西伯利亚蓼3-磷酸甘油醛脱氢酶的cDNA克隆及耐盐性分析
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摘要
本研究根据NaHCO_3胁迫下的西伯利亚蓼(Polygonum sibiricum Laxm.)消减文库中的ETS序列,利用RACE技术,克隆出3-磷酸甘油醛脱氢酶(GAPDH)的全长cDNA序列,命名为Ps GAPDH。通过生物信息学方法进行了初步的分析;利用Northern杂交技术对NaHCO_3不同胁迫时间下的地下茎、茎及叶的表达模式进行研究;将该基因构建到酿酒酵母(Saccharomyces cerevisiae,INVScI)表达载体pYES2,进行酿酒酵母的遗传转化,对转基因酵母的耐盐性进行了研究。主要实验结果如下:
(1)Ps GAPDHcDNA序列全长1331 bp,完整阅读框1014bp,编码337个氨基酸。氨基酸序列分子量为36.672 kDa,理论pI值是7.66,蛋白不稳定系数是24.31,属于稳定蛋白。氨基酸同源性比对显示草本植物西伯利亚蓼的GAPDH与其它草本植物的GAPDH基因同源序列相似性较高,最高达到96%;与落叶灌木也具有高的同源性,最高达到93%;且它与其它物种中存在于细胞质的GAPDH基因的氨基酸序列同源性高达96%。推测此基因属于胞质中的GAPDH基因。蛋白比对显示该基因编码蛋白有2个保守的功能域,一个为行使糖运输和代谢的GAPDH功能域;另一个是NAD(P)结合功能域。
(2)Northern印记杂交检测显示,NaHCO_3胁迫处理的西伯利亚蓼的地下茎、茎及叶片具有不同的表达模式。在叶部胁迫4-24 h过程中,Ps GAPDH表达减少,表现受抑制,48 h后趋于正常。而在茎部在2-8 h,随着胁迫时间的增加表达量增加,表现为诱导,而随后的24-48 h胁迫后表达量趋于正常。而在地下茎中,Ps GAPDH的表达量有微弱的增强。
(3)将Ps GAPDH基因与酵母表达载体pYES2连接,获得了pYES-GAPDH重组质粒。将pYES-GAPDH质粒转化到酵母菌INVScI中。随机挑选3个INVScI(pYES-GAPDH)单菌落,经PCR扩增和提取菌液质粒双酶切证明Ps GAPDH基因已构建到pYES2载体上;对INVScI(pYES-GAPDH)和INVScI(pYES2)菌株进行半乳糖诱导,RTPCR证明该基因已经在酵母中成功表达,对转基因酵母进行NaHCO_3和NaCl胁迫。结果表明,INVScI(pYES-GAPDH)重组酵母与INVScI(pYES2)相比,在10%NaHCO_3和4mol·L~(-1) NaCl胁迫下具有较高的存活率。说明,来源于西伯利亚蓼的GAPDH基因赋予酵母细胞一定的抗盐能力,表现出抗逆性的功能。
Polygonum sibiricum Laxm.can grow in a high salt stress environment.In this study,the primers which were used to amplify the full length eDNA of the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) were designed according to the expressed sequence tag(EST) sequence of the GAPDH gene acquired from suppression subtractive hybridization library of the stem of P.sibiricum.The full length eDNA ofPs GAPDHgene was cloned by using rapidamplification of cDNA ends(RACE) technology.Then the nucleotide and protein sequence were compared using bioinformation.Northern blotting of Ps GAPDH expression in different tissues of P.sibiricum,which was stressed under 3%of NaHCO_3 was analyzed.The Ps GAPDH gene was inserted into yeast(Saccharomyces cerevisiae) expression vector(pYES2), then the pYES2-GAPDH was transferred into yeast cells for functional analysis.The main results were as follows:
(1) The results showed that the cDNA of the Ps GAPDHgene was 1331 bp,encoding 337 amino acids residues and encoding a protein with a predicted molecular mass of 36.672 kDa, and theoretieal pI of 7.66,protein is stable which nonstabilization quotiety is 24.31.The deduced amino acid sequence shows high homology to the eytosolie GAPDH from other higher plants.The highest amino acid identities between sequences of different herbaceous plant species was 96%and of different shrubby plant species was 93%.Ps GAPDH is a tetrameric NAD-binding enzyme involved in glycolysis and glyconeogenesis.C-terminal domain is a mixed alpha/antiparallel beta fold,N-terminal domain is a Rossmann NAD(P) binding fold.
(2) The expression of this form of Ps GAPDH in the rhizome,stem and leaves of P. sibiricum under the stress of NaHCO_3 was studied on the mRNA level by northern blot hybridizations.These experiments showed that the Ps GAPDH mRNA levels in leaves was decreased from 4-24 h treated by NaHCO_3 and after 48 h,the mRNA levels became normal. The Ps GAPDH mRNA levels in stems was strongly increased after 2h and became normal after 48 h.The Ps GAPDH mRNA levels in rhizome slightly increased during stress of NaHCO_3.
(3) The recombinant plasmid pYES2-GAPDH was constructed by inserting Ps GAPDH gene(cDNA) into yeast expression vector pYES2,pYES2-GAPDH was transferred into yeast, and confirmed by PCR and enzyme digest detection of 3 randomly picked INVScI(pYES2-GAPDH) lines.The expression of foreign Ps GAPDH gene in yeast which is induced by galactose was investigated by RT-PCR,the yeast harboring empty pYES2 vector strain as the control.The results from biofunctional analyses with Ps GAPDH yeast transformants showed that GAPDH yeast transformants had significantly higher resistance to different salt stresses. INVScI(pYES2-GAPDH) had more salt-resistance ability than INVScI(pYES2) and the former survival rate was higher than that of the latter under the stress of 10%NaHCO_3 and 5 mol·L~(-1) NaCl.This indicated that the high ability of salt-tolerance of INVScI(pYES2-GAPDH) might be related to the expression of pYES2 gene.
