闫丽华+卢楠楠+和亚男+尹海波+谢先芝

摘要：光敏色素是植物重要的光受体之一，主要感受红光和远红光。水稻光敏色素基因家族包括3个成员，即PHYA、PHYB 和PHYC。我们已有研究表明PHYB在水稻生长发育和胁迫反应中具有重要作用。然而目前缺乏有效的水稻PHYB蛋白质抗体，极大地制约着PHYB基因作用机制的解析。在本研究中，我们比较了水稻光敏色素家族3个成员氨基酸序列同源性，选取了PHYB蛋白质C端的特异多肽。在此基础上，分离了特异多肽对应的cDNA序列，将其克隆在原核表达载体pET16b上，利用异丙基硫代-β-D-半乳糖昔（IPTG）诱导、纯化获得了PHYB蛋白质特异性抗原，通过免疫新西兰大白兔，制备了PHYB蛋白的多克隆抗体。免疫印迹检测表明该抗体具有很高的特异性。本研究为深入解析PHYB基因在水稻生长发育中的作用机制提供了必要保障。

关键词：水稻；光敏色素；原核表达；抗体

中图分类号：S511：Q78 文献标识号：A 文章编号：1001-4942（2017）08-0001-06

Abstract Phytochrome （PHY） is one of the important photoreceptors in plant. Phytochrome family mainly senses red and far-red lights. Rice phytochrome gene family is composed of three members known as PHYA， PHYB， and PHYC. Our previous studies has suggested that PHYB plays important roles in regulating rice growth and development as well as stress responses. However， absence of effective PHYB antibody greatly hinders the dissection of mechanisms of PHYB functions in rice. In this study， we selected the peptide specific to PHYB protein by comparing the amino acid identity among three rice phytochrome members. The cDNA fragment encoding specific PHYB peptide was isolated and cloned into prokaryotic expression vector pET16b. The PHYB antigen was obtained after isopropyl thio-beta-d-galactose（IPTG）induction and purification of expressed protein. The polyclonal PHYB antibody was prepared by immunizing New Zealand rabbits. Western blotting assays revealed that PHYB polycolonal antibody prepared in this study was specific to detect PHYB protein in rice extracts. The results from this study were necessary for comprehensively exploring the functional mechanism of PHYB in rice.

Keywords Rice； Phytochrome； Prokaryotic expression； Antibody

对高等植物而言，光不仅是光合作用的能量来源，也是一种重要的环境信号因子[1]。高等植物利用光敏色素（phytochromes）、隐花色素（cryptochromes）和向光素（phototropins）等光受体感受其生长环境中光质、光强、光周期等条件的变化，调节自身的生长发育，以最大限度适应周围环境。其中，光敏色素主要感受红光（red light，R）和远红光（far-red light，FR）[2]。水稻中光敏色素基因家族包括3个成员：PHYA、PHYB 和PHYC[3-6]。图位克隆和全基因组检索显示，PHYB基因位于水稻的第3染色体短臂，且为单拷贝。Takano等[7]通过伽马射线诱导突变体筛选得到了多个phyB突变体。通过比较野生……