稻瘟病菌3_磷酸甘油醛脱氢酶基因_gpd_的快速克隆_英文_

稻瘟病菌3_磷酸甘油醛脱氢酶基因_gpd_的快速克隆_英文_ ( ) 24 5: 468, 474, 1998 浙 江 农 业 大 学 学 报 Jo u rna l o f Zh e jiang A g r icu ltu ra l U n ive r sity - - 3- Ra p id C lon in g Fulllen g th cD NA of Glycera ldehydepho spha te D e- () hydrogena se Gen e gpdf rom M a g n ap o r th e g r isea 1112 ( ; ; ; 1 ,L u Guo do n gW an g Zo n gh u aX ie L ian h u iZh en g X u eq in D ep a r tm en t of P lan t P rotec t ion , 350002, ; 2 F uj ian A g r icu l tu ra l U n iv e rs ity F u z h ou C h inaN a t iona l K ey B iotech nology L abora tory f or T rop i2 ), , 571737, ca l C rop sC h inese A cad em y of T rop ica l A g r icu l tu ra l S c iencesH a inan C h ina A bstrac t H e re w e de sc r ibed a m o re sim p le and eff ic ien t m e tho d to c lo ne gene reg io n be tw een a 351 2sing le sho r t sequence in a cDN A m o lecu le and it s unk now n ’ o r ’ endT h e fu llleng th cDN A o f () 232g lyce ra ldeh ydep ho sp h a te deh yd ro gena se gene gp do f M ag nap or th e g r isea w a s c lo ned by u sing 1 th is new m e tho dTw o p r im e r s w e re syn th e sized ba sed o n th e co n se rved sequence s o f gp d gene s 12 1 1 5f rom o th e r f ilam en to u s fung iP p r im e r w a s a sp ec if ic sequence th a t is nea r to ’ end o f th e 2 3gene s and P p r im e r w a s th e com p lem en ta ry sequence o f a sp ec if ic sequence th a t nea r to ’ end o f 1 2? th e gene sL iga t io n p ro duc t s o f d scDN A pB lue sc r ip t SK w e re u sed a s tem p la te s d irec t ly in PCR 1 31 exp e r im en t sT h e ’ reg io n o f cDN A w a s am p lif ied by u sing sp ec if ic p r im e r P and th e standa rd se2 1 752 quenc ing p r im e r T T h e ’ reg io n o f cDN A w a s am p lif ied by u sing sp ec if ic p r im e r P and th e se2 1 353, quenc ing p r im e r T T h e ’ and ’ f ragm en t s o f cDN A w e re c lo ned and sequenced re sp ec t ive ly 1 011 and th e fu ll sequence o f cDN A co n ta in ing bp nuc leo t ide s w a s o b ta ined af te r com b in ing th e se 1 1 tw o sequence sT h e hom o lo gy o f nuc leo t ide and am ino ac id sequence s be tw een M g r isea and o th e r 12 6114% - 8616% 6416% - 8613% 1 100%f ilam en to u s fung i a re and re sp ec t ive lyA pp ro x im a te ly 1am ino ac id hom o lo gy a re fo und in func t io na l reg io n o f th e enzym e am o ng d iffe ren t fung i ( ) ; 232;Key words M ag nap or th e g r iseaG lyce ra ldeh ydep ho sp h a te deh yd ro gena se gene gp d c lo n ing m e tho d 111( 鲁国东; 王宗华; 谢联辉; 郑学勤 1 福建农业大学植保系, 福州 350002; 2 中国热带农业科学院)热带生物技术国家重点实验室, 海南 571737 () () () 稻瘟病菌 3- 磷酸甘油醛脱氢酶基因 的快速克隆 英文Λ 浙江农业大学学报, 1998, 24 5: 468gpd , 474 摘 要本文描述了一种简便而有效的克隆基因未知 5和 3末端序列的, 并用此方法克 ’ ’ () 隆了稻瘟病菌的 32磷酸甘油醛脱氢酶基因 Λ即通过比较1 2 种真菌的已知 基因序列, 寻找 gp dgp d 内部的保守区段, 并合成一对特异性引物Λ 引物 为靠近基因 5端的一个特异性序列, 引物1 P ’ 则与 3端的一段特异性序列互补Λ 以双链 2 载体连接物为模板, 利用基 2 ?P ’ cDN A pB lue sc r ip tSK 因的特异性引物 和载体上的测序引物 扩增出包含 末端的序列; 利用特 1 7, 3P T PCR cDN A ’ 异性引物 和标准测序引物 扩增出包含 末端的序列Λ 将两片段分别克隆、测序和合 2 3, 5P T cDN A ’ () 并后, 得到全长 1 011 的稻瘟病菌 32磷酸甘油醛脱氢酶基因 的编码区序列Λ 该序列及其相bp gp d 205225: 1998R ece ived ? 