水稻ISA1基因的克隆与分析英文

C lo n i n g a n d A n a lys is o f I SA 1from O ryza s a tiva

FANG J ie 2h o n g

13

,ZHAN G M i n g 2zh o u

13

,L IU J u n 1

,W ANG Xu e 2ya n 1

,SUN C h u an 2xi n

2

1.C o ll ege o f Life Sci e nce,C hi n a J il iang U n i ve rs i ty,Han gzhou 310018;

2.Bo t a ny D epa rt m en t,Sw ed i s h Un i ve rs it y o f Ag ricultura l Science s,

SE 275007

Ab s t ra ct [O bject i ve]The a i m wa s to cl o ne I SA1fr om O ryza sa ti va and ana l yze i t s exp re s si o n s i tua ti on i n di fferen t tis sue s and di ffe rent endo 2sp er m fil li ng s t a ge.[M ethod]W it h j apo n i ca rice cultiva r n i ppo nba re as t e s t m a t e ri al,the exp re s s i o n p a tt e rn s o f ISA1i n d i ffere nt ti s s ues an d d i f 2fere nt endo spe r m fi lli ng s tage w as ana l yzed by u s i ng sem i 2qua ntitat i ve R T 2PC R techn i que.[R esul t]The fu ll l eng t h op en rea di n g fragm en ts o f I SA1encode d 811am ino aci d res i d ues .The hom o l o go u s ali gnm en t and phy l o gene ti c ana l ys is showed t h at t h e s i m i l a ri ti e s of nuc l eo ti de aci d se 2quence s and de duced am i no ac i d se quence s o f I SA1of ri ce t o t ho se o f som e o the r p lant s we re 66.8%-83.0%and 69.0%-82.5%,re spe c 2ti ve l y,and had a re l at i ve l y h i gh deg ree o f s i m i lari ty w it h ba rley and whe at .The sem i 2q uant i ta ti ve R T 2PCR analys i s i nd i ca ted tha t I SA1e xp re ss ed w i th a p ea k value a t 12d afte r po lli na ti on i n endo spe r m bu t no t i n roo t,s t em o r leaf .[Conclu si on ]The study l a i d a fou nda ti o n fo r furt h er s tudy o n the exp re ss i on a nd reg ul a t o ry m echa nis m of I SA1g ene.Key w o rds O ryza sa ti va;I SA1;Gene cl o ning;Sem i 2quan tit a ti ve R T 2PC R

R D , M ,S y Z j N S F (Y36Y363)32z z j @j The rice sta rch is compo sed by am yl o se a nd am yl o pe c 2

ti n .The am yl o se is com p rise d of dehydra ted glycos i de through li ne a r α2D 21,42gluc an cha i ns The am yl ope c ti n ha s branche s whi c h a re l oc a ted be t wee n e ve ry 20t o e ve ry 30de 2hydra ted glycos i de.The se b ranche s a re connec te d t h r o ugh α2D 21,62gluco si d i c linka ge s unde r s t a rch bra nchi ng e nzy m e.F i na ll y,the sta rc h de branching e nzy m e i s u se d to m odify branche s a nd synthe si z e am yl ope c tin w ith ce rta i n struc ture

a nd func ti on [1]

.The Iso am yl a se l 1(ISA1),whi c h be l ongs t o sta rc h de bra nc hi ng enzy m e ,t a ke s de na ture d am yl o pe c ti n,glycoge n a nd am yl o pe c ti n a na l o gue s a s subs tra t e s t o remove α2D 21,62gl ucos i di c bo nd s i n am yl op ec tin synthe sis,so it i n 2

fl ue nce s the p r opo rti ons o f am yl o se and am yl opec ti n [2]

.

Mo re a nd m o re re sea rche s have dem on stra ted tha t I SA1plays ve ry i m po rtan t role s i n s ta rch syn t he s i s.A t p re sen t,the studi e s o f sys t em a ti c exp re s si o n a nd tran sc ri p ti ona l re gu l a ti o n of ri ce ISA1gene a re se l dom.The rice ISA1ge ne o f ri c e is c l oned a nd the exp re ssi on pa tte rns o f I SA1i n d i ffe re nt tissue s a nd endo spe rm s a t d i ffe re nt f i lli ng stage s w e re a na l yz ed,which l a ys founda ti o n s fo r furthe r studyi ng I SA1ge ne exp re s 2si on and regul a ti on m e cha nism.

