Nghiên cứu khả năng sinh tổng hợp iaa của chủng rh

NGHIEN CQU KHA N A N G SINH T O N G HOP lAA CUA CHUNG RH NGUYEN THI KIM QUY, NGUYEN THI HOAI HA, PHAM V A N T Y Trung tarn Cong nghe Sinh hge, DHQG, Ha Ngi Md dau Vice sir dung phan bdn vi sinh se lam tang giau dit mot cich ed hieu qua va ton kem ban vice su dung cac loai phan hda hge, nhiing loai cd the gay hai cho mdi trudng vi lim can kiet cic ngudn nang lugng khdng the tii tao Nhieu nghien eiiu da tap trung cbgn Igc cic vi sinh vat cd ich ggi la "vi sinh vat hiru hieu", gdm bdn hgp vi sinh vat, sdng edng sinh vdi nhu tu nhien, bao gdm cic vi sinh vat ehuyen hda photphat khd tan (ehuyen hda thiie an kbd tieu dh tieu), vi khuan phan giii chit him co, vi khuan lactic sinh bacterioxin khing vi sinh vat gay benh vi cic vi khuin cd nang sinh chat kich thich sinh trudng thue vat Chiing bd trg phit trien vi phit buy tic dung nhieu mat phue vu cho vice thiic diy sinh trudng ciia cay, xir ly miii hdi Cic vi sinh vat cd phan bdn sinh hge tiet mot sd chit kich thieh sinh trudng, chit hoi hge ehdng nam, ciing nhu lam tang su mim eua hat vi su sinh trudng ciia xh Vice sir dung phan bdn vi sinh se lam tang giau dit mot cich ed hieu qui va dd ton kem ban viee sir dung cic loai phan hoi hge, nhflng loai cd the gay hai cho mdi trudng va lim can kiet cic ngudn nang lugng khdng the tii tag Vi vay viec nghien ciiu, tuyen cbgn vi sinh vat cd boat tinh cao, vdi cic ehiie nang khae cung ly lieh eu the de lam phan bdn cho cay trdng la rit cin tbiet, nhim xay dung mot nen ndng nghiep sach, an toan va bin virng Day la phuong hudng rit quan trgng de giim 168 bdt tic hai ciia cic chit hoi hoc giim thieu d nhidm mdi trudng va giam gia sin phim phai chi phi qua nhieu ngoai te nhap khiu thud'c hoi hge Vdi mong mudn gdp phin edng nghe san xuit che phim phan htm ea vi sinh da cbiic nang va hoin tbien ban, cbiing tdi da tien hinb dt tii "Nghien eifu khd ndng sinh tong hgp lAA eua ehung Rh" Nguyen lieu va phi/dng phap 2.1 VI sinh vat Chiing vi khuan phan lap tir dit dugc giiJ vi bio quin tai Bao ting Gidng chuan Vi sinh vat, Trung tam Cdng nghe Sinh hge - Dai hge Qudc gia Hi Ngi 2.2 Trinh ti/cac doan mdi dCing nghien cuu 27f 780f 920r 5' - agagtttgatcctggctcag - 3' 5' - GATTAGATACCCTGGTAG - 3' 5'-GTCaaTrCCTTTGAGTTT-3' 1525r 5' - aaaggaggtgatccagec - 3' 10 X Taq buffer dNTP mix 2.5mM Taq polymeraza (5U) 2.3 M6I trwdng (YEM, g/l) [64] K2HPO4-l,0, MgS047 H2O-O, 4, NaCl-0,2, Mannitol-20,0 Kbit trung d 12rC/15phut, pH 7;2 2.4 Phuong phap xac dinh lAA (indole - - acetic - acid) [44,45,62] Ket qua va thao luan 3.1 Tuyen chon ehung Rhizoblum sinh lAA sau 42 gid nuoi bang phuong phap sac ky ban mong Thieu hut boocmon sinh trudng li van de ma cay trdng thudng gap phii Dieu niy cd the khic phue dugc neu bd sung vao dit viing rd cic chiing vi khuan cd boat tinh sinh cac chit kich thich sinh trudng cao 169 c 26 il I 24 21 III 21 •a, < < 18 16 17 B X O H m d lUffng lAA (m|/1) Ky hieu chiing Hinh Tuyen chon cac chiing vi khuan c6 kha nang sinh lAA Nhieu nghien eiiu da tim hieu kha nang cd dinh nita ciia vi khuan vung r6 Rhizobium [4, 5] Trong nghien eiiu niy chiing tdi quan tam den kha nang hinb lAA ciia chiing Tir cic miu dit trdng ngd vi dau xung quanh Ha Ngi ehung tdi tuyen cbgn dugc 10 ehung cd nang sinh tdng hgp lAA cao Axit indon axetie (lAA) li