 8 7 2 1 1 30 36,7 26,7 23,3 6,7 3,3 3,3 100 T l chng c HTKN so vi tng s (%) 13,7 10,0 8,6 2,6 1,3 1,3 37,5
T l cc chng x khun c hot tnh khng nm phn chia theo nhm mu ca Shirling v Gottlieb [37]. Kt qu c th hin trn bng 3.2 v hnh 3.2.
Hnh 3. 2. T l cc chng x khun c hot tnh khng nm theo nhm mu
-40Kt qu trn bng 3.2 cho thy: s lng chng c hot tnh nhiu nht l nhm trng chim 36,7%, sau l nhm xm - 26,7%, nhm xanh 25,3%, nhm hng - 6,7% v t nht l cc nhm nu v lc u chim 3,3%. Kt qu ny ca chng ti cng ph hp vi nhng nghin cu ca mt s tc gi trc y [14],[18]. ng thi chng ti cng nhn thy kh nng i khng ca cc chng x khun vi 3 chng nm gy bnh trn ch c s khc nhau (bng 3.3). Bng 3. 3. Tnh i khng ca x khun vi 3 chng nm gy bnh trn ch Chng XK c HTKN S lng T l (%) CT - 1A 9 30 3 chng nm gy bnh ch CT - 2E 16 53 CT - 5X 13 43 Khng c 3 chng 4 13,3
Trong tng s 30 chng c hot tnh khng nm, s chng khng nm CT - 2E l cao nht, c 16 chng (chim 53% ), tip theo l s chng khng nm CT - 5X, c 13 chng (chim 43%) v t nht l chng khng nm CT - 1A c 9 chng (chim 30%).
Hnh 3.3. T l cc chng x khun c hot tnh khng nm
-41ng ch trong s , c 4 chng c kh nng khng c c 3 chng nm, chim t l 13,3%, cc chng cn li phn ln ch khng c 1 n 2 chng nm. T l cc chng khng nm c th hin trn hnh 3.3. 3.2.2. Tuyn chn cc chng x khun c HTKN cao Sau khi tuyn chn s b c 30 chng c hot tnh khng nm, chng ti chn ra 2 chng c hot tnh khng nm mnh nht c k hiu l: 1 v R2. bc ny, cc chng c nui lc trong mi trng dch th ISP - 4 thu dch nui cy. Kt qu kim tra hot tnh ca dch nui cy theo phng php c l vi cc chng nm nh trn c th hin trn bng 3.4 v hnh 3.4. Bng 3. 4. Hot tnh khng nm ca 3 chng x khun Chng x khun 1 R2 Hot tnh khng nm (D- d, mm) CT - 1A 12 12 CT - 2E 14 16 CT - 5X 15 17
Kt qu trn bng 3.4 cho thy: mc d c 2 chng x khun la chn u vn gi c hot tnh khng nm, song mc khng i vi cc chng nm c s khc nhau. Kh nng khng nm CT - 1A ca c 2 chng x khun u yu nht v kh nng khng chng CT - 5X ca 2 chng x khun l cao nht. Chng ti tip tc tin hnh nghin cu cc c im sinh hc v phn loi ca 2 chng x khun 1 v R2. Hai chng x khun ny thuc hai nhm mu khc nhau. Chng 1 thuc nhm mu trng v chng R2 thuc nhm mu nu.
-42-
D1 R2 D1 C C R2
VSV kim nh: CT - 5X
VSV kim nh: CT - 2E
Hnh 3.4. Hot tnh khng nm ca 2 chng x khun la chn 3.3. c im sinh hc v c im phn loi ca 2 chng XK 1 v R2 3.3.1. c im hnh thi Chng R2 c cung sinh bo t dng xon, b mt bo t c dng gai. Chng 1 c cung sinh bo t dng thng, b mt bo t x x.
Hnh 3. 5. Cung sinh bo t v b mt bo t chng R2
-43-
Hnh 3.6. Cung sinh bo t v b mt bo t chng 1 3.3.2.c im nui cy X khun c nui cy trn cc mi trng khc nhau quan st kh nng sinh trng ca x khun v mu sc ca h khun ty. Kt qu cho thy x khun rt a dng v mu sc khi nui cy trn cc mi trng khc nhau. Ty theo thnh phn ca mi trng nui cy m mu sc khun ty c cht v khun ty kh sinh hay sc t ha tan tit ra khc nhau. Cc c im nui cy ca 2 chng R2 v 1 c trnh by trn bng 3.5 cho thy: i vi chng 1: khi nui cy trn cc mi trng ISP - 1, ISP - 2, ISP - 3, ISP - 5, ISP - 7 mu sc ca KTKS v KTCC u c mu trng. Trn mi trng ISP - 4 mu sc HSCC c mu xm v HSKS c mu trng. mi trng ISP - 6 mu sc ca HSKS c mu vng v HSCC c mu trng. i vi chng R2: mu sc ca HSCC v HSKS c a dng hn khi nui cy trn cc mi trng ISP khc nhau.
