拟南芥LecRK Ⅳ.3胞内激酶域的原核表达、纯化及鉴定  

Prokaryotic Expression,Purification and Identification of the Intracellular Kinase Domain of Arabidopsis Lectin-receptor Like Kinase Ⅳ.3

作　　者：赵记龙 许大金 任旭萌 张胜伟 ZHAO Jilong;XU Dajin;REN Xumeng;ZHANG Shengwei(Key Laboratory of Molecular and Celular Biologs of Ministry of Educatione College of Life Sciences,Hebei Normal University,Hebei Shjiazhuang 050024,China)

机构地区：[1]河北师范大学生命科学学院分子细胞生物学教育部重点实验室,河北石家庄050024

出　　处：《河北师范大学学报（自然科学版）》2022年第5期520-526,共7页Journal of Hebei Normal University：Natural Science

基　　金：国家自然科学基金(32100226);河北省自然科学基金(C2020205024);河北省高等学校科学技术研究项目(BJK2022036)。

摘　　要：类受体激酶在植物的生长发育、抵御生物以及非生物胁迫等过程中发挥着重要作用,被认为是植物中重要的跨膜信号分子.凝集素样类受体激酶(lectin-receptor like kinases,LecRKs)属于类受体激酶中的第二大亚家族,但是目前在植物领域中研究较少.笔者近期的研究结果表明,LecRKⅣ.3在植物应答盐胁迫、紫外线胁迫响应中发挥着重要作用.类受体激酶胞内域(intracellular domain,ID)的激酶活性对LecRKⅣ.3的功能至关重要,为了阐明LecRKⅣ.3是否具有激酶活性,首先克隆了LecRK Ⅳ.3ID,通过一步重组法构建至pET30a载体上,获得His-LecRKⅣ.3ID原核表达载体并转化至5种不同大肠杆菌表达宿主菌株中.以不同宿主菌生长情况来探索His-LecRK Ⅳ.3ID蛋白的最佳表达宿主,最终得到表达该蛋白的最佳宿主为OverExpressC43(DE3).此宿主获得的融合蛋白主要分布在上清液;Westernblot免疫印迹技术证明该蛋白能被Anti-His-HRP抗体识别;放射性同位素法检测到其具有激酶活性.该结果不仅为研究LecRK Ⅳ.3的生物学功能奠定了基础,也为将来纯化其他蛋白激酶提供理论参考.Receptor-like kinases play an indispensable role in plant growth and development, resistance to biotic and abiotic stresses, and are considered as important transmembrane signaling molecules in plants.Lectin-receptor like kinases(LecRLKs) belong to the second largest subfamily of receptor-like kinases, which have been rarely studied in plants.Our recent study showed that LecRK Ⅳ.3 plays an important role in plant response to salt stress and UV-B stress.Previous studies have shown that the kinase activity of the intracellular domain(ID) of receptor-like kinases is crucial for the function of LecRK Ⅳ.3.In order to elucidate whether LecRK Ⅳ.3 has kinase activity in vitro,LecRK Ⅳ.3ID was cloned and constructed into pET30 a vector by one-step recombination method.The prokaryotic expression vector His-LecRK Ⅳ.3ID was obtained and transformed into 5 different Escherichia coli expression host strains.The optimal expression host of His-LecRK Ⅳ.3 ID protein was explored based on the growth conditions of different host bacteria, and the optimal host for this protein was finally determined as OverExpress C43(DE3).The fusion protein obtained from this host was mainly distributed in the supernatant.Western blot showed that the LecRK Ⅳ.3 protein could be recognized by Anti-His-HRP antibody.LecRK Ⅳ.3 exhibited kinase activity when detected by radioisotope method.The results not only lay a foundation for studying the biological function of LecRK Ⅳ.3,but also provide a theoretical reference for the purification of other protein kinases in the future.

关 键 词：凝集素样类受体激酶 原核表达宿主 激酶活性 同位素 拟南芥 

分 类 号：Q784[生物学—分子生物学]