首页 棉铃虫磷酸甘油醛异构酶基因Hatpi的克隆及分析_英文_

棉铃虫磷酸甘油醛异构酶基因Hatpi的克隆及分析_英文_

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棉铃虫磷酸甘油醛异构酶基因Hatpi的克隆及分析_英文_棉铃虫磷酸甘油醛异构酶基因Hatpi的克隆及分析_英文_ Isola t ion and ana lysis of the tr io sepho spha te isom era se gene , H a tp i , from H e licove rpa a rm ige ra L IU Yan, Q I X iao2Peng, N IU B ao2Long, W EN G Hong2B iao, 3 CH EN J in2E, H E L i2H ua, SH EN W e i2Feng, M E...

棉铃虫磷酸甘油醛异构酶基因Hatpi的克隆及分析_英文_
棉铃虫磷酸甘油醛异构酶基因Hatpi的克隆及分析_英文_ Isola t ion and ana lysis of the tr io sepho spha te isom era se gene , H a tp i , from H e licove rpa a rm ige ra L IU Yan, Q I X iao2Peng, N IU B ao2Long, W EN G Hong2B iao, 3 CH EN J in2E, H E L i2H ua, SH EN W e i2Feng, M EN G Zh i2Q i()L abo ra to ry of En tomo2Mo lecu la r B io logy, Sericu ltu re R e sea rch In stitu te, Zhe jiang A cadem y of A gricu ltu ra l Sc ience s, H angzhou 310023 , Ch ina Ab stra ct: Helicoverpa a rm igera is an impo rtan t lep idop teran p est of m any c rop s inc lud ing co tton. Mo lecu la r analysis w ill p romo te p est con tro l in the fu tu re. The gene that encod ing trio sep ho sp hate isom erase in Helicoverpa ( a rm igera, Ha tpi, was mo lecu la rly cloned and ana lyzed in th is study. The cDNA of Ha tpi GenB ank acce ssion no. ) A Y736358 is 1 149 bp in length and encode s a 248 am ino acid p ro tein w ith the estim ated mo lecu lar m a ss of 26. 4 (βα) kD and isoe lectric po in t of 5. 82. structu re found in the deduced p ro tein struc tu re of H aTP I was sim ila r to 8 (tha t of o the r trio sep ho sp hate isom e rase. H igh iden tity was a lso found in the active catalytic site s L ys12 , H is94 , ) ( ) and Glu165 and p ep tide mo tifs A YEPVWA IGTG and GGA SL KPEF . R T2PCR ana lysis re su lts showed tha t Ha tpi wa s exp ressed in the em b ryo, larva, p up a and adu lt of H. a rm igera, sugge sting Ha tpi m ay p lay ro le s in va riou s developm en ta l stage s of H. a rm igera. Key word s: Helicovepa a rm igera; trio sep ho sp hate isom erise; gene; RACE; sequence analysis; exp ression ) et a l. , 2002; Gne re r et a l. , 2006. 1 INTROD UC T IO N Since DHA P is requ ired by the sn2glyce ro l23 2 ( α) p ho sp ha te dehyd rogena se 2GPDH 2glyce ro l ( ) TP I o r T MI is a Trio sep ho sp ha te isom e ra se ( ) p ho sp ha te oxida se GPO shu ttle, the TP I tha t key enzym e in the glyco lytic p a thway, wh ich ca ta lyze s + take s p a rt in the gene ra tion of NAD fo r fligh t the in te rconve rsion be tween d ihyd roxyace tone m u sc le ac tivity becom e s mo re impo rtan t in in sec ts ( ) DHA P and glyce ra ldehyde232p ho sp ha te p ho sp ha te ( ) Sack to r, 1970 . The tpi gene ha s been iden tified ( )G232P . Trio sep ho sp ha te isom e ra se p lays an from D rosoph ila , mo squ ito and B om byx m ori impo rtan t ro le in seve ra l m e tabo lic p a thways and is ( Tittige r et a l. , 1993; Tyshenko and W a lke r, 1997; (e ssen tia l fo r effic ien t ene rgy p roduc tion W ang et a l. , ) Zhong, 2001 . Ye t little is known abou t the tpi ) 999 . U P to now tpi gene ha s been we ll 1homo logou s gene in H. a rm igera. H e re, the H. ( cha rac te rized from a la rge num be r of o rgan ism s Koh l a rm igera tpi gene wa s c loned and ana lyzed. et a l. , 1994; Ap a ric io et a l. , 2003; Gaya th ri et a l. , ) ()2007. The ac tive ca ta lytic site s L ys, H is and Glu 2 M A TER IAL S A ND M ETHOD S ( and the p ep tide mo tifs A YEPVW S IGT and ) 2. 