苹果PGIP基因克隆及其在大肠杆菌中的表达
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摘要
多聚半乳糖醛酸酶抑制蛋白(polygalacturonase-inhibiting protein,PGIP)是一种胞外的细胞壁结合糖蛋白,它能够特异性结合病原真菌分泌的多聚半乳糖醛酸酶并抑制其水解活性,同时引发植物的防卫反应。研究PGIP基因及其表达产物抑菌活性对于该基因在植物抗病育种的应用具有重要意义。
本研究以“富士”苹果幼果为试材,通过RT-PCR克隆了PGIP cDNA,并进行测序、表达载体构建及原核表达,主要研究结果如下:
1通过RT-PCR获得了约1.1 kb的苹果PGIP cDNA片段,该片段与pMD18-T载体连接转化DH5α,对筛选的阳性克隆进行了测序,测序结果该cDNA长度为1 091 bp,该片段含有993 bp的完整开放阅读框,编码330个氨基酸残基组成的蛋白质,具有7个潜在的N-糖基化位点。预测该蛋白质分子量为37.6kD,理论等电点(pI)值为6.98,N端24个氨基酸残基构成的疏水区域为信号肽。该基因与GenBank中已登录的苹果PGIP基因的ORF核苷酸序列的同源性为99%,发现只有一个碱基发生了替换,49位点(T→C),导致编码氨基酸发生改变,亲水性的丝氨酸(Ser)→疏水性(Pro)脯氨酸,因此认为本研究所克隆的为苹果品种“富士”的PGIP基因,命名为MdPGIP。
2为进一步研究该基因在原核和酵母表达系统中能否编码蛋白质,确认该蛋白质的功能和活性,从PGIP的克隆载体pMD18-T质粒中分别扩增带有信号肽和不含有信号肽的目的片段,酶切回收的目的片段定向克隆到表达载体pET-32a中分别构建含有信号肽的原核表达载体(pET-32a-PGIP)和不含有信号肽的原核表达载体(pET-32a-PGIP-X)及含有信号肽的酵母表达载体(pPIC6α-PGIP)。
3 pET-32a-PGIP、pET-32a-PGIP-X转化BL21宿主菌中通过IPTG诱导表达,经SDS-PAGE分析表明获得了该基因的重组蛋白,重组蛋白以包涵体形式出现。
Polygalacturonase inhibiting proteins(PGIPs)are extracellular proteins located in plant cell wall. It is supposed to play an important role in the defense against plant pathogenic fungus. PGIPs can specially inhibit or reduce the hydrolytic activity of polygalacturonase (PGs) secreted by plant pathogenic fungus.Researchs on the PGIP gene and its expression product have significant meaning for PGIP gene-based plant disease resistance cultivar breeding.
Using RT-PCR method, the full-length cDNA of PGIP gene was cloned from the Malus domestica Borkh. cv.Fuji fruit. After further sequencing confirmation,the gene was sub-cloned into the expression vector and expressed in prokaryotic system.The main results of the experiment include the following conetents:
1 A cDNA fragment about 1.1 kb was amplified by reverse transcription polymerase chain reaction (RT-PCR) with a pair of primers designed according to the conserved sequences of apple PGIP genes in the GenBank. After cloning, the cloning vector pMD18-T was transformed into DH5α. The positive clones were picked out and sequenced.The results showed that the cloned cDNA was 1,091 bp, contains a full open reading frame of 993 bp encodiong 330 amino acid residues with a molecular mass of 37.6kD and showed 99% of identity to Malus pumila PGIP gene ORF. The deduced protein has a pI of 7.23, a hydrophobic region of 24 amino acid residues in the N-terminal which was considered to be a signal peptide, and the singal peptide sequence have seven potential N-glycosylation sites. Compared to the reported amino acid residue substitutions (49 T→C), which resulted in one amino acid residue substitutions(Ser→Pro). therefore,we get the conclusion that it is the apple PGIP gene abtained in this study and named as MdPGIP.
2 In order to identify the gene function and the activtiy of expresion products in prokaryotic and Eucaryotic expression, gene cDNA fragments with signal peptide and no signal peptide from pMD18-T vector plasmid was applified and cloned into expression vector pET-32 and pPIC6αrespectively, constructed prokaryotic expression vector pET-32a-PGIP with signal peptide, pET-32a-PGIP-X without signal peptide and yeast expression vector pPIC6α-PGIP with signal peptide.