-44Bng 3. 5: c im nui cy ca chng R2 v 1 Mi trng ISP - 1 ISP - 2 ISP - 3 ISP - 4 ISP - 5 HSCC Trng Trng Trng Xm Trng Trng Trng Chng 1 HSKS Trng Trng Trng Trng Trng Sc t Khng mu Khng mu Khng mu Khng mu Khng mu HSCC Nu Nu Trng ng Trng Trng ghi nu Nu Nu Chng R2 HSKS Trng Xm ghi Xm ghi Xm Trng ghi xanh Xm ghi (yu) Xm ghi Sc t Khng mu Khng mu Khng mu Khng mu Khng mu
ISP - 6 ISP - 7
Vng Trng
Khng mu Khng mu
Melanin Nu
* Kh nng hnh thnh sc t melanin Chng R2 v 1 c nui cy trn mi trng ISP - 6 nhit 300C. Sau 14 ngy quan st chng ti nhn thy:
Chng R2
Chng 1
Hnh 3.7. Kh nng hnh thnh sc t melanin ca 2 chng
-45i vi chng 1 mu sc ca mi trng khng c s bin i, tc l khng c kh nng hnh thnh sc t melanin. i vi chng R2 mu sc ca mi trng bin i t mu vng nht sang mu nu m, iu c ngha l chng R2 c kh nng hnh thnh sc t melanin trn mi trng c cha st. 3.3.3. c im sinh l - sinh ha * Kh nng ng ha cc ngun cacbon nh gi kh nng ng ha cc ngun cacbon khc nhau, chng ti tin hnh nui 2 chng R2 v 1 trn mi trng ISP - 9 c b sung cc ngun cacbon khc nhau vi nng 1%. Sau 7 - 14 ngy nui cy, kt qu c th hin trn bng 3. 6. Bng 3.6. Kh nng ng ha ngun cacbon ca 2 chng x khun 1 v R2 Mc sinh trng Chng 1 ++++ ++++ ++++ ++++ ++ + Chng R2 ++++ ++ ++ ++++ ++++ ++++ + +++
Ngun cacbon Glucose Saccharose Fructose Lactose Manitol Dextrin Cellulose Arabinose
Ghi ch: ++++: Sinh trng rt tt; +++: Sinh trng tt; ++: Sinh trng yu; +: Sinh trng rt yu; - :Khng sinh trng
-46Kt qu trn bng 3.6 cho thy: C 2 chng x khun nghin cu u c kh nng ng ha tt cc ngun cacbon khc nhau. Song chng R2 ng ha tt nht ngun cacbon l: Glucose, Lactose, Manitol, Dextrin v sinh trng yu nht trong mi trng c cha ngun cacbon Cellulose. i vi chng 1 c kh nng ng ha tt ngun cacbon Glucose, Saccharose, Fructose, Manitol v khng c kh nng ng ha 2 ngun cacbon l Lactose, Arabinose. * Nhit sinh trng thch hp Chng R2 v 1 c nui cc thang nhit khc nhau. Kh nng sinh trng ca 2 chng c th hin trn bng 3.7. Bng 3. 7: Nhit sinh trng thch hp ca 2 chng XK
Nhit Chng
250 + ++
280 +++ +++
300 +++ +++
320 ++ +
350 + +
400 + -
R2 1
Ghi ch: +++: Sinh trng tt; ++: Sinh trng bnh thng; +: Sinh trng yu; -: Khng sinh trng. Kt qu trn bng 3.6 cho thy, hai chng R2 v 1 c kh nng sinh trng trong khong nhit t 250 - 320C, chng R2 sinh trng tt nht nhit 280 - 300C v sinh trng yu nhit 350 - 400C. Chng 1 cng sinh trng tt nhit 280 - 300C v khng sinh trng nhit 400C. Nh vy nhit thch hp cho c 2 chng pht trin tt l 28 - 300C. * Kh nng chu mui Hai chng R2 v 1 c nui cy trong mi trng ISP - 1 c b sung NaCl cc nng 0,5; 3; 7; 9; 11; 12 v 0% i chng. Kt qu thu c bng 3.8.
-47Bng 3.8: Kh nng chu mui ca 2 chng R2 v 1 Nng NaCl (%) Chng R2 1 +++ +++ + ++ ++ + + + + + 0,5 3 5 7 9 12 0
Ghi ch: +++: Sinh trng tt; ++: Sinh trng bnh thng; +: Sinh trng yu; -: Khng sinh trng. Nng mui c nh hng n kh nng sinh trng ca x khun. Kt qu trnh by trn bng 3.8 cho thy: c 2 chng u c kh nng s dng nng mui ti 9% , nng mui cao hn thi c 2 chng u khng pht trin c. Nh vy nng mui 0,5% chng x khun R2 v 1 u c tc dng kch thch s sinh trng. * Kh nng sinh enzym ngoi bo
C R2 D1 R2
D1
CMC
Casein
R2 D1
Tinh bt Hnh 3.8. Hot tnh enzym ca cc chng x khun