1 Te st in sec ts GGA SL KPEF we re con se rved in a ll known TP Is () αβ W ang et a l. , 1999. /ba rre ls w ith e igh t p a ra lle l L a rvae of H. a rm igera we re co llec ted from βα2strand s su rrounded by a laye r of e igh t2he lice s a re ( )co tton fie ld s X iao shan, Zhe jiang P rovince, Ch ina ( recen tly found in subun it of TP I Gaya th ri et a l. , in 2005. Cu ltu re s we re m a in ta ined a t 25 ? w ith a ) ( ) 16 h?8 h ligh t ?da rk p ho top e riod and 40 %2007. In stab ility of the isom e ra se due to d iffe ren t m u ta tion s of the enzym e m ay unde rlie the ob se rved re la tive hum id ity in the labo ra to ry. L a rvae we re reduced ca ta lytic ac tivity. TP I defic iency is u sua lly rea red on an a rtific ia l d ie t and adu lts in sea led box ( a ssoc ia ted w ith seve ra l gene tic d iso rde rs such a s w ith 10 % so lu tion of honey in wa te r Bown et a l. , ) 1997 . ch ron ic haemo lytic anaem ia, ca rd iom yop a thy, 2. 2 Gen om ic D NA ex tra c t ion su scep tib ility to infec tion s, seve re neu ro logica l ( dysfunc tion and ea rly dea th Schne ide r, 2000; O lah Genom ic DNA wa s extrac ted from ind ividua ls ( ) ( Foundation item: N a tiona l Key Techno logie s R &D P rogramm e of Ch ina No. 2004D FBA0007 ; Zhe jiang Sc ience Fund fo r Young Scho la rs No. )2007R20006 In troduction of the first au tho r: L IU Yan, fem a le, bo rn in 1976 , PhD , m a jo r in en tomo2mo lecu la r b io logy, Te l. : 86 20571 286404173; E2m a il: m ayan ly @ sina. com 3 A u tho r fo r co rre spondence, Tel. & Fax: 86 20571 286404173; E2m ail: m engzq2011 @ sina. com R ece ived: 2009 201 214; A ccep ted: 2009 207 208 ( )T TaKaR a. The in se rtion s we re iden tified by PCR u sing a simp le o rgan ic extrac tion m e thod ou tlined by ( ) by Tob le r et a l. 2005 . App roxim a te ly 150 m g of amp lifica tion u sing M 13 fo rwa rd and reve rse p rim e rs adu lt wa s p laced in to 1. 5 mL cen trifuge tube s and befo re sequenc ing. 2. 4 RAC E m e thod μ( homogen ized in 500L of in sec t grind ing buffe r 10 μThe cDNA s we re syn the sized from 2 g of to ta l ( ) mmo l /L Tris2HC l pH 8. 0 , 100 mmo l /L ED TA , RNA u sing BD SMAR T RACE cDNA Amp lifica tion )0. 5 % SD S. The homogena te wa s d ige sted fo r 3 h ( ) Kit BD B io sc ience s. The 5 ′and 3 ′cDNA end s a t 55 ? w ith 0. 5 m g of p ro te ina se K. The we re ob ta ined by touchdown PCR w ith LA Taq DNA homogena te wa s then extrac ted th ree tim e s, first w ith ( ) u sing a un ive rsa l p rim e r po lym e ra se TaKaR a equa l vo lum e s of buffe red p heno l, second w ith equa l ( ) 2sp ec ific p rim e rs the genem ixtu re U PM and vo lum e s of buffe red p heno l / ch lo rofo rm / isoam yl ( ) GSP s : P3 , 5 ′2TCTCTCCGAAAA TTGTTCTC 23 ′ ( ) a lcoho l 25 ?24 ?1 and th ird w ith equa l vo lum e s of ( ) 5 ′end p rim e rand P4 , 5 ′2GGGCTAA TGTTGTAC ( ) ch lo rofo rm / isoam yl a lcoho l 24 ?1 . The DNA wa s () TCGCT23′3′end p rimeraccording to manufacture’s ( )then p rec ip ita ted w ith 2 ×vo lum e of 95 % v / v ( )p rotocol http: / /www. bdbio sciences. com / clontech / . ( e thano l and 1 /10 vo lum e of 3. 