隐花色素CRY2基因片段克隆与RNAi植物表达载体的构建.pdfVIP

分子植物育种，2008 年，第6 卷，第6 期，第1182-1186 页 Molecular Plant Breeding, 2008, Vol.6, No.6, 1182-1186 新思路、新技术、新方法 Novel Thinking Technology 隐花色素CRY2 基因片段克隆及RNAi 植物表达载体的构建 周波 孙梅 阎淑滑 李玉花* 东北林业大学花卉生物工程研究所, 哈尔滨, 150040 * 通讯作者, lyhshen@126.com 摘 要 隐花色素是植物向光反应的主要的光受体，介导蓝光/ 近紫外光调控的反应。在拟南芥中CRY2 调 节去黄化反应并控制开化时间。我们根据在GenBank 中已经发表的拟南芥(NM_100320) 、中国大白菜 (AY176689)、油菜(AJ889779) CRY2 基因的cDNA 保守序列设计并合成同源引物。通过RT-PCR 方法从“津 田”芜菁(Brassica rapa L. subsp. rapa “Tsuda”)总RNA 中扩增出CRY2 基因的cDNA 部分片段，采用gateway 技术中的BP 反应将其克隆到pDONR221 中。测序表明，该片段与拟南芥具有87%的同源性，与油菜和中国 大白菜的同源性达97% 以上。采用gateway 技术中的LR 反应，以植物表达载体pH7GWIWG2(I)为基础，构 建了由组成型启动子35S 调控的CRY2 基因的ihpRNAi 植物表达载体。 关键词 隐花色素CRY2 基因, RNAi, 植物表达载体 cDNA Cloning and Construction of RNAi Plant Expression Vector of the Cryptochrome Gene CRY2 Zhou Bo Sun Mei Yan Shuhua Li Yuhua * Institute of Flower Biotechnology, Northeast Forestry University, Harbin, 150040 * Corresponding author, lyhshen@126.com Abstract Cryptochrome is a group of flavin -type blue/ultraviolet-A light sensing photoreceptors that regulate various growth and developmental responses in plants. CRY2 regulates de-etiolation and flowering time in Ara － bidopsis. According to the conservative cDNA sequences of CRY2 in Arabidopsis (NM_100320), Chinese cabbage (AY176689) and Brassica napus (AJ889779), the homology primers were designed. Furthermore, the partial cDNA of CRY2 was amplified from total RNA of Brassica rapa L. subsp. rapa “Tsuda ”by RT-PCR. The obtained sequence shows 87% identity to CRY2 of Arabidopsis and over 97% identity to that of Chinese cabbage and Brassica napus. Moreover, the special fragment was constructed into the plant expression vector pH7GWIWG2(I) by Gateway cloning technology. The expression vector was regulated by 35S promoter and expected to transcript intron splicing hairpin CRY2 RNA and interfere to the inherent CRY2 gene. Keywords Cryptochrome gene CRY2, RNAi, Plant expression vector 隐花色素(cryptochrom