不结球白菜病程相关蛋白基因BcPR5的克隆及表达分析

[目的]本文旨在研究不结球白菜病程相关蛋白基因BcPR5的结构与表达特征。[方法]通过RACE技术，从抗病品种‘苏州青’叶片克隆到BcPR5基因的全长cDNA序列。采用RT-qPCR方法分析该基因在霜霉病菌诱导，水杨酸（SA）、茉莉酸（MeJ）和脱落酸（ABA）等激素处理条件下的表达模式。SDS-PAGE技术分析该基因的原核表达特征。[结果]BcPR5基因的cDNA全长为954 bp，其中开放阅读框长度为732 bp，共编码243个氨基酸，相对分子质量为26.1×103，理论等电点是9.3。氨基酸同源系统进化分析表明，不结球白菜BcPR5基因与同属植物的进化关系相近，其中与大白菜第6号染色体上的基因同源性最高（100%）。RT-qPCR分析表明，抗病品种‘苏州青’和感病品种‘矮脚黄’在霜霉病菌和非生物胁迫（SA、MeJ和ABA）诱导过程中，BcPR5基因的表达量均呈先升高后降低的趋势，且抗病品种高于感病品种。原核表达结果表明，该蛋白在终浓度为1.0 mmol？L-1的IPTG诱导4 h后能实现融合蛋白的高效表达。[结论]BcPR5在不结球白菜抗霜霉病防御反应中发挥着重要作用，研究结果为该基因的功能验证提供理论基础。
[Objectives]In present work,the characteristics of the sequence structure and expression pattern involving in pathogenesis-related protein gene(BcPR5)in non-heading Chinese cabbage(Brassica campestris ssp. chinensis),were investigated to explore its species-specific function in the tolerance of downy mildew defense.[Methods]The full length sequence of BcPR5 cDNA was cloned from the disease-resisted variety ‘Suzhouqing’ by performing the rapid amplification of cDNA ends(RACE)reactions. The RT-qPCR subsequently was done for investigating the expression pattern of BcPR5 gene under the induction of downy mildew and the treatment of plant hormones involving in MeJ,SA and ABA. Additionally,the prokaryotic expression of BcPR5 protein was performed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE).[Results]The full length of BcPR5 cDNA sequence is 954 bp,of which,732 bp ORF is included which encodes a functional protein by 243 amino acid with the molecular weight of 26.1×103 and isoelectric point of 9.3. The phylogenetic analysis of amino acid homologous suggested that the BcPR5 from the non-heading Chinese cabbage has the highest similarity(100%)with the homologous in Chinese cabbage. The results of RT-qPCR showed that the expression level of BcPR5 gene increased at first and then decreased during infection by downy mildew and abiotic stresses(SA,MeJ and ABA)in the resistance variety ‘Suzhouqing’ and the susceptible variety ‘Aijiaohuang’,and the transcription at higher levels in resistance variety than that of in susceptible variety. Prokaryotic expression results showed that efficient expression of the protein could be realized after induced with 1.0 mmol？L-1 IPTG for 4 h.[Conclusions]These results suggest that BcPR5 plays an important role in response to the tolerance of downy mildew defense in non-heading Chinese cabbage,and will provide a theoretical basis for further functional verification for BcPR5 gene