mot chit kich thich sinh trudng thue vat Hoocmon dugc mot vii vi sinh vat sin sinh tai viing tiep xiic vdi be mat Idng hiit, dd, cay trdng cd the su dung mot cich cd hieu qui Trude day ngudi ta cho ring nang kich thich sinh trudng ciia nhdm vi khuin niy la qui trinh cd dinh nita Nbung sau dd bing thue nghiem ngudi ta da cbiing minb ring nhiing dieu kien mi qui trinh cd dinh nita khdng di6n vin xiy su kich thich sinh trudng thue vat Ket qui cho thiy ea 10 ehung (Hinh 1) deu cd kha nang sinh lAA rd ret Tuy nhien, him lugng lAA ciia cic chiing vi khuan sinh la khdng gidng 13-27mg/l Dae biet cd cbiing ky hieu Rh ed nang tdng hgp lAA him lugng dat tir 27mg/l Viec tuyen cbgn cac chiing vi khuan chi la khau diu tien cic bude tuyen cbgn cic dac tinh ciia vi khuan Dich vi khuan dugc nudi 56 gid d nbiet 30°C 1ml dich miu dugc ly tam lanh 4°C vdi tde 10000vdng/15phut Loai bd dich 170 Hoa tan can vdi 100|il metanol 100% Diing mao quan chim toan bd dieb miu Idn sic ky ban mong va 5|il eiia lAA (Sigma) dugc dung lam thang chuin [45, 46] Viec phan tich lAA bang phep sic ky ban mong muc dich chiing tdi dung de tich chinb xic lugng lAA dich nudi ciia chiing Rh vi (Hinh 2) cbiing bieu hien rd bang boat tinh nang sinh lAA tuang ling vdi chim lAA chuin Tien hinh cao vet d vi tri sd li ciia chiing Rh Cbiet miu va tinh sach miu bing ^*''* lOOfil metanol dng nghiem cd niit xoay Lge qua ban Igc 0,45|am Ly tam Hinh Xac dinh lAA ciia chiing lanh 4"C d 10 OOOvdng/phiit Rh bang sac ky ban mong 20phut Bd can, liy dich ndi, cho bay hoi tdi miu kbd Bd sung lai 20)0,1 metanol vio miu vi giir d 4"C Sau dd, miu dugc dung cho viec xac dinh him lugng lAA cd miu tren sic ky long cao ap (HPLC) Viec thu phan doan lAA qua sic ky long cao ap (Hinh 3) ciia chiing li khdng gidng ve tbdi gian, chi sau 10 pbiit da xuit hien dinh lAA chiing Rh Cic phan doan thu dugc tien hinb dinh lugng lAA tren miy quang pbd Dinh lugng dich thu dugc d dng nghiem, cho vao dng 1:1 ml lAA, dng II (dd'i chiing): ml nude cat Them vao mdi dng 2ml thud'c thit Thdi gian (phiil) Van Urk Salkowski (0.5M FeCl^ 1ml, 35% HCIO4 50ml) phan irng Hinh Hinh hap phu lAA ciia dugc lie deu Dung dich cd miu chiing Rh tren bieu d6 HPLC hong nhat sau 15-30 pbiit 171 Chi sd OD dugc dd'i chi6'u vdi phuong trinh dd thi chuin de tinh ham lugng lAA cd dich nudi Dieu chiing tdi nhan thiy d day rit rd li cbiing Rh li 40mg/l Dieu niy phii hgp vdi nhieu nghien eiiu va ngoii nude ve kha nang sinh lAA eua cic chiing vi khuan viingri [6] 3.2 Dac diem hinh thai, sinh ly, sinh hoa eua ehung Rh Nhu chiing ta biet, the gidi vi sinh vat bao gdm mot sd lugng Idn cic loii khic nhau, chiing khdng the dugc gdp ehung lai vao mot nhdm phan loai nao dd Tit ea cic dd'i tugng deu li cic ea th6' hien vi, phan Idn li don bio, cbiing dugc nudi eiy va nghien cihi theo nbttng phuong phap tuang tu Chiing rit da dang ve hinh thii hge, ea cac loii gidng ve hinh thii ciing cd the khic rit rd ret ve cic dac diem sinh ly, sinh boa hge Vi vay, nhieu vi sinh vat dugc xep vao cac nhdm sinh ly khic khdng phu 'thude vio vi tri phan loai ma phu thude vao tac dgng ciia chiing len mot -cbit nao dd ciia ea chit hoae vao ban chit eua cic sin pham trao ddi chit dugc tao Do vay, ehung HA4 va Rh cung khdng nim khdi qui luat ehung dd Hai chiing niy dugc nudi tren cic mdi trudng dac trung cho