M a te ria ls a n d M e th o d s

Te s t m a te ria l

The O ryza sa tiva L .subsp.J apo ni ca wa s supp li e d by I n 2stitute of N uc l e a r 2Agri cultura l S c i e nce s,Zhe jiang Uni ve rsity;PCR p ri m e rs we re p rovi de d by S a ngon bi o tec h (S ha ngha i )Co .,Ltd;Tota l RNA e xtra c ti on ki t wa s se rve d by B i o teke (B e iji ng );pGE M 2T e a sy ve c t o r w a s bought from P rom e ga Compa ny;M 2ML V re ve rse tran sc ri p ti on re age nt,L A Taq en 2zy m e we re bought from Ta ka ra Com pa ny .O the r re a ge nts we re C hi ne se a na l ytica ll y pu re.E xp e ri m en ta lm e thod

To ta l RNA e xtrac tio n of e ndo sp e r m a nd firs t c ha in syn 2the s is of cDNA The e ndo spe r m ti s sue s a t 3t o 24d a fte r

po lli na ti on and r oo t,stem a nd l e ave a t 12da fte r po l lina ti on

we re co l lec te d by li qui d nitroge n fl a sh fre e ze r,and the n p re 2se rve d a t -80℃.The t o ta l RNA extra c ti on wa s i n ac co rd 2a nc e w i th the i ns truc ti on of the tota l RNA iso l a ti o n ki t .The RNA purity a nd conce ntra ti o n we re e sti m a te d by Aga rose ge l a nd UV de te c ti on The RNA samp le s with good i n teg rity a nd purity we re synthe sized the first cDNA cha i n by reve rse tra n 2scri p ti on .

C lone of ISA 1CDNA in r ice Ac co rding t o publi shed I SA1ge ne sequence of rice (GenB ank:AB0934026),the de 2si gned p ri m e r Iso aF1and Isoa R1(Isoa F1:5′2C G ATC G 2GAG ATTTC GCAATT 23′;I soaR1:5′2AGG ACCGCACAAC T 2TCAACAT A 23′)we re used t o amp li fy ISA1cDNA se que nce

i nc l udi ng w hole OR F [3-4]

.The Taka ra L A PCR rea gen t w a s u se d t o conduc t PCR re a c ti on i n 25μl system a nd the re a c ti on conditi on wa s l is t e d a s de ge ne ra ti on a t 94℃fo r 30s,a nne a 2li ng a t 59℃fo r 30m i n,exte nsi on a t 70℃fo r 2m in a nd 35c yc l e s .The ta rge t fragm e nts we re re co ve re d and purifi e d,a nd the connec te d w ith pGE M 2T ea sy vec t o r,tra nsfo r m a ti o n,a nd sequence d .

S e que nc e a na lys is The se que nce a na l ysis and hom o l o gy a l ignm e nt wa s use d by DNAs ta r .,while the a na l ysis of ge ne structu re a nd chrom o som e l oca ti on we re conduc ted by Gram ene da t a ba se.The BLAS T re trieva l o f nuc l e oti de se 2que nce a nd p ro te i n se que nc e we re by NCB I da taba se.The C l us t a l W com pa rison a nd NJ m e tho d we re use d t o construc t

phyl o ge ne ti c tre e by M EGA4[5]

.

S em i 2qua n tita ti ve RT 2PCR a na l ys is The sem i 2quantita tive RT 2PCR a na l ysis wa s used to e x p l o re the e x p re ssi on pa tte rns of I SA1gene in diffe re nt ti s sue s a nd i n e ndo spe r m a t di ffe ren t filli ng sta ge s .The t o ta l RNA extra cte d by a bove m e tho ds w a s unde rgone DNa se Ⅰtrea t m ent a nd de t e c ted by N ano dr op ,t he n the t o ta l RNA of the sam e conce ntra ti o n wa s co nduc te d re 2ve rse transc ri pti o n re ac ti o n.The equi va l e nt reve rse d cDNA tem p l a te w e re am plifi ed ISA1ge ne fra g m ent (I SA1F:5′2CGGGT ACAGGTTC G ATGGT ATGTT 23′;I SA1R :5′2G AGG 2T AGGTCACCTTGCCAATCAA 23′)by p ri m e rs I SA1F a nd

IS R ,f T F 2αf [6]

F 2αF F 2αR (F 2αF 5′2TTT T TTGGTGT

G G G T 23′;F 2αR 5′2G TT TT G TTT T GT 23′),Agricultura l B i o technol o gy

Agri cu l tu ral Sc i ence &Techno l o gy,2010,11(2):84-86C op yright κ2010,I nf o r m at i o n I n s ti tu t e of HAAS.All ri gh ts res erved.

e ce i ve d:ecem be r 92009Accep t ed:a rch 282010uppo rted b he iang P r o vinci a l atu ral ci e nce o unda ti o n

09017a nd 044.

C o rre spo n di n g au tho r .E m ail :m c l u c l u.e https://www.wendangku.net/doc/6615226059.html,

A1while the l e ng t h o am pli con wa s 212bp.he e E 1

wa s re e re nc e gene a nd the p ri m e rs we re eE 1

a nd e E 1e E 1:CAC C AA CA A e E 1:AC CC CAC A CA C A A so