白菜型冬油菜响应干旱胁迫差异蛋白质组学和HSC70-1基因克隆及表达分析  

Differential proteomics analysis of winter rapeseed (Brassica rapa) under drought and HSC70-1 gene cloning and expression

作　　者：米超 赵艳宁 刘自刚[1] 孙万仓[1] 邹娅 徐明霞 MI Chao;ZHAO Yan-ning;LIU Zi-gang;SUN Wan-cang;ZOU Ya;XU Ming-xia(Key Laboratory of Crop Genetics and Germplasm Enhancement of Gansu Province,Gansu Provincial Key Laboratory of Arid hind Crop Sciences,College of Agronomy,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃省干旱生境作物学重点实验室甘肃省作物遗传改良与种质创新重点实验室甘肃农业大学农学院,甘肃兰州730070

出　　处：《中国油料作物学报》2019年第2期166-175,共10页Chinese Journal of Oil Crop Sciences

基　　金：国家重点基础研究发展计划(2018YFD0100502-2);国家自然科学基金(31660404);甘肃省高校科研成果转化培育项目(2018D-13);甘肃省现代农业产业技术体系建设

摘　　要：为探索白菜型冬油菜抗旱机理,采用双向电泳结合液相色谱-质谱技术分析抗旱冬油菜品系DR-5差异蛋白质组变化,克隆其差异蛋白热激同源蛋白70(heat shock cognate protein 70)基因HSC70-1,研究干旱胁迫对白菜型冬油菜HSC70-1表达的影响。质谱鉴定出23个差异蛋白质,分别参与刺激响应(5)、糖/能量代谢(6)、脂代谢(2)、信号转导(1)、氨基酸/蛋白质代谢(2)、核酸代谢(1)、细胞骨架(1)、光合作用(2)、伴侣蛋白(3)。同源克隆得到1 911bp的HSC70-1基因完整开放阅读框,编码637个氨基酸残基。所编码的蛋白HSC70是稳定的疏水性蛋白质,含有HSP蛋白家族特有的核酸结合功能区及底物结合功能区,有5个活性口袋,氨基酸序列与白菜型油菜(Brassica rapa)HSC70氨基酸序列相似度达98%。实时荧光定量和半定量分析HSC70-1基因干旱胁迫下表达,发现HSC70-1在叶片中上调表达。本研究结果为冬油菜抗旱育种提供了理论基础。In this study,we analyzed the differential proteome of winter rapeseed(Brassica rapa)by two-dimensional gel electrophoresis(2 D-DIGE)technology combined with the liquid chromatography-mass spectrometry(LC-MS)technology to detect the changes of differential proteome in DR-5(drought-resistant Winter Rapeseed line)under drought stress,and cloned the heat shock cognate protein 70 gene(HSC70-1)and analyzed the expression of this gene under drought stress.In order to provide a theoretical base for drought resistance breeding of winter rapeseed,the effect of moderate drought stress on winter rapeseed was studied.We obtained the coding region sequence of HSC70-1 in winter rapeseed by homologous cloning,and analysed the physicochemical properties,secondary structure,tertiary structure,signal peptide,transmembrane structure,conserved region,active pocket and phylogenetic conservatism of this gene encoding protein HSC70 by bioinformatics analysis methods.The real-time fluorescence quantitative and semi-quantitative analysis found the changes of HSC70-1 gene expression levels under drought stress.There were 23 differentially expressed proteins successfully identified by Mass spectrometry,which were involved in stimulation response(5),glucose/energy metabolism(6),lipid metabolism(2),signal transduction(1),amino acid/protein metabolism(2),nucleic acid metabolism(1),cytoskeleton(1),photosynthesis(2)and chaperone protein(3).We obtained the 1 911 bp complete open reading frame of HSC70-1 gene via cloning,which encoded 637 amino acid sequences.HSC70 is a stable hydrophobic protein,and it contains the unique nucleic acid and substrate binding functional area of HSP protein family,it have five active pockets.The amino acid sequence was highly similar to HSC70 in Brassica rape,and the similarity was 98%,which is highly conserved.The expression of HSC70-1 in winter rapeseed leaves was up-regulated under drought stress,which improved the tolerance of drought stress in winter rapeseed.

关 键 词：白菜型冬油菜 干旱胁迫 蛋白质组学 热激同源蛋白70 HSC70-1 

分 类 号：S565.403[农业科学—作物学] S332.4