tirng loii nhu da md t i d phin phuong phip - Chiing Rh cd hinh que kich thude 0,5-0,9x1,20-2,0|j,m, pbd bien la da hinh nhirng dieu kien bit Igi Thudng ehiia nbung hat poly-|3-hydroxybutyrat, nhiing hat khtic xa tia sang quan sat dudi kinh hien vi phan pha Vi khuin Gram (-), hieu khi, cd nang di dgng Khuan lac hinb trdn, Idi, nhiy, thudng cd dudng kinh 2-4mm Cd kha nang ddng boa ngudn eacbonhydrat nhu: Mantoza, saccaroza, glucoza, fructoza, xyloza, raffinoza, galaetoza, khdng sinh trudng dugc d ndng NaCl 2%, khdng sinh HjS Mot dieu nhan thiy d day la ea ehung deu ed the tdn tai dang cyst gap dieu kien bat Igi xiy ra, ehung thude vi khuan Gram am Khuan lac eua chiing deu nhiy vi khd lay tich Han niia cbiing Rh deu cd nang ddng hoi cic loai dudng pbd bien dung cho cic vi khuan nhu: Mantoza, saccaroza, glucoza, fructoza 172 Hinh Hinh thai khuin lac ciia ehung Rh Hinh Hinh dang te bao ciia chiing Rh (X7.500) Can cii vio cic dac diem hinh thii (Hinh ) , sinh ly, sinh hoi, ddng dua vao khoi phan loai Bergeys [66], ehung tdi xic dinh cd kha nang cbiing Rh thude chi Sinorhizobium De khing dinh cbinb xac den loai ehung Rh thude loii nao chi Sinorhizobium chiing tdi tien hinb tich cbiet ADN ciia cbiing Rh ddng thdi tien hinb phin ling PCR M "2" Ket time qud trinh phdn ling sau 30 chu ky khue'eh dqi, ehung Rh tong hgp dugc doqn gen ed kich thude nam khodng tH 1500 - 2000bp (Hinh 6) San pham ndy dugc dung lam khuon cho viec phdn tich trinh tu gidi trinh turADN 16S Hinh Dien di dd tren gel agaro 10% sdn pham PCR ciia ehung Rh Chii thieh: M ADN chuin Ikb Ladder 1500 -2000 bp - Chung Rh Tien hinh phan tich trinh tu rADN 16S eua Rh tren may dgc trinh tu tu dgng 3100 Avant bing doan mdi dac bieu ciia 16S Chiing tdi 173 dung cac cap mdi thdng dung va pbd bi6'n ma cac phdng thi nghiem trfin the gidi vin dang dung, bao gdm cic doan 27F, 780F, 980R va 1525R Diing ehuang trinh Blast search ket qui phan tich trinh tu cho thiy doan gen dugc tdng hgp eua Rh ed dii li 493bp day la nhtrng doan gen bao tdn va cbgn lge qui trinh diing mdi cho phan tich trinh tu song song Khi so sinh Rh vdi trinh tu gen ma hoi cho 16S da dugc dang ky ngan hing dir lieu gen Nhat Ban Do tuang ddng rit cao dat 97% vdi trinh tu ciia ehung thude loai Sinorhizobium xinjiangensis vdi ky hieu AJ420774, day la nhirng chi so tuang ddng so sanh dugc phep chip nhan Tit cac thdng sd thu dugc eua Sinorhizobium xinjiangensis Rh se lam co sd va cin tbiet cho viec lap so dd cay phi he (Hinh 7) S.melilotiAYI 96963 Mdi quan he gin giira chiing Sinorhizobium xinjiangensis Rh vdi 14 trat tu nucleotit thude Rhizobium ngan hang dir lieu gen Nhat Ban Sir dung ehuang trinh dien toan MEGA 2.1 va cac thdng sd tien hoi cin tbiet cho thiy mdi quan he da dugc tbiet lap tren ea sd phan tich so sinh chudi trinh tu nucleotit doan gen rADN 16S dii493bp Cho thiy ehung Rh nim tren cung mot nhinh va cimg nhdm vdi loai Sinorhizobium xinjiangensis ky hieu AJ420774 S.frediiAY260149 S.frediiAY260150 S.melilotlD14509 R.melilotiX67222 R.fredilX67231 S.sahellX68390 Rh S.xlnjlangenslsAF250354 Rh.melilotiD12783 Rh.fredilD12792 S.fredliD14516 - S.kummerowlaeAF364067 Trong cic budc nghien eiiu thi viec lua cbgn cac dieu kien S.morelenseA Y559079 nudi ciy va cac yeu td inh hudng S.morelenseA Y024335 den qua trinh sinh trudng eua cic ehung vi khuan phan