(1)Ps GAPDHcDNA序列全长1331 bp,完整阅读框1014bp,编码337个氨基酸。氨基酸序列分子量为36.672 kDa,理论pI值是7.66,蛋白不稳定系数是24.31,属于稳定蛋白。氨基酸同源性比对显示草本植物西伯利亚蓼的GAPDH与其它草本植物的GAPDH基因同源序列相似性较高,最高达到96%;与落叶灌木也具有高的同源性,最高达到93%;且它与其它物种中存在于细胞质的GAPDH基因的氨基酸序列同源性高达96%。推测此基因属于胞质中的GAPDH基因。蛋白比对显示该基因编码蛋白有2个保守的功能域,一个为行使糖运输和代谢的GAPDH功能域;另一个是NAD(P)结合功能域。
(2)Northern印记杂交检测显示,NaHCO_3胁迫处理的西伯利亚蓼的地下茎、茎及叶片具有不同的表达模式。在叶部胁迫4-24 h过程中,Ps GAPDH表达减少,表现受抑制,48 h后趋于正常。而在茎部在2-8 h,随着胁迫时间的增加表达量增加,表现为诱导,而随后的24-48 h胁迫后表达量趋于正常。而在地下茎中,Ps GAPDH的表达量有微弱的增强。
(3)将Ps GAPDH基因与酵母表达载体pYES2连接,获得了pYES-GAPDH重组质粒。将pYES-GAPDH质粒转化到酵母菌INVScI中。随机挑选3个INVScI(pYES-GAPDH)单菌落,经PCR扩增和提取菌液质粒双酶切证明Ps GAPDH基因已构建到pYES2载体上;对INVScI(pYES-GAPDH)和INVScI(pYES2)菌株进行半乳糖诱导,RTPCR证明该基因已经在酵母中成功表达,对转基因酵母进行NaHCO_3和NaCl胁迫。结果表明,INVScI(pYES-GAPDH)重组酵母与INVScI(pYES2)相比,在10%NaHCO_3和4mol·L~(-1) NaCl胁迫下具有较高的存活率。说明,来源于西伯利亚蓼的GAPDH基因赋予酵母细胞一定的抗盐能力,表现出抗逆性的功能。
Polygonum sibiricum Laxm.can grow in a high salt stress environment.In this study,the primers which were used to amplify the full length eDNA of the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) were designed according to the expressed sequence tag(EST) sequence of the GAPDH gene acquired from suppression subtractive hybridization library of the stem of P.sibiricum.The full length eDNA ofPs GAPDHgene was cloned by using rapidamplification of cDNA ends(RACE) technology.Then the nucleotide and protein sequence were compared using bioinformation.Northern blotting of Ps GAPDH expression in different tissues of P.sibiricum,which was stressed under 3%of NaHCO_3 was analyzed.The Ps GAPDH gene was inserted into yeast(Saccharomyces cerevisiae) expression vector(pYES2), then the pYES2-GAPDH was transferred into yeast cells for functional analysis.The main results were as follows:
(1) The results showed that the cDNA of the Ps GAPDHgene was 1331 bp,encoding 337 amino acids residues and encoding a protein with a predicted molecular mass of 36.672 kDa, and theoretieal pI of 7.66,protein is stable which nonstabilization quotiety is 24.31.The deduced amino acid sequence shows high homology to the eytosolie GAPDH from other higher plants.The highest amino acid identities between sequences of different herbaceous plant species was 96%and of different shrubby plant species was 93%.Ps GAPDH is a tetrameric NAD-binding enzyme involved in glycolysis and glyconeogenesis.C-terminal domain is a mixed alpha/antiparallel beta fold,N-terminal domain is a Rossmann NAD(P) binding fold.
(2) The expression of this form of Ps GAPDH in the rhizome,stem and leaves of P. sibiricum under the stress of NaHCO_3 was studied on the mRNA level by northern blot hybridizations.These experiments showed that the Ps GAPDH mRNA levels in leaves was decreased from 4-24 h treated by NaHCO_3 and after 48 h,the mRNA levels became normal. The Ps GAPDH mRNA levels in stems was strongly increased after 2h and became normal after 48 h.The Ps GAPDH mRNA levels in rhizome slightly increased during stress of NaHCO_3.
(3) The recombinant plasmid pYES2-GAPDH was constructed by inserting Ps GAPDH gene(cDNA) into yeast expression vector pYES2,pYES2-GAPDH was transferred into yeast, and confirmed by PCR and enzyme digest detection of 3 randomly picked INVScI(pYES2-GAPDH) lines.The expression of foreign Ps GAPDH gene in yeast which is induced by galactose was investigated by RT-PCR,the yeast harboring empty pYES2 vector strain as the control.The results from biofunctional analyses with Ps GAPDH yeast transformants showed that GAPDH yeast transformants had significantly higher resistance to different salt stresses. INVScI(pYES2-GAPDH) had more salt-resistance ability than INVScI(pYES2) and the former survival rate was higher than that of the latter under the stress of 10%NaHCO_3 and 5 mol·L~(-1) NaCl.This indicated that the high ability of salt-tolerance of INVScI(pYES2-GAPDH) might be related to the expression of pYES2 gene.
引文
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