1994-2013 China Academic Journal Electronic Publishing House. All rights reserved. 16% 和 6416% - 8613% 的同源 86应的氨基酸序列与已知真菌的 基因序列有着 6114% - gp d 性Λ 特别在酶的活性功能区不同真菌间有着近乎相同的氨基酸序列Λ () 稻瘟病菌; 32磷酸甘油醛脱氢酶基因 ; 克隆方法 关 键 词gp d 中图分类435. 111; 78SQ 1 , 4 , D e sp ite th e deve lopm en t o f n um e ro u s cDN A c lo n in g st ra teg ie sth e m o st sim p le an d ( ) 1 rap id m e tho d is b a sed o n th e DN A po lym e ra se ch a in reac t io n PCR tech n iqu ePCR em 2 () , + p lo y s tw o o ligo n u c leo t ide p r im e r so n e com p lem en ta ry to a sequ en ce o n th e st ran d an d ) (- th e o th e r to a dow n st ream sequ en ce o n th e st ran d an d th e sequ en ce b e tw een th e se tw o 1 2 p r im e r s can b e am p lif ied an d th en iso la tedT h e fu lllen g th cDN A w ill b e o b ta in ed b y th is 35, 1 , m e tho d if th e tw o p r im e r s lie in ’ an d ’ en d o f th e cDN A re sp ec t ive lyH ow eve rit is gen2 1e ra lly d iff icu lt to de sign th e sp ec if ic p r im e r s th a t m a tch th e tw o en d s o f u n k now n cDN A T h e comm o n st ra tegy in vo lved th e co n st ru c t io n o f a cDN A lib ra ry sc reen ed w ith a p rev io u sly ch a rac te r ized hom o lo go u s p ro b e de r ived f rom a re la ted sp ec ie s o r th e u t ility o f a st ra tegy () te rm ed“rap id am p lif ica t io n o f cDN A en d s”RA C E , w h ich w a s b a sed o n o n e side PCR an d () 172requ ired dT adap to r an d tw o sp ec if ic p r im e r s de r ived f rom a sm a ll reg io n o f co n se rved 1 1 sequ n eceW e repo r t h e re a m o re sim p le an d eff ic ien t m e tho d to c lo n e gen e reg io n b e tw een 351 2a sin g le sho r t sequ en ce in a cDN A m o lecu le an d it s u n k now n ’ o r ’ en dT h e fu lllen g th () 232cDN A o f g lyce ra ldeh ydep ho sp h a te deh yd ro gen a se gen e gp d o f M a g n ap orth e g risea w a s 1c lo n ed u sin g th is n ew m e tho d () T h e gp d gen e en co de s g lyce ra ldeh yde232p ho sp h a te deh yd ro gen a se GPD w h ich ca ta ly2 22321,se s th e reve r sib le o x ida t io n an d p ho sp ho ry la t io n o f D g lyce ra ldeh ydep ho sp h a te to fo rm 321 d ip ho sp ho g lyce ra teT h e gp d gen e h ad b een repo r ted to b e co n st itu t ive ly exp re ssed a t h igh 1 5leve lsT h e ’ f lan k in g reg io n o f th is gen e h ad co n sequ en t ly b een u sed to co n st ru c t t ran sfo r2 5 1 m a t io n vec to r s fo r som e f ilam en to u s fu n g iGPD f rom a w ide va r ie ty o f p ro k a ryo t ic an d eu 2 , k a ryo t ic so u rce s show s a h igh deg ree o f co n se rva t io n a t am ino ac id an d n u c leo t ide leve lb u t 351 2 w e fo u n d th e co n se rva t io n in th e ’ an d ’ reg io n o f gen e is low So am p lif ica t io n o f fu ll 13535len g th cDN A d irec t ly b y ’ an d ’ hom o lo go u s p r im e r s is im po ssib leW e c lo n ed th e ’ an d ’ 12f ragm en t o f th is gen e b y u sin g tw