丹参病程相关蛋白基因PR10的克隆与表达分析
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国家自然科学基金(81173490,8130070);河南中医学院博士科研基金(BSJJ2011-07)


Cloning and Expression Analysis of Pathogenesis-related Protein 10 Gene of Salvia miltiorrhiza
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    摘要:

    病程相关蛋白(PR)的产生与积累是植物体应对生物或非生物胁迫的主要特征之一。该研究以人工培养的丹参幼苗为材料,通过分析丹参转录组数据,根据丹参病程相关蛋白基因PR10的序列设计特异性引物,采用逆转录聚合酶链式反应(RT-PCR)从丹参中获得PR10基因的开放阅读框(ORF),命名为SmPR10-1(GenBank注册号KF877034),并进行原核表达和纯化。结果表明:(1)SmPR10-1基因ORF为477 bp,编码158个氨基酸,其蛋白质分子质量为17.38 kD。(2)通过蛋白结构预测、序列多重比对和构建进化树等生物信息学分析,发现SmPR10-1基因具有保守序列(G-X-G-G-X-G)和(K-A-X-E-X-Y),其编码蛋白与葡萄等双子叶植物中的PR10蛋白同源性较高。(3)经异丙基β-D-硫代半乳糖苷(IPTG)诱导,含有表达载体pET32a-SmPR10-1的大肠杆菌(Escherichia coli BL21)可诱导表达融合蛋白;对影响蛋白表达的4个因素优化结果表明,SmPR10-1蛋白的最佳表达条件为:IPTG终浓度0.4 mmol/L、起始宿主菌密度A600为0.8、诱导温度30 ℃、诱导时间8 h,并得到纯化的SmPR10-1蛋白。该结果为进一步研究SmPR10-1基因在丹参抗病方面的生物学功能和培育丹参抗病品种奠定了基础。

    Abstract:

    The synthesis of pathogenesis-related proteins (PR) is one prominent feature of plant defense responses on abiotic or non-abiotic stress situations.According to the transcriptome data of Salvia miltiorrhiza,designing specific primers and using reverse transcription-polymerase chain reaction (RT-PCR),we isolated an open reading frame of pathogenesis-related protein 10 (PR10) from S.miltiorrhiza and named as SmPR10-1 (GenBank Accession No.KF877034).The results indicated that:(1)SmPR10-1 has an open reading frame (ORF) of 477 bp,which encoded a protein of 158 amino acid residues,with a predicted molecular mass of 17.38 kD.(2)Bioinformatic analysis indicated that SmPR10-1 protein showed the highest homology,69% identity,with PR10 protein from Vitis vinifera with the same conserved sequence (G-X-G-G-X-G) and (K-A-X-E-X-Y).(3)Escherichia coli BL21 cells were transformed with the expression vector pET32a-SmPR10-1 and used for prokaryotic expression.Meanwhile,the four factors,inducing IPTG concentrations,initial density of host bacterium (A600),inducing temperature and inducing time,which influenced protein expression,were optimized.The optimum expression conditions of SmPR10-1 were final IPTG concentration of 0.4 mmol/L,A600 of 0.8,and the inducing time of 8 h at 30 ℃.Finally,the recombinant SmPR10-1 protein was purified through Ni2+ affinity chromatography.The results of this study provided not only the first and fundamental information about SmPR10-1 gene,but also a candidate gene for future genetic engineering of Chinese medical herbs,S.miltiorrhiza,against pathogen attack.

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赵 乐,马利刚,王志霜,等.丹参病程相关蛋白基因PR10的克隆与表达分析[J].西北植物学报,2015,35(6):1078-1084

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  • 在线发布日期: 2015-08-09
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