S ha bi Trung tm Hc liu i hc Thi Nguyn http://www.lrc-tnu.edu.vn
-48Trong qu trnh sng, phn gii cc hp cht hu c phc tp thnh cc hp cht n gin c th hp thu c. X khun c kh nng tit vo mi trng cc enzym ngoi bo. Chng ti tin hnh kim tra kh nng ny ca 2 chng x khun nghin cu, kt qu c th hin trn hnh 3.8 cho thy c 2 chng x khun u c kh nng thy phn mnh casein, tinh bt v CMC. 3.3.4. Hot tnh khng sinh ca 2 chng R2 v 1 Hot tnh khng sinh ca x khun cng l mt trong cc ch tiu phn loi. Cng vi vic nghin cu kh nng khng nm gy bnh trn ch. Chng ti cng kim tra kh nng khng mt s chng nm kim nh gy bnh thc vt nh nu trong mc 2.1.1.3. Kt qu c th hin trn bng 3.9 v hnh 3.9. Bng 3.9: Hot tnh khng sinh ca 2 chng R2 v 1 vi 3 chng nm kim nh Chng x khun R2 1 Hot tnh khng sinh (D - d, mm) F. oxysporum 22 18 F. moniliforme 15 19 R. solani 17 0
Kt qu trn bng 3.9 cho thy: chng R2 c ph c ch cc VSV kim nh kh rng, c kh nng khng c 3 loi nm. Tuy nhin chng R2 c kh nng khng nm F. oxysporum l mnh nht vi vng c ch l 22 mm, cn vi nm R. solani l 17 mm v nm F. moniliforme l 15 mm (hnh 3.9). Chng 1 ch c hot tnh chng nm F. oxysporum v F. moniliforme m khng c kh nng khng nm R. solani.
-49Kh nng i khng cc chng VSV cng vi cc c im v hnh thi, sinh l, sinh ha c dng tham kho trong phn loi cc chng x khun ny. T cc kt qu nghin cu trn cho thy 2 chng x khun 1 v R2 khng ch c kh nng khng nm gy bnh trn ch m cng c kh nng khng c cc nm gy bnh thc vt nh F. oxysporum, F. moniliforme, R. solani.
F.oxysporum m
D1 D1 R2
VSV kim nh: F. oxysporum