o sp ec if ic hom o lo go u s p r im e r s w ith in gen e s de r ived f rom 5 , 7 ) f ilam en to u s fu n g ian d GenB an k da tab a sean d tw o gen e ra l sequ en c in g p r im e r s w ith in a 1p la sm id vec to r 1 M a te r ia l an d M e tho d s 111 M a ter ia l 852141 1v iru len t o n m an y r ice cu lt iva r s p ro v ided b y Iso la te B o f M g r isea w a s h igh ly , 1 5 B io tech no lo gy D ep a r tm en tC h in a N a t io n a l R ice R e sea rch In st itu teT h e ho st st ra in D H () 1 1an d vec to r pB lu e sc r ip t II SK A b b rpB So f E col i w e re p ro v ided b y N a t io n a l K ey B io tech2 1, no lo gy L abo ra to ry fo r T rop ica l C rop sC h in e se A cadem y o f T rop ica l A g r icu ltu ra l Sc ien ce s 470 浙 江 农 业 大 学 学 报24 卷 1 E n zym e s an d o th e r reagen t s w e re p u rch a sed f rom re la ted com p an ie s 112 m RNA ex tra c t ion 852141 T h e iso la te B w a s cu ltu red in yea st sta rch m ed ium an d th e m yce lia w e re co llec t2 19% 1 , 0ed w a sh ed w ith N aC l an d th en g ro u n d in liqu id n it ro gen T o ta l RN A w a s ex t rac ted 8 . w ith gu an id in ium ch lo r ide a s Sam b roo k e t a l su gge sted an d th e m RN A w a s p rep a red b y “”1 u sin g P rom egaPo lyA T rac t m RN A Iso la t io n Sy stem s 113 cD NA c lon in g 15 , T h e f ir st st ran d cDN A w a s syn th e sized u sin g o ligo dT a s p r im e ran d th e seco n d 1 st ran d cDN A w a s o b ta in ed b y rep lacem en t m e tho dT h e do u b le st ran d cDN A w a s p u r if ied 241 b y p a ssage th ro u gh a Sep h a ro se C lB co lum n an d added E coR I adap to rT h e cDN A w ith 1adap to r w a s th en liga ted in to th e E coR I site o f th e pB S vec to r 114 Pr im er s de s ign an d PCR am p l if ica t ion Tw o sp ec if ic p r im e r s w e re de sign ed an d syn th e sized b a sed o n th e co n se rved 5- 712 sequ en ce s o f gp d gen e s f rom f ilam en to u s fu n g i an d GenB an k da tab a se: P 1 53, 2 531 1 ’GA GT CCA C T GGT GT C T T CA C’P ’CCA C T C GT T GT C GTA CCA ’T h e P p r im e r 52 w a s a sp ec if ic sequ en ce th a t is n ea r to ’ en d o f th e gen e s an d th e P p r im e r w a s th e com p le2 1 3m en ta ry sequ en ce o f a sp ec if ic sequ en ce th a t is n ea r to ’ en d o f th e gen e sTw o stan da rd se2 : 3 53,qu en c in g p r im e r s co n ta in ed in pB S vec to r w e reT ’A T TA A CCC T CA C TA A A GGGA ’ 7 531T ’TA A TA C GA C T CA C TA TA GGG’ 2T h e cDN A c lo n e o r liga t io n p ro du c t s o f d scDN A pB S w e re u sed a s tem p la te d irec t ly in 1 11, 3PCR exp e r im en t sA s show n in F ig th e ’ reg io n cDN A o f gp d gen e w a s am p lif ied b y u s2 1 1 75in g th e sp ec if ic p r im e r P an d th e sequ en c in g p r im e r T T h e ’ reg io n o f cDN A w a s am p li2 1 2 3 f ied u sin g P an d T p r im e rT h e p ro cedu re s fo r PCR am p lif ica t io n w e re in it ia l den a tu ra t io n 94 ? ? ? 