0 mo l /L N aOAC pH The RT2PCR p roducts were purified directly from bands ) μ5. 2 . Genom ic DNA wa s re su sp ended in 100 L excised from agarose gels before cloned into pMD182T 0. 1 ×TE buffe r. ( ) TaKaRa and sequenced. Full length cDNA and 2. 3 Iso la t ion of RNA an d the RT2PC R ana ly sis (genome p rimers were P5 5′2A GCGGTGAA GTGAA GA To ta l RNA wa s sep a ra te ly from samp le s by u sing Trizo l reagen t acco rd ing to the m anufac tu re ’s ) (AGTTTAC23′and P6 5′2GTTTGA TTTTTAAGTTTCG μin struc tion. Fo r R T exp e rim en ts, 3 g of to ta l RNA )ATTG23′. PCR fragments were cloned and sequenced wa s dena tu red a t 65 ? fo r 5 m in fo llowed by qu ick at least tw ice per strand. μμch ill on ice in a 14L reac tion con ta in ing 1L o ligo 2. 5 Ana lysis of expression pa ttern of the H a tp i gene ( ) ( ) μdT Sangon B io. p rim e r, and 1 L of Fo r deve lopm en ta l ana lysis, to ta l RNA wa s 18 ( 10 mmo l /L dN TP m ixtu re 10 mmo l /L each dA TP, extrac ted from va riou s deve lopm en ta l stage s ) dGTP, dCTP and dTTP a t neu tra l pH . A fte r ( ) ( inc lud ing em byro 5 th day afte r ha tch, la rva 2 nd μ( add ition of 2 L 10 ×reac tion buffe r KeyGEN, ) ( day of the 3 rd in sta r ,p up a 3 rd day afte r )( ) p up a tionand adu lt 1 st day of ec lo sion of H.) μcn , 1 L AMV www. keygen tec. com. a rm igera re sp ec tive ly. R T2PCR wa s p e rfo rm ed a s ) μ( ) ( KeyGEN, 10 U n its, 1 L D TT 1 mo l /L and ( de sc ribed above u sing p rim e rs P7 5 ′2GTACTGGCA μ( μ) 0. 5 L RN a se inh ib ito r 40 U /L , reac tion the ) ( A GACA GCA TCTC23 ′and P8 5 ′2GTTTGA TTTTT te rm ina ting, wa s incuba ted a t 37 ? fo r 1 h. A fte r ) AA GTTTCGA TTG23 ′. H aactin we re app lied a s an the reac tion wa s hea ted a t 70 ? fo r 15 m in fo r in te rna l con tro l. Each reac tion wa s rep ea ted a t lea st inac tiva tion. The cDNA fragm en ts we re amp lified th ree tim e s. ( ) w ith Ex Taq DNA po lym e ra se TaKaR a u sing the 2. 6 Sequen ce ana ly s is )( H aactin GenB ank acce ssion no. : X97614. 1 ( The Sw iss2P ro t and EMBL da taba se h ttp: / / ( p rim e rs Fac tin 5 ′2CGCGACCTCACAG ACTACCT2 ) cn. exp a sy. o rg / too ls / b la st / we re u sed to b la st the ) ( 3 ′and R ac tin 5 ′2GGCCA GACTCA TCGTACTCC homo logs among TP Is. Phylogene tic tree ana lysis wa s ) T23 ′a s in te rna l con tro l. H igh ly con se rved fragm en t p e rfo rm ed u sing C lu sta lx, PH YL IP and TreeV iew of tpi wa s amp lified from H. a rm igera w ith softwa re s. M u ltip le sequence a lignm en t wa s ( degene ra te p rim e rs P1 5 ′2TYACTGSTGANA TN TC p e rfo rm ed by u sing C lu sta lW w ith defau lt p a ram e te rs ) ( VCCM GC 23 ′and P2 5 ′2A GDCTGVCTCCNCCWA ( se t a s in the C lu sta lW web se rve r a t EB I h ttp: / / ) CBAA GAAV 23 ′. The rm a l cyc ling cond ition wa s ) www. eb i. ac. uk / c lu sta lw / and GEN EDOC32. T M I95 ? fo r 5 m in fo llowed by 30 cyc le s of 95 ? 30 s, ba rre l struc tu re wa s ana lyzed by u sing 123D 58 ? 30 s and 72 ? 