C
R2 VSV kim nh: F. monilforme C
F. oxysporum
F. moniliforme
D1
VSV kim nh: R. solani

C R2
R. solani. Hnh 3.9. Hot tnh khng 3 chng nm kim nh ca 2 chng 1 v R2
-503.3.5. V tr phn loi ca hai chng x khun R2 v 1 * Phn loi chng R2 Bng 3.10. So snh c im phn loi ca chng R2 vi S. misawaensis c im phn loi Hnh dng cung Hnh thi Mi trng ISP - 6 sinh bo t B mt bo t Kh nng sinh melanin Mu KTCC Mi trng tinh bt Sc t tan Mi trng Czapek Glycerin Sinh khng sinh KTCC KTKS Sc t tan Vng nht Nu ghi Trng hng Melanin C Chng R2 Xon (S) C gai ngn C Loi chun S. misawaensis Xon (S) C gai ngn C Vng hoc vng nu Xm hoc xm trng Vng n vng nu Nu ghi Trng hng Melanin Acwaimycin
Vng nu
Mu KTKS
Xm
i chiu vi kha phn loi x khun ca Gause, 1983 [49]. Chng ti nhn thy chng R2 c nhiu c im ging vi loi S. misawaensis. Kt qu so snh cc c im ca chng R2 vi loi S. misawaensis c trnh by trn bng 3. 10. Chng R2 c cung sinh bo t dng xon, b mt bo t c gai ngn. Khi nui cy trn cc mi trng tinh bt mu sc KTCC t mu trng n
-51vng nu, cn mu sc KTKS bin i thnh mu xm hoc xm trng. Trn mi trng Czapek Glycerin mu sc ca KTKS t mu trng bin i thnh mu trng hng, cn KTCC bin i thnh mu nu ghi. Chng R2 c kh nng sinh sc t melanin trn mi trng c cha st. Khi so snh cc c im phn loi ca chng R2 vi loi chun S. misawaensis trong kha phn loi ca Gauze, 1983. Chng ti nhn thy chng R2 c nhiu c im ging vi loi ny v hnh thi, cung sinh bo t, b mt bo t, c im nui cy, kh nng hnh thnh sc t melanin v CKS. Nh vy, chng R2 rt c th thuc loi S. misawaensis, nhm Cinereus, seri: chromogenes. Chng ny c Hamada et Okami, m t vo nm 1968c [28]. * Phn loi chng 1 c im phn loi ca chng 1 c th hin trn bng 3.11 cho thy: chng 1 c cung sinh bo t dng thng, b mt bo t x x. Khi nui cy trn mi trng Gause - I, mu sc ca KTCC t mu vng nht bin i thnh mu vng nu, mu sc KTKS c mu trng. Trn mi trng Gause - II, mu sc ca KTKS t mu vng bin i thnh mu nu, KTCC khng c mu. Trn mi trng Czapek Glucoza, mu sc ca KTCC t mu trng bin i thnh mu nu vng, mu ca KTKS c mu trng nh kem. Chng 1 khng c kh nng hnh thnh sc t melanin. i chiu vi kha phn loi ca Gauze, 1983, chng ti nhn thy chng 1 c nhiu c im ging vi loi Actinomyces brunneofungus v cc c im hnh thi, cung sinh bo t, kh nng hnh thnh sc t melanin v c im nui cy, kh nng hnh thnh CKS chng nm.
-52Nh vy, c th chng 1 thuc loi A. brunneofungus, nhm Albus, seri: albocolorabus. Chng ny c Krasilnikov et al, 1971[48]. Bng 3.11. So snh c im phn loi ca chng 1 vi A.brunneofungus c im phn loi Hnh dng cung Hnh thi Mi trng ISP - 6 sinh bo t B mt bo t Kh nng sinh melanin Mu KTCC Gauze - I Mu KTKS Sc t tan Mu KTCC Gauze - II Mu KTKS Sc t tan Mu KTCC Czapek gluco Mu KTKS Sc t tan Sinh khng sinh Chng 1 Thng (RF) X x Khng Vng nu Trng Khng Khng mu Nu Khng Nu vng Trng nh kem Yu C Loi chun A.brunneofungus Thng v ln sng X x Khng Vng nu Trng Khng c Khng mu Nu Khng Nu hoc nu vng Trng nh kem Yu Flavofungina m t vo nm
3.4. Kh nng sinh tng hp CKS ca 2 chng x khun la chn 3.4.1. La chn mi trng ln men thch hp Trong cng ngh ln men, mi trng ln men ng vai tr quan trng. Mt mi trng ln men tt phi l mi trng va thun li cho chng sinh