4 , 30 94 50 , 50 90 , a t fo r m in cyc le s o f den a tu ra t io n a t fo r san n ea lin g a t fo r s 72 ? ? 1150 , 72 7 an d ex ten sio n a t fo r sfo llow ed b y a f in a l ex ten sio n a t fo r m in 115 C lon in g an d sequen c in g of cD NA s 2T h e PCR p ro du c t s w e re c lo n ed in pB S TA vec to r acco rd in g to th e m e tho d o f M a rch u k e t 9 1 2 a l P la sm id s w ith cDN A in se r t s w e re f ir st sc reen ed b y th e p la te s co n ta in in g IP T G an d X 1 , ga lan d th en iden t if ied u sin g re st r ic t io n en zym e sT h e p la sm id DN A s w ith co r rec t in se r t s 8 1 w e re p rep a red b y th e a lk a lin e ly sis m e tho d an d p u r if ied w ith P E G T h e DN A s w e re se2 () 2370qu en ced in A B I A DN A sequ en ce r P e rk in E lm e ra s th e m e tho d o f supp lie r’ s recomm en2 1 da t io n 2 R e su lt s an d D iscu s s io n 211 Am p l if ica t ion an d c lon in g , 52A f te r th e am p lif ica t io n reac t io n th e ’en d cDN A p ro du c t s n am ed a s C 5 f ragm en t w a s 1 040 32exp ec ted to b e abo u t bp in len g th an d th e ’en d cDN A p ro du c t s n am ed a s C 3 f ragm en t ) (800 11 w a s exp ec ted to b e abo u t bp in len g th acco rd in g to th e c lo n in g st ra tegy F igan d th e ? 1994-2013 China Academic Journal Electronic Publishing House. All rights reserved. 1 size o f gp d gen e s f rom o th e r fu n g iDN A s w ith th e exp ec ted size w e re iso la ted f rom aga ro se 1 ƒge ls an d c lo n edP la sm id s w ith cDN A in se r t s w e re p ick ed o u t an d de tec ted b y B am H IH in d I2 1 , II d ige st io n C 5 P 2 C 5 P 5 an d C 5 P 7 c lo n e s w ith C 5 f ragm en t an d C 3 P 15 an d C 3 P 20 w ith C 3 f rag2 1m en t w e re re sp ec t ive ly co n f irm ed af te r re st r ic t io n an a ly sis 11 F igThe c lon in g stra tegy of gpd gen e 212 D NA sequen ce an d deduced aa sequen ce 2 se1 T h e C 3 P 20 w a s su b c lo n ed in to C 31 P 5 an d C 32 P 6 b y E coR I d ige st io n A ll c lo n e s w e re 2 qu en ced in bo th d irec t io n an d th e fu lllen g th cDN A sequ en ce o f gp d gen e w a s o b ta in ed af te r 1 12 com b in in g an d an a ly sin g th e se sequ en ce sF ig show s th e sequ en ce s o f co d in g reg io n an d it s 1 1 011 336dedu ced am ino ac id o f gp d gen eT h e reg io n co n ta in s bp n u c leo t ide s an d en co de s 472 浙 江 农业大学学报24 卷 1 am ino ac id s an d a te rm in a l co de, TA A 1 2 11 F igNuc leo t ide an d deduced am in o ac id sequen ce s of gpd gen e f rom M gr sea -1011 337To ta l n uc leo t ide s an d en cod in g a puta t ive aa pro te in 1 1 T h e gp d gen e sequ en ce o f M g risea show s h igh hom o lo go u s to o th e r k now n gp d gen e s 1 12 T h e hom o lo gy o f n u c leo t ide an d am ino ac id sequ en ce s b e tw een M g risea an d o th e r f ila2 16% 6416% - 8613% ( ) 8614% - 1 61an d m en to u s fu n g i a re re sp ec t ive ly da ta u n show n Inp a r t icu la r, in p a r t s o f th e GPD po lyp ep t ide k now n to b e e ssen t ia l fo r en zym a t ic ac t iv ity th e ? 