1 m in. The la st cyc le wa s a fina l ( Th read ing h ttp: / /123 d. nc ifc rf. gov / run123D +. exten sion a t 72 ? fo r 5 m in. Amp lified p roduc ts ) h tm land the refe rence re su lt of M ethanoca ldococcus we re sep a ra ted by ge l e lec trop ho re sis on 1. 2 % ) ( jannasch ii TP I Gaya th ri et a l. , 2007 . aga ro se ge ls a t 100 V fo r app roxim a te ly 1 h u sing ( 1 ×TA E buffe r 40 mmo l /L Tris ace ta te and 3 RESUL TS ) 2 mmo l /L ED TA in wa te r. A fte r e lec trop ho re sis, TMthe ge l wa s sta ined fo r 30 m in in 0. 01 % SYBR 3. 1 Iso la t ion an d D NA sequen ce ana ly s is of tp i ( ) Green I nuc le ic ac id ge l sta in FMC B io. . gen e of H. a rm ige ra Fo llow ing p u rifica tion of PCR p roduc ts by B io sp in A 510 bp DNA fragm en t of tpi cDNA wa s () Ge l Extrac tion Kit B ioF lux B io. , the sequence s of ob ta ined from H. a rm igera la rva l by u sing degene ra te p u rified PCR fragm en ts we re TA 2c loned in to pMD 18 2 p rim e rs. A fte r 3′and 5′RACE amp lifica tion, a 655 bp 3′end and a 459 bp 5′end cDNA fragm en ts we reop en read ing fram e of Ha tpi cDNA is 747 bp , wh ich ( ) encode s a 248 am ino ac id p ro te in F ig. 1 w ith a ob ta ined re sp ec tive ly. Fu ll2length cDNA and genom ic ca lcu la ted mo lecu la r m a ss of 26. 4 kD and the DNA of H. a rm igera tpi we re acqu ired by ove rlaying the c loned sequence s and sequenced w ith the p rim e r isoe lec tric po in t of 5. 82. ( ) p a ir A Y736358 fo r fu ll2length amp lifica tion. The H a tpi gene con ta ined 5 exon s and 5 in tron s. The 1 st in tron is loca ted in 5 ′2un tran sla ted region genom e sequence of H. a rm igera tpi a re cDNA and () ( ) U TR F ig. 2: A and the o the r 4 in tron s fe ll 1 149 bp and 2 239 bp in length, re sp ec tive ly, of be tween Gly75 and Glu76 , L ys111 and V a l112 and wh ich the cDNA wa s nam ed Ha tpi and registe red in ( ) in te rrup t V a l38 and Gln180 F ig. 2: B . ( ) GenB ank GenB ank acce ssion no. A Y736358 . The N uc leo tide s a re num be red on the left of each from NCB I to inve stiga te the evo lu tiona ry line. The deduced am ino ac id sequence is shown ( ) re la tion sh ip s w ith tha t of H. a rm igera F ig. 4 .be low the nuc leo tide sequence and num be red from The p hylogene tic ana lysis showed tha t the se enzym e s ( the first m e th ion ine. The degene ra te p rim e rs P1 , we re d ivided in to 6 c lade s acco rd ing to the ir ) P2 , m a rked w ith con se rved region a re shown w ith evo lu tiona ry re la tion sh ip s. TP Is from m amm a l we re ( c la ssed in to the 1 st c lade. The 2 nd c lade con ta ined unde rline and a rrow. Gene2sp ec ific p rim e rs 5 ′and ) tho se from fish, frog and ch icken. TP Is from in sec ts 3 ′end p rim e rs u sed in RACE we re fram ed and inc lud ing H aTP I in the p re sen t p ap e r we re group ed d irec ted by a rrow. The in itia tion and te rm ina tion ( ) codon s a re shadowed. to the 3 rd c lade a rrowhead m a rked, F ig. 4 . TP Is 3. 