水稻热休克蛋白HSP70基因克隆、表达分析及原核表达  

Cloning, expression analysis and prokaryotic expression of heat shock protein HSP70 gene in rice

作　　者：杜巧丽 蒋君梅 陈美晴 宁娜 任明见[1] 李向阳[2] 谢鑫[1] Du Qiaoli;Jiang Junmei;Chen Meiqing;Ning Na;Ren Mingjian;Li Xiangyang;Xie Xin(Key Laboratory of Agricultural Microbiology,College of Agriculture,Guizhou University,Guiyang 550025,Guizhou Province,China;Key Laboratory of Green Pesticides and Agricultural Bioengineering,Ministry of Education,Guizhou University,Guiyang 550025,Guizhou Province,China)

机构地区：[1]贵州大学农学院,农业微生物特色重点实验室,贵阳550025 [2]贵州大学,绿色农药与农业生物工程教育部重点实验室,贵阳550025

出　　处：《植物保护学报》2021年第3期620-629,共10页Journal of Plant Protection

基　　金：国家自然科学基金(31801691);贵州省科技计划(黔科合支撑(2019)2408号)。

摘　　要：为明确水稻热休克蛋白70(heat shock protein 70,HSP70)基因在逆境胁迫下的表达特征,以日本晴粳稻水稻品种为材料,通过基因克隆方法获得水稻HSP70基因cDNA全长序列,并采用生物信息学的方法分析其序列特征,利用实时荧光定量PCR(quantitative real-time PCR,RT-qPCR)技术测定不同水稻组织、不同激素、不同温度以及不同逆境下HSP70基因的相对表达量,同时对其原核表达条件进行优化。结果表明,水稻HSP70基因全长1 950 bp,预测蛋白大小为71.18 kD,等电点为5.10,亚细胞定位于细胞质内,有较强的亲水性。水稻HSP70蛋白序列与香蕉HSP70蛋白序列的亲缘性较高;水稻HSP70基因表达具有组织特异性,在茎中的相对表达量最高,在根中最低;低温胁迫后,水稻HSP70基因在12 h内基因相对表达量相对稳定,在24 h时相对表达量达到峰值,为9.60;高温胁迫后,在较短时间内水稻HSP70基因显著上调并达到峰值,为79.10;激素吲哚乙酸、脱落酸和油菜素内酯均能诱导水稻HSP70基因表达;但在甘露醇模拟干旱和NaCl盐胁迫处理下,水稻HSP70基因的相对表达量呈现先升后降的趋势。水稻HSP70蛋白的最佳诱导温度是30℃,异丙基-β-D-硫代吡喃半乳糖苷最佳诱导浓度为1.2 mmol/L。In order to clarify the expression characteristics of heat shock protein 70(HSP70) gene in rice under adversity stresses, the full-length HSP70 gene cDNA sequence in rice(Oryza sativa subsp. japonica cv. Nipponbare) was obtained through gene cloning, and its sequence characteristics were analyzed using bioinformatics methods. Quantitative real-time PCR(RT-qPCR) technology was used to analyze the expression profile of this gene in different tissues of rice treated with different hormones, different temperatures and different stresses. The results showed that the HSP70 gene in rice was 1 950 bp in length, which was predicted to have a molecular weight of 71.18 k D, an isoelectric point of 5.10, and a subcellular location in the cytoplasm with strong hydrophilicity. The HSP70 protein sequence in rice was closely related to the HSP70 protein sequence in banana. The expression of HSP70 gene in rice was tissue-specific, in that the relative expression level was the highest in the stem and the lowest in roots;after low temperature stress, the relative expression of HSP70 gene in rice was relatively stable within12 h, and the relative expression reached a peak of 9.60 at 24 h;after high temperature stress, the relative expression of HSP70 gene was significantly up-regulated and reached the peak of 79.10 in rice within a short period of time. The expression of HSP70 gene in rice was induced by the hormones indole-3-acetic acid, abscisic acid and brassinolide. However, under mannitol simulated drought and sodium chloride salt stress, the relative expression of HSP70 gene in rice showed a trend of increasing first and then decreasing. The results showed that the optimal temperature and isopropyl-β-D-thiopyranogalactone concentration were 30℃ and 1.2 mmol/L, respectively, for prokaryotic expression of rice HSP70 protein.

关 键 词：热休克蛋白 水稻 基因克隆 表达分析 

分 类 号：S511[农业科学—作物学]