1994-2013 China Academic Journal Electronic Publishing House. All rights reserved. 151 178 1 100% hom o lo gy is a lm o st Su ch a s th e re sidu e s su r ro u n d in g th e ca ta ly t ic C y san d H iso f + 34 ( th e ac t ive site an d th e re sidu e s in vo lved in N A D b in d in g no tab ly tho se a ro u n d A sp an d 5- 7101 P h e1 T h e GPD s f rom th e a scom yce te fu n g iM 1 g risea , A sp e rg il lu s n id u la n s, G lom e re l2 la c in g u la ta , C la v icep s p u rp u rea , C och l iobolu s h e te ros t rop h u s, C ry p h on ec t r ia p a ra s it ica , an d P od osp ora a n se r in a a re m o re c lo se ly re la ted to each o th e r th an th e gen e f rom th e b a sid2 1 , , iom yce teO n th e o th e r h an dth e gen e f rom th e b a sid iom yce te fu n g i U s t ila g o m ay d is , , P h a n e roch a e te ch ry sosp or ium S ch iz op hy l lum com m u n ean d A g a r icu s bisp oru s a re a lso m o re 1 , , c lo se ly re la ted to each o th e rGPD f rom C u rv u la ria lu n a ta an im p e rfec t fu n gu sis ve ry , c lo se ly re la ted to tho se f rom th e a scom yce te sco n f irm in g th a t it s p lacem en t m ay b e in th a t 1 g ro up Com p a r iso n o f th e am ino ac id sequ en ce s o f GPD h a s b een u sed in th e c la ssif ica t io n o f10- 111 b ac te r ia o r to gen e ra te p h y lo gen ic re la t io n sh ip am o n g fu n g i 213 Codon usa ge () 1 4864, ƒM o st co do n s a re u t ilized in gp d gen e f rom M g risea b u t th ey a re h igh ly b ia sed 1 76% tow a rd s tho se en d in g in p y r im id in eO f a ll co do n s h ave a p y r im id in e in th a t po sit io n () : 61% , : 15% , : 21% , : 3% 1 CT GA T h is b ia s is sim ila r to th a t fo u n d fo r o th e r h igh ly ex 2 p re ssed gen e s in f ilam en to u s fu n g i b u t c lea r ly d iffe ren t f rom th a t in h igh ly exp re ssed gen e s o f 5, 6 1 Sacch a rom yce s ce rev isiae 214 Con c lus ion s 21411 , In th is stu dyw e de sc r ib ed an a lte rn a t ive to o th e r cDN A c lo n in g m e tho d s th a t is ad2 1 23, van tageo u s in m an y re sp ec t sA sin g le gen e sp ec if ic p r im e r is u sed to gen e ra te ’en d c lo n e s , , 521w ho se sequ en ce s can b e u sedif n ece ssa ryto de sign th e p r im e r s requ ired fo r ’en d c lo n in g 1 T h e p ro to co l is rap id an d m o re eff ic ien t th an o th e r m e tho d sO th e r po ten t ia l app lica t io n s o f 1 , th is p ro to co l a re po ssib leFo r ex am p lep r im e r s b a sed o n am ino ac id sequ en ce m igh t b e ade2 1 : qu a te fo r cDN A c lo n in gIt sho u ld a lso b e u sed to c lo n e gen e f rom genom ic lib ra ryT h e ge2 nom ic DN A can b e c leaved b y o n e re st r ic t io n en zym e an d c lo n ed in to pB S vec to r to co n st ru c t 1 an en r ich ed lib ra ryT h e DN A f ragm en t th a t co n ta in a im gen e can b e am p lif ied b y u sin g a 1sin g le sp ec if ic p r im e r w ith in th e