桃PGIP基因的克隆、原核表达及酵母载体的构建
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摘要
多聚半乳糖醛酸酶抑制蛋白(polygalacnironase-inhibitingproteins,PGIPs)是一种具有LRR抗病基因序列结构的细胞壁结合蛋白。当病原真菌侵染植物时,它能与病原真菌分泌的endo-PG专一性结合,抑制其活性,延缓病原真菌对植物细胞壁的降解,引发植物的防卫反应。克隆桃PGIP基因并确定其功能是该基因研究利用的基础。
本试验以桃叶片为试材,克隆了多聚半乳糖醛酸酶抑制蛋白基因cDNA全长并进行原核表达及酵母表达载体的构建,主要研究结果如下:
1.根据已发表的一些核果的PGIP基因序列设计引物,由桃叶片提取DNA及RNA进行扩增、克隆、测序,得到全长为1192bp的PGIP基因序列及7条1045bp的cDNA序列。经过Clustal W比对分析,7条cDNA序列的相似性在93%到99%之间。利用MEGA 4软件邻接法构建系统进化树,将7条cDNA序列分为2组。
2.构建了PGIP基因大肠杆菌表达质粒pET-32a-PGIP,并进行大肠杆菌中的融合表达,证明该基因片段可编码一条多肽链。
3.构建了含有PGIP基因cDNA的酵母表达载体pPIC6αB-PGIP,可用于蛋白质真核表达和活性检测。
Polygalacturonase-inhibiting proteins(PGIPs) belong to a kind of protein family which have the structure of Leucine-rich repeat(LRR) and are primarily localized in the cell wall. It is believed to play an important role in the defense against plant pathogenic fungi. PGIPs can specially bind and inhibit or reduce the hydrolytic activity of polygalaeturonases(PGs) secreted by plant pathogenic fungi, and limit the growth of plant pathogens, as well as eliciting defense responses in plant. Therefore, the basic research of cloning and proving the function of PGIPs are important for the further reach.
In this study, full length PGIP cDNA sequence was cloned from the leaves of Prunus persica. And fusion expression in E.coli was completed and a yeast expression vector was constructed. Main results of this study were as follows:
1. Through polymerase chain reaction(PCR) with specific primers based on the submitted PGIP sequences from other plants of Prunus, one fragment about 1192bp and 7 fragments about 1045bp were obtained from the total DNA and RNA extracted from the leaves of Prunus persica. 7 cDNA sequences were analyzed and compared with Clustal W, the analysis showed that the homology shared 93%~99%. Phylogenetic trees were constructed by MEGA 4 to analyze their evolutionary relationship. These 7 sequences were divided into 2 groups.
2. The prokaryotic expression vector pET-32a-PGIP was constructed and fusion expression was completed in E.coli BL21. It proves this cloning fragment can encode a polypeptide chain.
3. The yeast expression vector pPIC6αB-PGIP was constructed which can be used to express PGIP proteins and make the activity assay.
本试验以桃叶片为试材,克隆了多聚半乳糖醛酸酶抑制蛋白基因cDNA全长并进行原核表达及酵母表达载体的构建,主要研究结果如下:
1.根据已发表的一些核果的PGIP基因序列设计引物,由桃叶片提取DNA及RNA进行扩增、克隆、测序,得到全长为1192bp的PGIP基因序列及7条1045bp的cDNA序列。经过Clustal W比对分析,7条cDNA序列的相似性在93%到99%之间。利用MEGA 4软件邻接法构建系统进化树,将7条cDNA序列分为2组。
2.构建了PGIP基因大肠杆菌表达质粒pET-32a-PGIP,并进行大肠杆菌中的融合表达,证明该基因片段可编码一条多肽链。
3.构建了含有PGIP基因cDNA的酵母表达载体pPIC6αB-PGIP,可用于蛋白质真核表达和活性检测。
Polygalacturonase-inhibiting proteins(PGIPs) belong to a kind of protein family which have the structure of Leucine-rich repeat(LRR) and are primarily localized in the cell wall. It is believed to play an important role in the defense against plant pathogenic fungi. PGIPs can specially bind and inhibit or reduce the hydrolytic activity of polygalaeturonases(PGs) secreted by plant pathogenic fungi, and limit the growth of plant pathogens, as well as eliciting defense responses in plant. Therefore, the basic research of cloning and proving the function of PGIPs are important for the further reach.
In this study, full length PGIP cDNA sequence was cloned from the leaves of Prunus persica. And fusion expression in E.coli was completed and a yeast expression vector was constructed. Main results of this study were as follows:
1. Through polymerase chain reaction(PCR) with specific primers based on the submitted PGIP sequences from other plants of Prunus, one fragment about 1192bp and 7 fragments about 1045bp were obtained from the total DNA and RNA extracted from the leaves of Prunus persica. 7 cDNA sequences were analyzed and compared with Clustal W, the analysis showed that the homology shared 93%~99%. Phylogenetic trees were constructed by MEGA 4 to analyze their evolutionary relationship. These 7 sequences were divided into 2 groups.
2. The prokaryotic expression vector pET-32a-PGIP was constructed and fusion expression was completed in E.coli BL21. It proves this cloning fragment can encode a polypeptide chain.
3. The yeast expression vector pPIC6αB-PGIP was constructed which can be used to express PGIP proteins and make the activity assay.
引文
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