Đại học quốc gia hà nội Tr-ờng đại học khoa học tự nhiên *** TRN TH THY LINH NGHIấN CU V NNG CAO KH NNG SINH TNG HP CC CHT Cể HOT TNH KHNG NM T CHNG Serratia marcescens DT3 LUN VN THC S KHOA HC H Ni 2016 Đại học quốc gia hà nội Tr-ờng đại học khoa học tự nhiên *** TRN TH THY LINH NGHIấN CU V NNG CAO KH NNG SINH TNG HP CC CHT Cể HOT TNH KHNG NM T CHNG Serratia marcescens DT3 Chuyên ngành: Sinh hc thc nghim LUN VN THC S KHOA HC Ging viên h-ớng dẫn: TS Th Tuyờn TS Lờ Hng ip H Ni - 2016 Lun thc s Khoa Sinh hc M U Hng nm trờn th gii, bnh cõy gõy nhng tn tht to ln cho sn xut nụng nghip Chỳng phỏ hy n 537,3 triu tn cỏc loi nụng sn ch yu, chim 11,6% tng sn lng nụng nghip trờn th gii Trong cỏc loi bnh cõy thỡ bnh nm gõy chim khong 83%, ú bnh nm Fusarium v Rhizoctonia chim t l tng i ln Nm bnh Fusarium v Rhizoctonia gõy bnh trờn nhiu loi cõy rau qu v cõy lng thc nh lc c chua, khoai tõy, c phờ, tiờu Chỳng cú kh nng tn ti t mt thi gian di, phỏt sinh v gõy hi t giai on cõy v kộo di cho ti thu hoch nu khụng ỏp dng cỏc bin phỏp phũng tr trit Bin phỏp phũng tr cỏc bnh hi cõy trng ph bin nht cho n l s dng cỏc loi thuc húa hc.Mc dự cú u im l ph tỏc dng rng, hiu qu v tỏc dng nhanh nhng thuc húa hc ngy cng bc l rừ nhng nhc im nh hiu qu ngy cng kộm v gõy ụ nhim mụi trng Chớnh vỡ th vic s dng cỏc ch phm sinh hc phũng tr cỏc bnh cõy trng vi sinh vt gõy ang l xu hng ch yu Cỏc ch phm ny ang c s dng rng rói nhm to mt nn nụng nghip hu c an ton v bn vng Serratia l mt ging trc khun Gram õm, k khớ tựy nghi, h Enterobacteriaceae.Chỳng cú kh nng sinh cỏc cht cú hot tớnh khỏng nm ó c s kim soỏt nhiu bnh hi cõy trng khỏc nhau.Trờn th gii ó cú nhiu cụng trỡnh nghiờn cu ng dng Serratia phũng tr nm bo v cõy trng, nhiu sn phm c sn xut v thng mi húa.Tuy nhiờn, kt qu t c nghiờn cu v s dng cỏc ch phm sinh hc cũn hn ch Chi phớ thuc bo v thc vt trờn th gii ch chim 1,9% tng giỏ tr ca cỏc loi thuc bo v thc vt Vỡ vy, vic tng cng nghiờn cu phỏt trin ch phm sinh hc dit nm l cn thit Xut phỏt t nhu cu thc t chỳng tụi thc hin ti: Nghiờn cu v nõng cao kh nng sinh tng hp cỏc cht cú hot tớnh khỏng nm t chng Serratiamarcescens DT3 nhm thc hin cỏc ni dung nghiờn cu: Trn Th Thựy Linh K22 Lun thc s Khoa Sinh hc Chn lc c mụi trng nuụi cy chng Serratia marcescensDT3 cú kh nng sinh tng hp cỏc cht cú hot tớnh khỏng nm cao nht Ti u mụi trng v cỏc iu kin nuụi cy lm tng kh nng sinh tng hp cỏc hot cht khỏng nm Tỏch chit, tinh sch hot cht cú hot tớnh khỏng nm t chng Serratia marcescen DT3 Trn Th Thựy Linh K22 Lun thc s Khoa Sinh hc CHNG TNG QUAN TI LIU 1.1 Vi khun Serratia 1.1.1 Khỏi quỏt v chng Serratia marcescens Serratia marcescens l mt mi bn loi ó c cụng nhn ca chi Serratia Serratia marcescens c phỏt hin vo nm 1819 ti í Cỏc chng vi khun Serratia marcescens phõn b t, nc, thc vt Chỳng phỏt trin nhit t 5400C, pH t 5-9 Chỳng l tỏc nhõn gõy bnh cho ngi v ng vt nhiờn bờn cnh ú thỡ mt s chng Serratia marcescens li c s dng cỏc nghiờn cu v y hc, quõn s, nụng nghip[31] Serratia marcescensl cỏc vi khun hỡnh que Mng cú cu trỳc c trng ca vi khun