青稞核糖体蛋白基因HbRPL19的克隆、序列分析及原核表达  

Cloning,Sequence Analysis and Prokaryotic Expression of Ribosomal Protein Gene HbRPL19 from Tibetan Hulless Barley(Hordeum vulgare L. var. nudum HK. f.)

作　　者：徐齐君[1,2] 扎桑 王玉林[1,2] 原红军 曾兴权[1,2] 尼玛扎西 XU Qi-jun;ZHA Sang;WANG Yu-lin;YUAN Hong-jun;ZENG Xing-quan;Nimazhaxi(Tibet Academy of Agriculture and Animal Husbandry Sciences,Tibet Lhasa 850002,China;Barley Improvenment and Yak Breeding Key Laboratory of Tibet Autonomous Region,Tibet Lhasa 850002,China;Research Institute of Agriculture Resource and Environment,Ti-bet Academy of Agriculture and Animal Husbandry Sciences,Tibet Lhasa 850002,China)

机构地区：[1]西藏自治区农牧科学院农业研究所,西藏拉萨850002 [2]省部共建青稞和牦牛种质资源与遗传改良国家重点实验室,西藏拉萨850002 [3]西藏自治区农牧科学院,西藏拉萨850002

出　　处：《西南农业学报》2018年第6期1111-1115,共5页Southwest China Journal of Agricultural Sciences

基　　金：西藏财政专项(2017CZZX001/2;2015CZZX001;2018CZZX 001);西藏科技重大专项(Z2016B01N01)

摘　　要：【目的】克隆青稞核糖体蛋白基因HbRPL19,分析其序列特征并在大肠杆菌中诱导目的蛋白,为后续的研究奠定基础。【方法】根据鉴定得到的青稞HbRPL19的序列设计了引物,从青稞叶片的c DNA中扩增得到目的片段,并对目的基因序列进行了测序鉴定和序列分析,同时构建了原核表达载体p ET28a-RPL19,转化入大肠杆菌BL21(DE3)后利用IPTG诱导外源蛋白质表达并检测。【结果】成功扩增出了青稞核糖体蛋白基因HbRPL19的CDS全长序列,大小为654 bp。根据生物信息学分析的结果,HbRPL19编码的蛋白质含有217个氨基酸,相对分子质量为24.47 KD,理论等电点(p I)为10.42,总平均亲水性(GRAVY)为-0.353,不稳定系数为53.46,存在典型的SH3-like结构域和核糖体蛋白L19结构域。系统进化分析表明,HbRPL19与小麦和山羊草的RPL19具有较近的亲缘关系。蛋白表达结果显示,重组蛋白主要以包涵体形式存在于沉淀中。【结论】获得了青稞抗寒相关基因HbRPL19的序列和蛋白。[ Objective] The present paper was conducted to clone the ribosomal protein gene HbRPL19, analyze the sequence characteristics and induce the target protein in E. coli, which laid the foundation for the subsequent research. [ Method] The primers were designed accord- ing to the sequence of the identified Tibet barley ribosomal protein gene HbRPL19, and target fragment was amplified from the cDNA of the barley leaf. After that, the target sequence was sequenced and analyzed. Prokaryotic expression vector pET28a -RPL19 was constructed and transformed into E. coli BI21 （ DE3 ）. At the same time, positive bacteria was induced by IPTG to express and detect foreign proteins. [ Result] The full CDS of the barley ribosomal protein gene HbRPL19 was amplified and the size was 654 bp. According to the results of bioinformatics analysis, HbRPL19 encodes a protein containing 217 amino acids with a relative molecular mass of 24.47 KD, a theoretical i- soelectric point （pI） of 10.42, a total average hydrophilicity （GRAVY） of - 0. 353, an unstable coefficient of 53.46, a SH3-1ike domain and a ribosomal protein L19 domain. Pbylogenetic analysis showed that HbRPL19 had a close genetic relationship with RPL19 of wheat and Aegilopstauschii. The results of protein expression showed that the recombinant protein was mainly precipitated in the form of inclusion bodies. [ Conclusion] The sequence and protein of the cold-resistant gene HbRPL19 was obtained in this study.

关 键 词：青稞 核糖体蛋白 RPL19 序列分析 原核表达 

分 类 号：S512.3[农业科学—作物学]