-53trng tt va cho hiu sut khng sinh cao. la chn mi trng ln men p ng c c hai iu kin trn, chng ti s dng 5 loi mi trng ln men c s l: A - 4H, A - 4, Gause - I, Gause - II v ISP - 4. Qu trnh ln men c tin hnh trong bnh nn dung tch 250 ml c cha 25ml mi trng. Sau 120 gi nui lc nhit 280C - 300C, thu dch ln men, xc nh HTKS bng phng php c l. Kt qu c th hin bng 3.12 v hnh 3. 12 Bng 3.12: Hot tnh khng sinh ca 2 chng x khun trn cc mi trng ln men khc nhau Chng XK Mi trng A - 4H A-4 R2 Gauze - I Gauze - II ISP - 4 A - 4H A-4 1 Gauze - I Gauze - II ISP - 4 Sinh khi (mg/ml) 8,34 0,12 4,6 0,15 5,51 0,23 7,52 0,21 3,19 0,15 9,24 0,23 4,43 0,18 6,48 0,33 7,25 0,22 2,78 0,26 HTKS (D-d,mm) 22,33 1,42 19,52 0,48 17,65 0,58 13,93 0,13 12,18 0,15 21,34 0,21 18,72 0,17 16,23 0,44 9,14 0,34 7,25 0,14
-54-
Hnh 3.10: nh hng ca mi trng n kh nng tng hp CKS Kt qu trn bng 3.12 v hnh 3.10 cho thy: Trong 5 mi trng ln men c 2 chng nghin cu u cho sinh khi ln nht trong mi trng A - 4H. Vi chng R2 l 8,0 mg/ml, chng 1 l 9,0 mg/ml. ng thi dch ln men ca mi trng A - 4H cng c hot tnh khng sinh mnh nht (21mm). T kt qu ny chng t cc thnh phn trong mi trng ln men c nh hng nhiu n kh nng hnh thnh CKS ca x khun nh nhiu nghin cu trc khng nh [14],[17]. Vi mc ch l la chn mi trng ln men thch hp cho qu trnh sinh tng hp CKS, cn c vo kt qu ny chng ti nhn thy mi trng A - 4H l thch hp nht v c s dng cho cc nghin cu tip theo. 3.4.2. nh hng ca ngun cacbon Cc hp cht cacbon va l ngun dinh dng li va l ngun cung cp nng lng cho c th. VSV ni chung v x khun ni ring c kh nng s dng c nhiu ngun cacbon khc nhau. nghin cu nh hng ca ngun cacbon ln kh nng sinh trng v hnh thnh CKS, ng thi xc nh c ngun cacbon thch hp nht cho 2 chng R2 v 1, chng ti tin hnh kho st mt s ngun cacbon thng thng c s dng ln men CKS.
-55Bng 3.13. nh hng ca ngun cacbon ln kh nng sinh tng hp CKS ca 2 chng R2 v 1 Cc ch tiu Sinh khi HTKS K hiu chng R2 1 R2 1 Glucose Ngun cacbon Tinh bt Lactose 13,58 0,56 11,55 0,15 20,23 0,61 16,34 0,74 Sacarose 18,93 0,57 17,26 0,34 21,22 0,78 18,29 0,65
17,44 0,22 16,23 0,42 18,53 0,45 15,75 0,33 15,24 0,36 18,21 0,32 17,53 0,16 16,82 0,58
Kt qu trnh by trn bng 3.13 cho thy: c 2 chng x khun R2 v 1 u c kh nng s dng c c 4 ngun ng nghin cu. Tuy nhin nh hng ca cc ngun cacbon ny ln s sinh trng v kh nng to khng sinh ca cc chng c s khc nhau. i vi chng R2 ngun cacbon sacarose cho sinh khi ln nht v HTKS mnh nht. Chng 1 ngun cacbon Glucose cho sinh khi ln nhng sacarose c HTKS mnh nht.
Hnh 3.11: nh hng ca ngun cacbon n kh nng tng hp CKS
-563.4.3. nh hng ca ngun nit Nit l ngun dinh dng khng th thiu i vi VSV ni chung v x khun ni ring. Trong mi trng ln men, ngun nit c nh hng nhiu n s tng hp CKS ca x khun [3]. Ngun nit c s dng trong mi trng ln men x khun c th dng v c hoc hu c. V vy chng ti tin hnh kho st nh hng ca c 2 ngun nit ny n kh nng sinh tng hp CKS ca 2 chng x khun nghin cu. Bng 3.14. nh hng ca ngun nit ln kh nng sinh tng hp CKS ca 2 chng R2 v 1 Nng Ngun nit
Bt u tng Cao nm men (NH4 )2SO4 KNO3
Sinh khi (mg/ml) R2