2 Expre ssion pa ttern of the H a tp i gen e in p a ra site s, such a s nem a toda, trem a toda and To exam ine the exp re ssion of H a tpi gene in ce stoda, we re c la ssed in to the 4 th c lade. The 5 th va riou s stage s, sem i2quan tita tive R T2PCR u sing to ta l c lade con sisted of TP Is from fungi and a lgae. The RNA ob ta ined from the em b ryo, la rva, p up a and 6 th c lade comp rised TP Is from p lan ts. Th ree adu lt we re p e rfo rm ed. R e su lts showed tha t H a tpi wa s ch lo rop la st TP Is we re c lo se r to fungi and a lga l exp re ssed in a ll samp le s bu t no sign ifican t d iffe rence ( ) enzym e s a ste risk m a rked , wh ich sugge sted tha t ( ) wa s found in the exp re ssion strength F ig. 3 . the se ch lo rop la st TP Is m ay be from a lgae o r fungi genom e. Phylogene tic s of TP I reflec ted the evo lu tiona ry of o rgan ism s to a grea t exten t, wh ich ind ica ted tha t tpi gene cou ld be an idea l cand ida te in fu rthe r evo lu tiona ry re la ted re sea rch. ( βα ) ba rre l and ca ta lytic re sidue s a re 8 con se rved in repo rted TP I fam ily, and the re is no excep tion fo r them in H aTP I, who se con se rved ( ) ca ta lytic re sidue s L ys12 , H is94 and Glu165 and F ig. 3 Exp re ssion of H a tpi gene in d iffe ren t deve lopm en ta l stage s of H elicoverpa a rm igera ( the p ep tide mo tifs A YEPVWA IGTG and 1 - 4 stand fo r the exp re ssion leve l of Ha tpi gene in em b ryo, larva, p up a ) GGA SL KPEFa re m a rked in F ig. 5 , re sp ec tive ly.and adu lt, resp ec tive ly. Am ino ac id comp a rison s revea led 68 % - 95 % iden tity be tween the se TP Is. 3. 3 Phy logen e t ic ana ly sis of TP I pro te in s TP Is from d iffe ren t o rgan ism we re co llec ted F ig. 5 A lignm en t of the deduced am ino ac id sequence s of in sec t TP Is ( ) ( ) ( ) Q09 KA2 , B la ttella germ an ica Ge rm an cock roach ; Q8M PF2 , Tenebrio m olitor yellow m ea lwo rm ; Q643R1 , B om byx m ori silk mo th ; H aTP I, ( ) ( ) Helicoverpa a rm igera; P30741 , C u lex ta rsa lis encep halitis mo squ ito; Q17HW 3 , A edes aegypti ye llowfeve r mo squ ito; Q7 PXW 5 , A nopheles gam biae ( ) str. PEST; O77458 , D rosoph ila yakuba fru it fly. Gray and b lack shad ing ind ica te the con se rved and iden tica l re sidue s, re sp ec tive ly. Con se rved (βα) barre l and ca talytic re sidue s a re shown w ith unde rline and a ste risk, re sp ec tive ly. 8 F ig. 4 Phylogene tic tree of the deduced am ino ac id sequence s of 59 TP Is from 52 euka ryo tic o rgan ism s The d istance tree wa s ca lcu la ted u sing the M EGA p rogram , wh ich was based on a C lu sta l a lignm en t of the sequences afte r p hylogene tic ana lysis. B ranch ( ) length s a re p ropo rtiona l to p e rcen tage sequence d iffe rence. N um be rs ind ica te the boo tstrap va lue s. I, M amm a lia: P60175; Pan trog lody tes ch imp anzee; ( ) ( ) ( P60174 , H om o sapiens; Q5R928 , Pongo pygm aeus o rangu tan ; P15426 , M acaca m u la tta rhe su s m acaque; Q60HC9 , M acaca fascicu la ris c rab ) ( ) ( ) ( ) ea ting m acaque cynomo lgu s monkey; P00939 , O ryctolagus cun icu lus rabb it; P54714 , C an is fam ilia ris dog; Q6 SA19 , R a ttus norverg icus ( ) ( ) ( ) ( ) ( ) ra t; P48500 , R a ttus norveg icus ra t ; P17751 , M us m uscu lus mou se ; Q1W 5B8 , S us scrofa p ig ; Q5 E956 , B os tau rus bovine . II, V ( ) ( ) ( e rteb ra ta, Eu te leo stom i: P00941 , L a tim eria cha lum nae coe lacan th; P00940 , Ga llus ga llus ch icken; Q90XF8 , X iphophorus m acu la tus sou the rn p ) ( ) ( ) ( ) latfish; Q90XG0 , B rachydan io rerio zeb rafish D an io re rio ; Q90XG1 , A cipenser brevirostrum sho rtno se stu rgeon ; Q7 ZWN5 , X enopus laevis () ( ) ( ) ( ) ( ) A frican c lawed frog; Q7 ZWB0 , B rachydan io rerio zeb rafish D an io re rio ; Q90XF9 , B rachydan io rerio zeb rafish D an io rerio ; Q589R5 , ( ) ( ) ( ) O ryzias la tipes m edaka fish Jap anese ricefish ; Q90XF7 , X iphophorus m acu la tus sou the rn p la tyfish . III, A rth ropoda, H exapoda, In sec ta: ( ) ( ) ( ) Q09 KA2 , B la ttella germ an ica Ge rm an cock roach ; Q8M PF2 , Tenebrio m olitor yellow m ea lwo rm ; Q643R1 , B om byx m ori silk mo th ; H aTP I, ( ) ( ) Helicoverpa a rm igera; P30741 , C u lex ta rsa lis encep halitis mo squ ito; Q17HW 3 , A edes aegypti ye llowfeve r mo squ ito; Q7 PXW 5 , A nopheles gam biae ( ) ( ) str. PEST; P29613 21 , D rosoph ila m elanogaster fru it fly; O77458 , D rosoph ila yakuba fru it fly. IV , N em a toda, Trem a toda and Ce stoda: Q10657 , ( ) Caenorhabd itis elegans; Q60 S70 , Caenorhabd itis briggsae; Q45XG1 , O rien tobilha rzia tu rkestan icum ; P48501 , S ch istosom a m anson i b lood fluke ; ( ) ( ) ( ) Q27775 , S ch istosom a japon icum b lood fluke; Q86 ER7 , S ch istosom a japon icum b lood fluke; Q9 GTX8 , Taen ia solium po rk tap ewo rm . V , Fungi, ( ) Euglen ida: Q2H977 , C haetom ium g lobosum ; Q6V EG6 , Eug lena g racilis; Q5 S7 Y4 , A stasia longa euglenop hycean a lga . V I, P lan ts: Q69 K00 , ( ) ( ) ch lo rop la st of O ryza sa tiva; Q9M4 S8 , ch lo rop la st of F raga ria ananassa strawbe rry; P48496 , ch lo rop last of S pinacia oleracea sp inach ; Q0H294 , ( ) ( ) P teris vitta ta Ch ine se ladde r b rake ; Q8LR75 , O ryza sa tiva; Q3HRV9 , S olanum tuberosum po ta to ; Q5JZZ3 , Phaseolus vu lga ris va r. nanus; ( ) ( ) ( ) Q38 IW 8 , G lycine m ax soybean; P21820 , Coptis japon ica Jap ane se go ld th read; Q38J I4 , S olanum tuberosum po tato; Q6 T379 , S olanum chacoense ( ) ( ) ( ) Chaco po ta to; P48495 , Petun ia hybrida p e tun ia; P48491 , A rabidopsis tha liana; Q0JQ P8 , O ryza sa tiva; P12863 , Z ea m ays m a ize; P46226 , ( ) () ( ) S eca le cerea le rye; Q9 FS79 , T riticum aestivum whea t; P34937 , Hordeum vu lga re ba rley. Th ree ch lo rop last TP I tha t are c lo se r to fungi and alga l enzym es a re m a rked w ith aste risk. Gaya th ri P, B ane rjee M , V ijaya lak shm i A , A zeez S, B a la ram H , B a laram P, M u rthy MRN , 2007. Struc tu re of trio sep ho sp ha te ( ) 4 D ISC USS IO N isom e rase T MI from M ethanoca ldococcus jannasch ii. A cta C rysta llog r. D B iol. C rysta llog r. , 63: 206 - 220. Gne re r JP, Krebe r RA , Ganetzky B , 2006. w asted aw ay, a D rosoph ila The H a tpi gene repo rted in p re sen t study m u ta tion in trio sep ho sp ha te isom e ra se, cau ses p a ralysis, con ta in s th ree ca ta lytic re sidue s L ys12 in loop1 , neu rodegene ra tion, and ea rly death. P roc. N a tl. A cad. S ci. USA , 103: 14 987 - 14 993. ( ) H is94 in loop4 and Glu165 in loop6 F ig. 5 , two Kap an DD , F lanagan N S, Tob le r A , Pap a R , R eed RD , Gonza lez JA , p ep tide mo tifs A YEPVWA IGTG a round the glu tam ic R e strepo MR , M a rtinez L , M a ldonado K, R