RcHSP1718, E1coli BL21, 2 S tate Key L aboratory of Genetic Engineering, School of L ife Science,
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1 2009, 36 ( 12) : Acta Horticulturae Sinica RcHSP1718 1, 2, 1, 2, 2, 2, 2, 2, 23 ( 1, ; 2,, ) : RcHSP1718, Southern 38 /3 h, ( SchlossMannieim) (Radio) (Las Vegas) (Cap rice de Meiland) ( Girl), ( KordesπPerfecta) (H iogi) ( Pfalzer Gold), RcHSP1718, E1coli BL21, RcHSP1718, ph, RcHSP1718 : ; RcHSP1718 ; ; : S : A : X (2009) The Expression of R ch SP1718 Gene Im prov ing Tolerance to Ab iotic Stress in Recom b inan t E1coli Cells J IANG Chang2hua 1, 2, HU Yong2hong 1, XU J ian2yao 2, ZHANG Hao 2, SH I J in2lei 2, L IM in 2, WANG Ya2 qiong 2, and M ING Feng 23 ( 1 Shanghai B otanical Garden, Shanghai , China; Fudan U niversity, Shanghai , China) 2 S tate Key L aboratory of Genetic Engineering, School of L ife Science, Abstract: R chs P1718 is a high temperature ( HT) induced exp ressing gene, blot indicate that there are several cop ies of R chsp1718 in genome DNA of Rosa hybrida L. more strongly exp ressed in varieties resistant to HT, by RT2PCR and Southern R chs P1718 is such as SchlossMannieim, Radio, Las Vegas, Cap rice de Meiland, Girl, meanwhile weakly or not exp ressed in varieties sensitive to HT, such as KordesπPerfecta, H iogi, Pfalzer Gold. The results indicate that this gene is possibly relative to the tolerance in R osa hybrida L. to HT. In order to study its possible function, R chs P1718 gene has been trans2 ferred into Escherich ia coli BL21. Recombinant RcHSP1718 was overexp ressed in the recombinant E1coli cells which showed imp roved viability under different abiotic stress conditions, such as, high salt concentration, heavy metal and oxidative stress, as compared w ith control cultures. Those results would p rovide molecular i2 dentification p rincip le to selecting varieties of R osa hybrida L. w ith strong tolerance to HT. Key words: R osa hybrida L. ; R chs P1718; induced exp ression; abiotic stress : ; : : [ (2008) 1024 ]; 863 ( 2008AA10Z116) ; ( 08QH14003 ) ; ( ) ; (J ) 3 Author for correspondence ( E2mail: huyonghong68@ hotmail1com)
2 (R osa hybrida L. ),, ( heat shock p roteins, HSP) HSP ( Sun et al., 2002) Neta2 Sharir (2005) HSP21 O shsp26 (Lee & V ierling, 2000) (Molecular chaperones), HSP (W aters et al., 1996) HSP1619 ( Yeh et al., 1997) CsHSP1715 ( Soto et al., 1999) (2006) (, 2007;, 2008a) 38 /3 h,, 22D,,, R chsp1718 cdna, 465 bp ( EF053229), 154, shsp, RcHSP nieim, , ( Schloss Man2 SM ) (Radio, RA ) (Las Vegas, LA ) (Cap rice de Mei2 land, ME) ( Girl, GI) ; ( KordesπPerfecta, KP) (H iogi, H I) ( Pfalzer Gold, GO ) (, ) Southern R chsp1718 CTAB, DNA, 15 g DNA D ra B gl, 251 bp DNA, Amersham B iosciences (RPN3540) 113 R chsp1718 Southern 38 /3 h,, RNAout ( ) RNA, PrimeScrip t TM cdna 1 R chs P1718 ORF 5 3 : RT Reagent Kit ( TaKaRa) 5 2ATGTCGCTTATC2 CCAAATTT23 ; : 5 2TTACCCAGAAATTTCAAT23 PCR : 94 4 m in; s, s, 72 1 m in, 30 ; m in 114 R chsp R chsp1718 ORF, Sac, S al cdna, PCR PCR pet32a Sac Sal, T4 pet32a2rchsp1718 E1coli DH5, R chs P1718 cdna, pet32a2 RcHSP1718 pet32a ( ) E1coli BL21, - 80
