| 摘要: |
| 【目的】 从富士苹果幼果果实中克隆多聚半乳糖醛酸酶抑制蛋白(PGIP)基因，并进行原核表达，为进一步研究PGIP的生物学功能奠定基础。【方法】 根据Genbank中已经发表的苹果PGIP基因序列设计引物，采用RT -PCR从苹果果实中扩增PGIP基因cDNA,回收目的基因片段并连接到pMD18-T载体，鉴定后进行测序。然后将PGIP全长cDNA和去信号肽的cDNA导入到pET-32a(+)表达载体中，分别获得融合表达质粒pET-PGIP和pET-PGIP-X，将其分别转化大肠杆菌BL21感受态细胞，用不同终浓度IPTG进行诱导表达，收集表达产物并进行SDS-PAGE电泳。对pET-PGIP-X基因工程菌表达产物的可溶性进行检测。【结果】 苹果PGIP cDNA序列长为1 091 bp，编码区为993 bp，可编码330个氨基酸残基，将其命名为MdPGIP；重组表达质粒pET-PGIP和pET-PGIP-X在宿主大肠杆菌中分别表达出分子质量约49.6和46.1 ku的融合蛋白，pET-PGIP-X表达产物以包涵体的形式存在。【结论】 成功克隆了苹果PGIP基因，并在大肠杆菌中获得高效表达。 |
| 关键词: 苹果 多聚半乳糖醛酸酶抑制蛋白 基因克隆 内源多聚半乳糖醛酸酶 |
| DOI： |
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| 基金项目:国家自然科学基金项目(30671447) |
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| Cloning of PGIP gene from Malus domestica M.and its fusion expression in E.coli |
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| Abstract: |
| 【Objective】 In order to lay a foundation for the study of the biological function of polygalacturonase-inhibiting protein (PGIP),PGIP gene was cloned from apple fruit and its prokaryotic expression was induced by IPTG.【Method】 A pair of specific primers were designed according to the conserved sequences of apple PGIP genes in GenBank.The cDNA of apple PGIP gene was amplified from fruit by RT-PCR and cloned into plasmid of pMD18-T,then identified by sequencing.The full-length cDNA and the cDNA excluding signal peptide were respectively constructed into expression plasmid pET-32a(+),and the recons were respectively named pET-PGIP and pET-PGIP-X.Then the recombinant plasmid pET-PGIP and pET-PGIP-X were transformed into host bacterium BL21 and their expression were induced by different concentrations of IPTG.Expression products were analyzed by SDS-PAGE.The pET-PGIP-X expression product was detected by solubility analysis.【Result】 The results of sequencing and bioinformatical analysis showed that this fragment was 1 091 bp,including a 993 bp complete ORF,encoding 330 amino acid,which was named MdPGIP.The results of SDS-PAGE showed that the recombinant plasmid pET-PGIP and pET-PGIP-X could express interest protein and the molecular weight of which was 49.6 ku and 46.1 ku.Signal peptide excised protein expressed by pET-PGIP-X mainly existed in the form of inclusion bodies.【Conclusion】 Apple PGIP gene was successfully cloned and high level expression existed in E.coli. |
| Key words: Malus domestica Mill polygalacturonase-inhibiting protein gene cloning Endo-polygalacturonase |