itschoff C, H ecke l D G, ac id re sidue s and GGA SL KPEF a round the C 2 M cm illan WO , 2006. Loca liza tion of m u llerian m im ic ry gene s on a den se linkage m ap of Helicon ius era to. Genetics, 173: 735 - 757. te rm inu s, wh ich a re con sisten t w ith p reviou s study Koh l L , Ca llen sM , W ie renga R K, Opp e rdoe s FR , M iche ls PAM , 1994. ( ) Sch lieb s et a l. , 996; W ang et a l. , 1999 . The se 1Trio se2p ho sp ha te isom e rase of L eishm an ia m ex icana m ex icana re su lts ve rified tha t TP I is a h igh ly con se rved c lon ing and cha rac teriza tion of the gene, ove rexp re ssion in Escherich ia coli and ana lysis of the p ro te in. Eu r. J. B iochem. , 220: glyco lytic enzym e among d iffe ren t o rgan ism s. B e ing 331 - 338. a m em be r of the hou sekeep ing gene s, tpi is requ ired Logsdon JM , Tyshenko M G, D ixon CD , Jafa ri J , W a lke r V K, Pa lm e r ( fo r ce ll grow th and m a in tenance B rown et a l. , JD , 1995. Seven new ly d iscove red in tron po sition s in the trio se2 ) p ho sp ha te isom e ra se gene: Evidence fo r the in tron s2la te theo ry. 1985 . Tha t is con sisten t w ith the exp re ssion of P roc. N a tl. A cad. S ci. USA , 92: 8 507 - 8 511. H a tpi in a ll exam ined in the p re sen t study. M a rec F, neven L G, Rob in son A S, V reysen M , Go ld sm ith MR , A fte r the 21 in tron po sition s repo rted random ly N aga ra ju J , F ranz G, 2005. D eve lopm en t of genetic sexing stra in s ( ) in the tpi cod ing region Logsdon et a l. , 1995 , in L ep idop te ra: F rom trad itional to tran sgen ic app roache s. J. Econ. En tom ol. , 98: 248 - 259. none of the iden tified sequence va ria tion s affec ted O lah J , O ro sz F, Ke se ru GM , Kova ri Z, Kovac s J , Ho llan S, O vad i J , the am ino ac id compo sition of the TP I p ro te in, hence 2002. Trio sep ho sp ha te isom e ra se defic iency: a neu rodegene rative they a re un like ly to imp ac t the ca ta lytic ac tivity of m isfo ld ing d isea se. B iochem. S oc. T rans. , 30: 30 - 38. Sack to r B , 1970. R egu la tion of in te rm ed ia ry m e tabo lism , w ith sp ecia l ( ) the enzym e Sun et a l. , 2008 . Ye t in the fou r refe rence to con tro l m echan ism s in in sec t fligh t m u scle. A dv. Insect sha red a in tron s of H a tpi gene, on ly one in tron Physiol. , 7: 267 - 347. ( ) po sition be tween L ys111 and V a l112 tha t wa s Sch lieb s W , Thank i N , E ritja R , W ie renga R , 1996. A c tive site ( ) p rop ertie s of monom e ric trio sep ho sp hate isom e rase monoT MI a s summ a rized in the 21 known in tron po sition s deduced from m u tationa l and struc tu ra l stud ie s. P rotein S ci. , 5: 229 () Logsdon et a l. , 1995 . R e la tion sh ip s be tween th is - 239. va ria tion and the func tion of H a tpi rem a in unc lea r Schne ide r A S, 2000. Trio sep ho sp ha te isom e ra se deficiency: H isto rica l p ersp ective s and mo lecu lar a sp ec ts. B a illières B est P ract. R es. un til now. A recen t study in D rosoph ila revea ls tha t C lin. Haem a tol. , 13: 119 - 140. the p a thogene sis of TP I defic iency cou ld re su lt from Se igle JL , Ce lo tto AM , Pa llad ino MJ , 2008. D egrada tion of func tiona l p ro tea som a