3 The expression vector pET-32a-PGIP、pET-32a-PGIP-X were transformed into host bacterium BL21 were expressed by IPTG inducing. Expression products were analized by SDS-PAGE, The result showed that the gene expression products were mostly existed as inclusion body.
本研究以“富士”苹果幼果为试材,通过RT-PCR克隆了PGIP cDNA,并进行测序、表达载体构建及原核表达,主要研究结果如下:
1通过RT-PCR获得了约1.1 kb的苹果PGIP cDNA片段,该片段与pMD18-T载体连接转化DH5α,对筛选的阳性克隆进行了测序,测序结果该cDNA长度为1 091 bp,该片段含有993 bp的完整开放阅读框,编码330个氨基酸残基组成的蛋白质,具有7个潜在的N-糖基化位点。预测该蛋白质分子量为37.6kD,理论等电点(pI)值为6.98,N端24个氨基酸残基构成的疏水区域为信号肽。该基因与GenBank中已登录的苹果PGIP基因的ORF核苷酸序列的同源性为99%,发现只有一个碱基发生了替换,49位点(T→C),导致编码氨基酸发生改变,亲水性的丝氨酸(Ser)→疏水性(Pro)脯氨酸,因此认为本研究所克隆的为苹果品种“富士”的PGIP基因,命名为MdPGIP。
2为进一步研究该基因在原核和酵母表达系统中能否编码蛋白质,确认该蛋白质的功能和活性,从PGIP的克隆载体pMD18-T质粒中分别扩增带有信号肽和不含有信号肽的目的片段,酶切回收的目的片段定向克隆到表达载体pET-32a中分别构建含有信号肽的原核表达载体(pET-32a-PGIP)和不含有信号肽的原核表达载体(pET-32a-PGIP-X)及含有信号肽的酵母表达载体(pPIC6α-PGIP)。
3 pET-32a-PGIP、pET-32a-PGIP-X转化BL21宿主菌中通过IPTG诱导表达,经SDS-PAGE分析表明获得了该基因的重组蛋白,重组蛋白以包涵体形式出现。
Polygalacturonase inhibiting proteins(PGIPs)are extracellular proteins located in plant cell wall. It is supposed to play an important role in the defense against plant pathogenic fungus. PGIPs can specially inhibit or reduce the hydrolytic activity of polygalacturonase (PGs) secreted by plant pathogenic fungus.Researchs on the PGIP gene and its expression product have significant meaning for PGIP gene-based plant disease resistance cultivar breeding.
Using RT-PCR method, the full-length cDNA of PGIP gene was cloned from the Malus domestica Borkh. cv.Fuji fruit. After further sequencing confirmation,the gene was sub-cloned into the expression vector and expressed in prokaryotic system.The main results of the experiment include the following conetents:
1 A cDNA fragment about 1.1 kb was amplified by reverse transcription polymerase chain reaction (RT-PCR) with a pair of primers designed according to the conserved sequences of apple PGIP genes in the GenBank. After cloning, the cloning vector pMD18-T was transformed into DH5α. The positive clones were picked out and sequenced.The results showed that the cloned cDNA was 1,091 bp, contains a full open reading frame of 993 bp encodiong 330 amino acid residues with a molecular mass of 37.6kD and showed 99% of identity to Malus pumila PGIP gene ORF. The deduced protein has a pI of 7.23, a hydrophobic region of 24 amino acid residues in the N-terminal which was considered to be a signal peptide, and the singal peptide sequence have seven potential N-glycosylation sites. Compared to the reported amino acid residue substitutions (49 T→C), which resulted in one amino acid residue substitutions(Ser→Pro). therefore,we get the conclusion that it is the apple PGIP gene abtained in this study and named as MdPGIP.
2 In order to identify the gene function and the activtiy of expresion products in prokaryotic and Eucaryotic expression, gene cDNA fragments with signal peptide and no signal peptide from pMD18-T vector plasmid was applified and cloned into expression vector pET-32 and pPIC6αrespectively, constructed prokaryotic expression vector pET-32a-PGIP with signal peptide, pET-32a-PGIP-X without signal peptide and yeast expression vector pPIC6α-PGIP with signal peptide.
3 The expression vector pET-32a-PGIP、pET-32a-PGIP-X were transformed into host bacterium BL21 were expressed by IPTG inducing. Expression products were analized by SDS-PAGE, The result showed that the gene expression products were mostly existed as inclusion body.
引文
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