12,6 0,25
Hot tnh khng sinh (D-d,mm) 1
(%)
1 1 1
1
1
11,2 0,32
R2
21,63 0,58 20,32 0,15
13,52 0,23 13,28 0,17 19,78 0,26 19,73 0,52 9,73 0,18 7,82 0,52 8,01 0,22 17,65 0,44 16,82 0,38
8,56 0,24 16,92 0,32 14,340,17
Ngun nit hu c c chng ti s dng l cao nm men v bt u tng, ngun nit v c l nitrat kali (KNO3) v mui sunfat amon ((NH4 )2SO4.). Cc ngun nit ny c b sung vo mi trng A - 4H. Qu trnh ln men c tin hnh trong bnh nn 250 ml c cha 25 ml mi trng. Sau 120 gi nui lc, thu dch ln men v xc nh HTKS. Kt qu c trnh by trn bng 3.14 v hnh 3.12.
-57T kt qu trn cho thy c 2 chng x khun nghin cu u sinh trng tt trong 2 mi trng c ngun nit hu c l bt u tng v cao nm men. Song cha r c s khc nhau r rt gia 2 ngun nit ny.
Hnh 3.12 : nh hng ca ngun nit n kh nng tng hp CKS Cng nh s sinh trng, HTKS ca 2 chng nghin cu cng cao hn trong 2 mi trng c bt u tng v cao nm men. Cc kt qu ny khng nh u th ca ngun nit hu c ln kh nng sinh trng cng nh sinh tng hp CKS ca x khun ni chung. iu ny c th gii thch trong cc ngun nit hu c, ngoi thnh phn protein cn cha cc cht cn thit cho qu trnh tng hp CKS. Kt qu nghin cu ny ca chng ti cng ph hp vi mt s kt qu cng b trc y [14]. 3.5. Phn loi cc chng x khun theo phng php sinh hc phn t 3.5.1. Kt qu tch chit DNA tng s ca cc chng x khun Trong nghin cu ny, chng ti tin hnh tch chit DNA t 2 chng x khun l 1 v R2. Trc khi tin hnh tch chit DNA, cc chng x khun c nui cy trn mi trng tng sinh thu sinh khi t bo. DNA c tch chit theo phng php ca Ausubel v cs c ci tin [39]
-58c trnh by trn. Kt qu tch chit c kim tra bng cch in di trn gel agarose 0,8% v c trnh by trong hnh 3.13. 1 R2
Hnh 3.13. nh in di DNA tng s ca 2 chng x khun Trn c s kt qu thu c trong hnh 3.13 cho thy DNA c tch ra t c 2 chng x khun nu trn. Tng ng vi mi chng, sn phm in di ch c mt bng duy nht chng t DNA khng b t gy trong qu trnh tch chit. Hm lng DNA thu c l tng i ln v c sch cao. Nh vy, c th ni rng DNA thu c trong qu trnh tch chit t c cc yu cu ra. Sn phm DNA p ng tt cho cc nghin cu khuch i gen 16S - rRNA bng phn ng PCR. 3.5.2. Kt qu nhn gen 16S - rRNA bng phn ng PCR to tin cho vic nh loi 2 chng x khun c phn lp nu trn, trong nghin cu ny chng ti tin hnh khuch i gen 16S - rRNA. y l mt gen ch th c dng ph bin trong nh loi VSV. Cp mi c thit k khuch i cho phn ng PCR cho php khuch i trnh t trn vn ca gen 16S - rRNA, m bo tt cho cc nghin cu tch dng v
-59xc nh trnh t y ca gen ny. Kt qu khuch i gen c trnh by trong hnh 3.14.
Hnh 3.14. nh in di sn phm PCR ca 2 chng x khun nghin cu M. Maker X174 ct bng Hind III 3,4. chng 1. 5,6. Chng R2 Da trn kt qu in di hnh 3.14 cho thy c 2 chng x khun 1 v R2 u c s nhn ln ca gen 16S - rRNA. Sn phm PCR c c hiu cao, khng c cc sn phm ph. Mt khc, da trn DNA maker chun cho thy sn phm PCR c kch thc khong 1,5 kb tng ng vi kch thc theo tnh ton l thuyt trong qu trnh la chn mi. Nh vy c th kt lun l on DNA c nhn ln chnh l gen 16S - rRNA. Kt qu ny l c s quan trng chng ti c th tip tc tch dng v xc nh trnh t y ca gen 16S 2 chng nghin cu.
