pBI121表达载体及其转化植株的快速鉴定  

Rapid method to identify expression vectors and tansgenic plants based on pBI121 plasmid

作　　者：王华新[1] 曹家树[1] 向珣[1] 余小林[1] 叶纨芝[1] 

机构地区：[1]浙江大学蔬菜科学研究所,农业部园艺植物生长发育与生物技术重点开放实验室,浙江杭州310029

出　　处：《浙江大学学报（农业与生命科学版）》2008年第2期137-142,共6页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金资助项目(30370975);浙江省重大科技资助项目(2005C12019-02);宁波市科技计划资助项目(2004A410018).

摘　　要：以双元质粒pBI121为基础分别构建正义、反义和RNA干涉(RNAi)植物表达载体.根据pBI121质粒多克隆位点两侧的DNA序列设计1对通用引物,以表达载体的重组质粒DNA为模板进行PCR扩增,使用限制性内切酶BamHⅠ酶切PCR产物以验证扩增条带的真实性,从而可以快速鉴定pBI121表达载体的重组克隆.这种方法也可用于快速鉴定上述表达载体的转基因植株,其检测结果与常规鉴定方法完全一致.此外,可以根据需要对表达载体的阳性PCR扩增产物进行测序,测序结果能够精确地显示出重组pBI121表达载体中目的片段的插入方向以及插入序列是否出现碱基突变.本研究为各种pBI121表达载体及其转基因植株的鉴定提供了一种快速、有效的方法.To screen rapidly positive clones, recombinant plant expression vectors constructed based on the binary vector pBI121, containing sense, antisense or RNA interference （RNAi） cassette respectively, were amplified by PCR with a universal primer pair designed according to the flank sequence of multiple clone site in pBI121, and PCR products with predicted sizes were further verified by digestion with the restriction enzyme BamHⅠ. The PCR amplification-digestion with BamHⅠ method was used for screening transgenic plants carrying the recombinant pBI121 expression vector, and detecting results of the method were consistent with the traditional means for identification of transgenic plants. In addition, PCR products amplified from positive clones harboring recombinant expression vectors may be sequenced to precisely demonstrate whether interesting fragments were inserted into pBI121 in a right orientation and whether mutation existed in the inserted sequences. These results show that a rapid and effective method to identify the expression vectors and their transgenic plants containing pBI121 plasmid was developed.

关 键 词：pBIl21质粒 植物表达载体 转基因植株 鉴定方法 

分 类 号：Q789[生物学—分子生物学]