水稻类受体蛋白激酶FTPK1原核表达及其活性检测.docVIP

水稻类受体蛋白激酶FTPK1原核表达及其活性检测 　　摘要：类受体蛋白激酶（receptor-like kinase， RLKs）是植物信号分子的受体，它能够通过胞内激酶域对底物进行磷酸化或去磷酸化，从而对下游靶基因的转录及表达进行调控，参与胞内信号转导，在植物生长发育及环境应答过程中发挥作用。本研究以水稻“中花11”cDNA为模板，通过PCR技术获得类受体蛋白激酶基因FTPK1的全长序列，进一步构建原核表达载体pET-28a-FTPK1后，经1 mmol/L IPTG诱导12 h，获得88 kD的诱导表达蛋白条带，显示FTPK1可在大肠杆菌中诱导表达。表达产物经Ni柱纯化及透析后，获得纯化的融合蛋白。FTPK1激酶活力检测显示，其可磷酸化蛋白底物His和MBP，具有一定的磷酸化活性。本研究为进一步制备FTPK1特异性抗体，深入解析其理化特性及生物学功能奠定了基础。 　　关键词：水稻；类受体蛋白激酶；FTPK1；原核表达；蛋白纯化；激酶活力检测 　　中图分类号：S511.0353文?I标识号：A文章编号：1001-4942（2018）05-0007-07 　　Abstract Receptor-like protein kinases （RLKs） are receptors of signaling molecules of plants. It can be phosphorylated or dephosphorylated through the intracellular kinase domain， thus regulating the transcription and expression of downstream target gene， participating in the intracellular signal transduction， and playing a role in plant growth and environmental response. In this study， the cDNA of rice variety Zhonghua 11 was used as template to obtain the full-length sequence of FTPK1 gene by PCR， and the pET-28a-FTPK1 prokaryotic expression vector was constructed. An 88 kD fusion protein could be induced by 1 mmol/L IPTG for 12 hours， indicating that FTPK1 could be induced in E. coli. The purified protein product of FTPK1 could be obtained after purification and dialysis by Ni column. The detection of protein kinase activity showed that FTPK1 could phosphorylate the substrates of His and MBP， which showed that FTPK1 has phosphorylation activity. This research shed some lights for further preparation of the FTPK1 specific antibody， and lay solid foundation for analysis the physical and biochemical functions of FTPK1. 　　Keywords Rice； Receptor-like protein kinase； FTPK1； Prokaryotic expression； Protein purification； Kinase activity detection 　　所有生命体在细胞水平上都是通过各种受体接收和处理信息来维持机体的正常生命活动，这些受体不仅能够识别来自环境和临近细胞的信号，还能够激活其下游的信号级联途径。在动物中，典型的一类受体是受体激酶（receptor kinases，RKs）。RKs有一个能够专一性识别配基的胞外结构域，其通过一个跨膜结构域连接着一个胞内激酶区。其激活过程依赖于磷酸化[1]。 　　植物类受体蛋白激酶（receptor-like kinases，RLKs）的结构类似于动物RKs[2-4]。由一个胞外区、