-60Chng 4 KT LUN V KIN NGH 4.1. KT LUN 1. T cc mu t khc nhau, phn lp v thun khit c 80 chng x khun thuc chi Streptomyces, trong s c 30 chng c hot tnh khng nm gy bnh trn ch cc mc khc nhau, chim t l 37,5%. Trong s cc chng c hot tnh khng nm, nhm trng chim 36,7%, xm - 26,7%, xanh - 23,3%, hng - 6,7%, nu - 3,3%, lc - 3,3%. 2. tuyn chn c 2 chng x khun l 1 v R2 c hot tnh mnh nht trong s 30 chng c hot tnh khng nm, khng c c 2 chng nm gy bnh trn ch l CT - 2E v CT - 5X, ng thi cng c kh nng khng cc nm kim nh. 3. tin hnh xc nh cc c im hnh thi, sinh l, sinh ha, khuch i gen 16S - rRNA nhn nh: - Chng R2 c th l loi Streptomyces misawaensis. - Chng 1 c th l loi Actinomyces brunneofungus. 4. xc nh c mt s iu kin ln men thch hp cho s sinh tng hp CKS ca 2 chng x khun. - Mi trng A - 4H thch hp cho ln men to cht khng sinh cho c 2 chng x khun 1 v R2. - Ngun cacbon thch hp nht tng hp CKS ca chng R2 l Sacarose v chng 1 l glucose. - Bt u tng v cao nm men u l nhng ngun nit hu c thch hp cho ln men CKS ca c 2 chng x khun ny.
-614. 2. KIN NGH V NHNG NGHIN CU TIP THEO 1. Tip tc nghin cu gii trnh t gene 16S ca hai chng x khun 1 v R2. 2. Tip tc nghin cu kh nng tng hp CKS ca hai chng 1 v R2. 3. Tch chit v xc nh bn cht ha hc ca cc CKS. 4. Tm hiu kh nng ng dng ca cc CKS th v cn c kho nghim trn din rng nh gi hiu qu s dng trong cng tc bo v thc vt.
-62CNG TRNH CNG B LIN QUAN N LUN VN Bi Th H, Vi Th oan Chnh (2008), "Kh nng khng nm gy bnh trn ch ca mt s chng x khun phn lp t t Thi Nguyn", Tp ch khoa hc v cng ngh, s 2 (46), tr. 92 - 96.
-63TI LIU THAM KHO
TING VIT 1. 2. B nng nghip v pht trin nng thn (2006), K thut trng,chm sc v ch bin ch, NXB Nng nghip, tr. 72 - 77. Ng nh Quang Bnh (2005), Vi sinh vt hc cng nghip, Vin sinh thi v ti nguyn sinh vt, Trung tm khoa hc T nhin v cng ngh Quc gia, H Ni, tr.53 - 71. 3. 4. Nguyn Vn Cch (2004), Cng ngh ln men cc cht khng sinh, NXB khoa hc v k thut, H Ni. Chi cc bo v thc vt Ph Th, S dng an ton, hiu qu thuc bo v thc vt trn cy ch, 07/2003, tr. 24 - 30. 5. Nguyn Hong Chin (2000), Nghin cu chng x khun Streptomyces V6 sinh cht khng sinh chng vi khun gy bnh ho xanh c chua, Lun vn thc s sinh hc, H Ni. 6. Vi Th oan Chnh (2000), Nghin cu kh nng nng cao hot tnh khng sinh ca chng Streptomyces rimosus R77 v Streptomyces hygroscopicus 5820 bng k thut dung hp t bo trn, Lun n TS sinh hc, Vin cng ngh sinh hc, H Ni. 7. Nguyn Ln Dng, Phm Th Trn Chu (1978), Mt s phng php nghin cu vi sinh vt hc - Tp III. NXB khoa hc v k thut, H Ni. 8. Nguyn Ln Dng, on Xun Mu, Nguyn Phng Tin, ng c Trch, Phm Vn Ty (1972), Mt s phng php nghin cu vi sinh vt hc, Tp I, NXBKHKT H Ni, 328 - 345. 9. Nguyn Ln Dng, Phm Vn Ty, Nguyn nh Quyn (1977), Vi sinh vt hc - tp II, Nh xut bn i hc v Trung hc chuyn nghip, H Ni.
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I HC THI NGUYN TRNG I HC S PHM ------ ------