Gram õm, di ng sinh bo t cú thnh t bo mng c cu to t mt lp peptidoglycan c bao bc bi mt lp mng bờn ngoi Cỏc mng ngoi cú lipopolysaccharides l mt loi phospholipid c bit gm cỏc axit bộo c gn vo mt dimer phosphate glucosamine.Mt glucosamine gn lin vi mt polysaccharides ct lừi m m rng n cỏc polysaccharides O Cỏc mng ngoi cng l mt phng tin iu tit s hp th cht dinh dng v loi tr cỏc c t Chng Serratia marcescen DT3 c phõn lp t mu t Vit Nam Ngnh: Proteobacteria Lp: Gamma Proteobacteria B: Enterobacteriales H: Enterobacteriaceae Chi: Serratia Loi: Serratia marcescens Hỡnh 1.1 Hỡnh nh chng Serratia marcescens DT3 trờn mụi trng LB c Trn Th Thựy Linh K22 Lun thc s Khoa Sinh hc 1.2 Bnh cõy trng nm Fusarium v Rhizoctoniagõy 1.2.1 c im sinh hc ca nm Fusarium v Rhizoctoniahi cõy trng Fusarium l mt h ln ca nm si phõn b rng rói t v gn vi thc vt Hu ht cỏc loi u sng kớ sinh vụ hi v l thnh viờn tng i phong phỳ ca cng ng vi sinh vt t Fusarium oxysporum l phõn tỏn rng nht ca cỏc loi Fusarium c tỡm thy trờn ton th gii.F oxysporum khụng cú giai on sinh dc in hỡnh, nhng sn xut ba loi bo t vụ tớnh: bo t nh, bo t ớnh ln v bo t hu.Bo t nh l cỏc bo t sn xut nhiu nht, nú cú hỡnh bu dc, hỡnh elip hoc thn c nh hỡnh v sn xut trờn si nm trờn b mt.Bo t ớnh ln, cú 3-5 t bo v nhn hai u hoc cnh cong, c tỡm thy trờn bo t phõn trờn b mt ca cõy b bnh Bo t hu c hỡnh thnh n l hay theo cp nhng ụi c tỡm thy cỏc cm hoc chui ngn Nú l bo t trũn cú vỏch dy c sn xut trờn mt si nm hoc bo t ớnh ln.Bo t hu khụng ging nh cỏc bo t khỏc cú th tn ti t mt thi gian di[4,19] Hỡnh 1.2 Hỡnh thỏi nm F oxysporum trờn a thch PDA Nm F oxysporum l mt tỏc nhõn gõy bnh t rt ph bin vi lỏ cõy mm, lm cht v phõn hy cht hu c cõy.Nú tn ti t nh si nm v cỏc loi bo t nhng thng tn ti t nh bo t hu Lõy lan mm bnh theo hai cỏch c bn lõy lan khong cỏch ngn qua bn nc, bng thit b trng trt v khong cỏch di qua Trn Th Thựy Linh K22 Lun thc s Khoa Sinh hc KT LUN Trong mụi trng ó kho sỏt l mụi trng NA, NYD, LB v mụi trng bt u tng 2% chỳng tụi ó chn c mụi trng LB nuụi chng S marcescens DT3 cho hot tớnh khỏng nm mnh Dch chit ngoi bo nng 50%, ó c ch c 71%- 81% s sinh trng v phỏt trin ca chng nm F oxyporum v R solani Ti u mụi trng v mt s iu kin nuụi cy chng S marcescens DT3 cho hot tớnh khỏng nm cao nht Mụi trngLB sau ti u bao gm: 1,25% pepton , 1% Nacl, 0,5% cao nm men vi thi gian nuụi cy 28h, pH ti u 7, nhit 280C.Hot tớnh khỏng nm mụi trng ti u ó tng so vi mụi trng trc ti u lờn 15 ln i vi nm R solani v 2,6 ln i vi nm F oxyporum ó tinh sch c protein cú hot tớnh khỏng nm vi lng phõn tkhong 56 kDa t chng S marcescens DT3 Proteinkhỏ bn vi nhit c cỏc nhit 800C 30 phỳt khụng lm nh hng n kh nng c ch sinh trng v phỏt trin ca nm F oxyporum v R solani Proteinase K khụng nh hng n hot tớnh khỏng nm vi nng t 0,5- àg c bit protein tinh sch cú kh nng khỏng nm F oxyporum mnh KIN NGH Hon thin quy trỡnh tinh sch protein cú hot tớnh khỏng nm quy mụ lờn men ln ng dng to sn phm sinh hc cú kh nng khỏng nm phc v phỏt trin nụng nghip bn 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