lap dugc li mot viec rit quan trgng vi khdng Hinh Cay pha he dua tren phan tich trinh tu rADN 168 ciia chiing Rh va cac the thieu loai CO quan he gdn H 174 Vdi muc dich nghien eiiu phdi trdn bdn hgp cic cbimg vi khuan che pham phan hiJu ca da chiie nang, thi khdng the khdng nghien ciiu ky dac tinh ciia tirng cbiing dugc Do vay, ehiing tdi ciing tien hinh nghien ciiu tiing chiing cd ket hgp phan tich cic chiing vdi mong mudn tim dugc cac dieu kien thieh hgp cho sinh trudng va tdng hgp lAA ciia chiing Rh Ket luan Tir cic miu dit trdng dau da phan lap dugc cbiing vi khuin Rh cd nang kich thich sinh trudng thue vat Dua vao phuong phip phan loai truyen thdng theo khoi phan loai eua Bergey's va phuang phip phan loai hien dai dua tren phan tich trinh tu rADN 16S, ehung vi khuan Rh thude loai Sinorhizobium xinjiangensis Chiing Rh cd kha nang sinh chat kich thich sinh trudng thue vat dat 45mg/l Summary Study on Indole 3-Acetic acid production of bacteria Rh Nguyen Thi Hoai Ha, Nguyen Thi Kim Quy, Pham Van Ty Center of Biotechnology, Vietnam National of University Hanoi The strain of Rh was isolated from soil cell are rods, non sporing, non-notile, catalase negative Metabolision was predominantly fermentative, lactic acid being formed from glucose homofermentatively, xylose was fermented The analysis of 16S rDNA sequence shows that this strain Rh was identified as Sinorhizobium xinjiangensis lAA was produced Tai lieu tham khao Hoben H J., Somasegaran P., Handbook for Rliizobia, SpringerVerlag, New York, pp 1-6 Holt J G., N R Krieg, P H A Sneath, J T Staley, and S T Williams (1994), Bergey's Manual of Systematic Bacteriology, Williams and Wilkins Baltimore, Maryland, pp 235-242 Lynch, J M (1990), "Some consequences of microbial 175 Rhizosphere competence for plant Rhizosphere, ed J M Lynch, pp 1-10 and soil" In The Michalczuk L., Ribnicky D M., Cooke T J and Cohen J D (1992), "Regulation of indole-3-aeetic acid biosynthesis in carrot cell cultures" Plant Physiol, (100), pp 1346-1353 Ribnicky D M., Cohen J D., Hu W S and Cooke T J (2002), "An auxin surge following fertilization in carrot: Its significance for plant totipotency", Planta, (214), pp 505-509 Ridge R W and B G Rolfe (1985), ''Rhizobium sp degradation of legume root hair cell wall at the site of infection thread origin", Appl Environ Microbiol, 50, pp 717-720 Vincent, J M (1970), A Manual for the Practical study of the Root-Nodule Bacteria, Burgess and Son Ltd Great Britain, pp 45 Visser E J W., Cohen J D., Barendse G W M., Blom C W P M., Voesenek L A C J (1996), "An ethylene-mediated increase of sensitivity to auxin induces adventitious root formation in flooded Rumex palustrisSm Plants", Plant Physiol, (112), pp 1687-1692 176 ... khuan Rh thude loai Sinorhizobium xinjiangensis Chiing Rh cd kha nang sinh chat kich thich sinh trudng thue vat dat 45mg/l Summary Study on Indole 3-Acetic acid production of bacteria Rh Nguyen... thii (Hinh ) , sinh ly, sinh hoi, ddng dua vao khoi phan loai Bergeys [66], ehung tdi xic dinh cd kha nang cbiing Rh thude chi Sinorhizobium De khing dinh cbinb xac den loai ehung Rh thude loii... cbiing Rh li 40mg/l Dieu niy phii hgp vdi nhieu nghien eiiu va ngoii nude ve kha nang sinh lAA eua cic chiing vi khuan viingri [6] 3.2 Dac diem hinh thai, sinh ly, sinh hoa eua ehung Rh Nhu chiing