LUN VN THC S SINH HC

THI NGUYN - 2008

I HC THI NGUYN TRNG I HC S PHM ------ ------

Chuyn ngnh: Sinh hc thc nghim M s: 60.42.30

LUN VN THC S SINH HC

NGI HNG DN KHOA HC TS. VI TH OAN CHNH

THI NGUYN - 2008

Thi nguyn, ngy 15 thng 9 nm 2008 Tc gi

Chng 3 - KT QU V THO LUN

NHNG CH VIT TT TRONG LUN VN

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

Chng 1 TNG QUAN TI LIU

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

Trong : SSM : Mc ging nhau gia hai c th A, B (%)

Trong : S J : mc ging nhau gia hai chng A, B (%)

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

Chng 2 NGUYN LIU V PHNG PHP NGHIN CU

S ha bi Trung tm Hc liu i hc Thi Nguyn

- Cc dng c thy tinh ca Trung Quc, c, Vit Nam...

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

Chu k nhit 3 pht 1 pht 1 pht 1 pht 30 giy

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

Chng 3 KT QU V THO LUN

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

Hnh 3. 2. T l cc chng x khun c hot tnh khng nm theo nhm mu

S ha bi Trung tm Hc liu i hc Thi Nguyn

Hnh 3.3. T l cc chng x khun c hot tnh khng nm

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

VSV kim nh: CT - 5X

VSV kim nh: CT - 2E