l degrada tion of the app a ren tly func tiona l trio se p ho sp hate isom e ra se p ro te in unde rlie s suga rk ill p a tho logy. Genetics, 179: 855 - 862. ( ) enzym e Se igle et a l. , 2008 . Mo reove r, tpi had Sun GL , Shen W , W en JF, 2008. Trio sep ho sp ha te isom e ra se gene s in been loca ted on Z ch romo som e in L ep idop te ra in sec t ( ) two trop h ic mode s of eugleno id s Euglenop hyceae and their ( ) H elicon ius era to Kap an et a l. , 2006 , wh ich m ay p hylogene tic ana lysis. J. Euka ryot. M icrobiol. , 55: 170 - 177. fac ilita te the fu tu re p e st con tro l by con struc ting Tittiger C, W hya rd S, W a lke r V K, 1993. A nove l in tron site in the trio sep ho sp ha te isom e ra se gene from the mo squ ito Cu lex ta rsa lis. () sp ec ific tran spo sa l stra in s T W , Zo r tran sgen ic N a tu re, 361: 470 - 472. () line s a s tha t in B om byx m ori M a rec et a l. , 2005 . Tob le r A , Kap an D , F lanagan N S, Gonza lez C, Pe terson E, J iggin s CD , Mo re in te re st cou ld be focu sed on in the fu tu re. John son JS, H eckel D G, M cM illan WO , 2005. F irst2genera tion linkage m ap of the wa rn ingly co lo red bu tte rfly Helicon ius era to. Referen ce s Hered ity, 94: 408 - 417. Tyshenko M G, W a lke r V K, 1997. Towa rd s a reconc ilia tion of the in tron s Ap aric io R , Ferre ira ST, Po lika rpov I, 2003. C lo sed confo rm a tion of the early o r la te view s: Trio sep ho sp ha te isom e ra se gene s from in sec ts. active site loop of rabb it m u sc le trio sep ho sp ha te isom e ra se in the B ioch im ica et B iophysica A cta, 1 353: 131 - 136. ab sence of sub stra te: Evidence of confo rm a tional he te rogene ity. J. W ang YX, Tan S, Hoo i SC, 1999. Iden tifica tion and cha rac teriza tion of M ol. B iol. , 334: 1 023 - 1 041. a nove l ra t trio sep ho sp ha te isom e ra se gene in rem nan t ileum afte r Bown D P, W ilk in son H S, Ga tehou se JA , 1997. D iffe ren tia lly regu la ted m a ssive sm a ll bowe l re sec tion. D ig. D is. S ci. , 44: 25 - 32. inh ib ito r2sen sitive and in sen sitive p ro tea se gene s from the Zhong BX, 2001. P ro te in da tabank fo r seve ra l tissue s de rived from five p hytop hagou s in sec t p e st, H. a rm igera , a re m em bers of comp lex in sta r of silkwo rm. A cta Genetica S in ica, 28: 217 - 224. m u ltigene fam ilie s. Insect B iochem. M ol. B iol. , 27: 625 - 638. 棉铃虫磷酸甘油醛异构酶基因 H a tp i 的克隆及分析 3岩 , 齐晓朋 , 牛宝龙 , 翁宏飚 , 陈金娥 , 何丽华 , 沈卫锋 , 孟智启 刘 ()浙江省农业科学院蚕桑研究所 , 杭州 310021 摘要 : 棉铃虫 H elicoverpa a rm igera是一种严重危害棉花等经济作物的鳞翅目害虫 , 开展分子水平研究对防控害虫将 ( ) 具有 重 要 参 考 意 义 。本 研 究 利 用 RACE rap id amp lifica tion of cDNA end s技 术 克 隆 了 棉 铃 虫 磷 酸 甘 油 醛 异 构 酶 ( )( ) trio sep ho sp ha te isom e ra se基因 H a tpi GenB ank登录号为 A Y736358 。该基因 cDNA 全长为 1 149 bp , 编码 248 个氨 (βα) 基酸 , 预测等电点为 5. 82, 分子量为 16. 4 kD。H aTP I含有磷酸甘油醛异构酶类蛋白的典型 结构 、保守的活性 8 (())位点 L ys12 , H is94和 Glu165和小肽序列 A YEPVWA IGTG和 GGA SL KPEF等 。R T2PCR 检测分析发现 H a tpi在棉铃 虫卵巢 、幼虫 、蛹 、成虫均有 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 达 , 提示该基因可能在棉铃虫的不同发育阶段均起作用 。 关键词 : 棉铃虫 ; 磷酸甘油醛异构酶 ; 基因 ; RACE; 序列分析 ; 表达 ( ) 中图分类号 : Q966 文献标识码 : A 文章编号 : 0454 26296 2009 1020952207 ()责任编辑 : 邓艳
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