3 12 : RcHSP BL21 LB ( Amp 100 mg L - 1 ), r m in - 1 ; 1%, 1 h OD 600, SDS2PAGE 1% r m in - 1 OD , IPTG ( 1 mmol L - 1 ), h, r m in m in,, ; r m in m in, ; G250 OD 595, ;, 5 m in,, 6 g; 1215% SDS2PAGE W estern IPTG, h 2 h OD , h, H is, IgG 115 BL BL21 1 ml IPTG 2 h OD , r m in m in, (OD ml 10 9 ) ; 100 L 1 ml, 100 L 1 ml, 6 ( 10 3 ) ; 100 L LB ( Amp 100 mg L - 1 ) ; d 37, IPTG 2 h OD , h 1 ml ; OD 600, OD ;, 37, BL21 pet32a2rchsp1718 (H1 H2, ) pet32a ( EV ) BL21 IPTG 2 h OD , BL21 (W T) LB r m in m in,, 0D LB Z, 37, ; LB LB : mmol L - 1 L icl; mmol L - 1 NaCl; mmol L - 1 Na 2 CO 3 ; mmol L - 1 CdCl 2 ; mol L - 1 H 2 O Southern D ra B gl DNA, Southern blot RcHSP1718, ( 1) 1 Southern blot RcHSP1718 Fig. 1 Southern blot analysis of RcHSP1718 in genome of Rosa
4 R ch SP1718 RT2PCR, (25 ) R chsp1718, (38 ) SM RA LA ME GI, KP GO, H I ( 2) 2 RT2PCR RcHSP1718 NT. ; HT. H I. ; ME. ; GO. ; RA. ; LA. ; GI. ; F ig. KP. ; SM. 2 RT2PCR test for expression of RcH SP1718 in var ieties of Rosa hybrida L. under d ifferent tem perature treatm ent NT. Normal temperature; HT. H igh temperature. H I. H iogi; ME. Cap rice de Meiland; GO. Pfalzer Gold; RA. Radio; LA. Las Vegas; GI. Girl; KP. KordesπPerfecta; SM. SchlossMannieim. 213 R chsp , pet32a pet32a2rchsp1718 BL21 37, S,, R chsp1718 BL21, RcHSP pet32a2rchsp1718 F ig. 3 The growth curve of tran sgen ic and em pty vector E1coli stra in under culture of norma l cond ition
5 12 : RcHSP R chsp1718 E1coli SDS2PAGE ( 4 ), BL21 ( pet32a2rchsp1718 ) IPTG 015 h 3916 kd, RcHSP1718 ( 6 ), BL21 (pet32a, EV ),, 2 h F ig. 4 SD S2PAGE RcHSP SD S2PAGE ana lysis of E1coli BL21 harbor in pla sm id pet32a2rchsp1718 pet32a2rchsp1718,, ( H is) W estern blot ( 5) 37, IPTG h (pet32a2rchsp1718), 6 h 50, IPTG h, IPTG, ,,, (pet32a2rchsp1718), 1 h 7019%, 5 h 415% 1 h 1119%, 4 h,, RcHS P1718 E1coli BL21 ( 6, A) d,,, ( pet32a2rchsp1718), 12 d 33%, 65%, R chsp1718 E1coli BL21 ( 6, B)
6 , L icl (W T) ( EV ) R chsp1718 (H1; H2 ) 4, L icl 300 mmol L - 1, H1 H2 40%, W T F ig. 7 7 V iab ility a ssay of E1coli under various stresses
7 12 : RcHSP EV 10% ; 800 mmol L - 1 CdCl 2 W T EV, H1 H2,, ; NaCl 800 mmol L - 1, H1 H2, W T EV ; Na 2 CO 3 20 mmol L - 1, H1 H2, 50%, W T EV, 20% ; 200 mol L - 1 H 2 O 2 4, W T EV, 20%, H1 H2, 80%, (pet32a2rchsp1718) RcHSP1718, RcHSP1718 BL21 ph, RcHSP ( shsp) ( Sun et al., 2002),, R chsp1718, HSP21 (Neta2Sharir et al., 2005) O shsp26 (Lee & V ierling, 2000), R chsp1718,, (, 2007;, 2008b) CsHSP1715, CsHSP1715,, ( Soto et al., 1999) R chsp1718, E1coli BL21,, ph shsp ( Yeh et al., 1997), R chsp1718 shsp (Bgsl et al., 2006; Goloubinoff & de Los R ios, 2007),,, ATP HSP70 ( Ehrnsperger et al., 1997; Lee & V ierling, 2000; Sung et al., 2001) HSP60 (Bukau & Horwich, 1998) HSP100 ( Yang et al., 2006),, (Mogk et al., 2003) RcHSP1718,, E1coli BL21 RcHSP1718 ; (J iang et al., 2009;, 2009), RcHSP1718,, References BgslBenjam in, Grimminger Valerie, W alter Stefan The molecular chaperone H sp104 - A molecular machine for p rotein disaggregation. J Struct B iol, 156 (1) : Bukau B, Horwich A L The H sp70 and H sp60 chaperone machines. Cell, 92: EhrnspergerM, Grgber S, GaestelM, Buchner J B inding of non2native p rotein to H sp25 during heat shock creates a reservoir of folding intermediates for reactivation. EMBO J, 16: Goloubinoff P, de Los R ios P The mechanism of H sp70 chaperones: ( Entrop ic) pulling the models together. Trends B iochem Sci, 32 (8) : Hu Yong2hong, Jiang Chang2hua, Q in Jun, Mo Jian2bin Research for effects of high temperature on several morphological, physiologi2
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