津田芜菁隐花色素CRY1 RNA干涉载体的构建  

Cryptochrome CRY1 RNA Interference Vector of Tsuda Turnip Built by Gateway Clone Technique

作　　者：孙梅[1] 周波[1] 马光[1] 刘明雪[1] 李玉花[1] 

机构地区：[1]东北林业大学花卉生物工程研究所,哈尔滨150040

出　　处：《分子植物育种》2013年第3期437-442,共6页Molecular Plant Breeding

基　　金：国家自然科学基金项目(30730078)资助

摘　　要：隐花色素CRY1在拟南芥光形态建成及光信号转导中起重要作用,在津田芜菁(Brassica rapaL. subsp. rapa ‘Tsuda’)中是否起相同作用尚未报道。本研究利用实验室已克隆得到的津田芜菁CRY1基因的cDNA全长序列,在NCBI进行序列比对,选取特异的片段。同时根据Gateway克隆技术特点,设计含有attB接头的引物。利用高保真的LA Taq DNA聚合酶,通过PCR方法在目的片段的两端加上attB序列。通过BP反应和LR反应将CRY1基因片段克隆到pH7GWIWG2(I)双元干涉载体。对重组载体pH7GWIWG2(I)-CRY1的鉴定结果表明成功构建了CRY1基因的干涉载体。利用Gateway克隆技术构建植物干涉表达载体简便易行,该结果为遗传转化研究其功能奠定了基础。The cryptochrome CRY1 plays an important role in developmental process ofphotomorphogenesis and light signal transduction in Arabidopsis thaliana, its function in Brassica rapa L. subsp, rapa Tsuda was not yet to be known. After blast analysis of the CRY1 gene full-length cDNA sequences ofBrassica rapa L. subsp, rapa ＇Tsuda＇ in NCBI, a unique fragment was screened in this research. According to the Gateway clone technology, we designed two pairs of primers containing attB adapters. LA Taq DNA polymerase which can reduce the non- specific binding greatly was used during two PCR in which adding attB sequence to the cloned gene. By the BP and LR recombination reaction, the PCR product containing attB was inserted into interference expression vector pH7GWIWG2（I）. The plant interference expression vector pH7GW1WG2（I）-CRY1 was successfully constructed. The results showed that it was easy to construct a plant interference expression vector by Gateway clone techno- logy. It also provided some basic information for genetic transformation with this gene and the study of its function.

关 键 词：芜菁 CRY1 Gateway克隆技术 RNA干涉 

分 类 号：S631.3[农业科学—蔬菜学]