gen e an d a sequ en c in g p r im e r w ith in vec to r 21412 T h e cDN A o f gp d gen e h ave b een c lo n ed an d sequ en ced f rom th e p lan t p a tho gen ic 1 1 21 337fu n gu s M g risea b y o u r n ew m e tho dT h e gen e en co de s a p u ta t ive aa p ro te in Co do n 1 u sage in th e gp d gen e is h igh ly b ia sed in favo r o f co do n s en d in g in C T h e dedu ced am ino ac id 1 , sequ en ce o f M g risea gp d is qu ite sim ila r to tho se o f o th e r sp ec ie se sp ec ia lly in ce r ta in re2 1 g io n s su ch a s th e ca ta ly t ic dom a in Ref eren ce s 1 , , 1 2: F ro hm an M A D u sh M K M a r t in G R R ap id p ro duc t io n o f fu llleng th cDN A s f rom ra re t ran sc r ip t sA m p lif ica2 21 , 1988, 85: 8998- 9002t io n u sing a sing le genesp ec if ic o ligo nuc leo t ide p r im e rP roc N a tl A cad S ci U S A 2 , 1 1 , 1993, 11: 306- 313G ib so n SSom e rv ille C Iso la t ing p lan t gene sT ib T ech 3 , 1 1L iang P P a rdee A B D iffe ren t ia l d isp lay o f euk a ryo t ic m e ssenge r RN A by m ean s o f th e po lym e ra se ch a in reac t io n , 1992, 257: 967- 971S cience 4 , , 1 1 , 1993, 259:L isit syn N iL isit syn N aW ig le r M C lo n ing th e d iffe rence s be tw een tw o com p lex genom e sS cience 474 浙 江 农 业 大 学 学 报24 卷 946- 951 5 , , , 1 2T em p le to n M D R ik k e r ink E H A So lo n S L et a lC lo n ing and m o lecu la r ch a rac te r iza t io n o f th e g lyce ra ldeh yde 3221 ,p ho sp h a te deh yd ro gena seenco d ing gene and cDN A f rom th e p lan t p a tho gen ic fungu s G lom erel la cing u la ta G ene 1992, 122: 225- 230 , , 21 232, 6 P un t P J D ingem an se M A J aco b sM e ijsing B J M et a lIso la t io n and ch a rac te r iza t io n o f th e g lyce ra ldeh yde 1 , 1988, 69: 49- 57p ho sp h a te deh yd ro gena se gene o f A sp erg il lu s n id u lansG ene 7 , 1 232Sm ith T L L eo ng S A Iso la t io n and ch a rac te r iza t io n o f a U st ilago m ayd is g lyce ra ldeh ydep ho sp h a te deh yd ro ge2 21 , 1990, 93: 111-117 na seenco d ing geneG ene ( ) 8 , , 1 221 : Sam b roo k J F r it sch E F M an ia t is T M olecu la r C lon ing A L abora tory M anu a l nd ed it io n N ew Yo rkCo ld , 1989Sp r ing H a rbo r L abo ra to ry P re ss 9 , , 1 2, M a rch uk D D rum n M Sau lino A Co n st ruc t io n o f T vec to r sa rap id and gene ra l sy stem fo r d irec t c lo n ing o f un2 1 1991, 19: 1154- 1158m o d if ied PCR p ro duc t sN u cl A cid s R es , , 1 1 10 L aw rence J GO chm an H H a r t l D D M o lecu la r and evo lu t io na ry re la t io n sh ip s am o ng en te r ic bac te r iaJ G en M i2 , 1991, 137: 191121921crobiol 11 1 2Sm ith T L D isp a ra te evo lu t io n o f yea st s and f ilam en to u s fung i ind ica ted by p h y lo gene t ic ana ly sis o f g lyce ra ldeh yde 321 , 1989, 86: 7063-p ho sp h a te deh yd ro gena se gene sP roc N a tl A cad S ci U S A 7066 () 责任编辑杜玲玲? 1994-2013 China Academic Journal Electronic Publishing House. All rights reserved.