矮牵牛花特异表达启动子PchsA的克隆及蓝色基因F3′5′H表达载体的构建  

Cloning of the Specific Expression Gene Promoter-PchsA of Petunia hybrida Flower and Construction of F3′5′H Gene Expression Vector

作　　者：李吉莲[1,2] 姚宁涛[1] 祝建波[1] 邓福军[2] 

机构地区：[1]石河子大学生命科学学院农业生物技术重点实验室,新疆石河子832003 [2]新疆农垦科学院棉花所,新疆石河子832000

出　　处：《安徽农业科学》2009年第17期7897-7899,共3页Journal of Anhui Agricultural Sciences

基　　金：新疆生产建设兵团重大科技攻关项目(2006GG20);新疆生产建设兵团科技攻关项目(2006GG03);兵团博士资金"转基因棉花杂交棉育种技术新体系的建立"项目

摘　　要：[目的]克隆花特异表达启动子PchsA,将此启动子与蓝色基因"F3′5′H"构建新的表达载体,拟以该启动子驱动"F3′5′H"在其他花色中特异表达。[方法]根据GenBank报道的矮牵牛启动子的序列设计并合成一对特异引物,以蓝紫色矮牵牛总DNA为模板,用PCR仪扩增目的片段。[结果]PCR扩增出的启动子DNA片段长约550 bp,回收后连接到PGM-T质粒载体上,经转化、筛选确定重组子,酶切鉴定后送上海生工生物工程公司测序,得到片段长度为553 bp。经DNAMAN软件分析和GenBank上BLAST序列比对,显示序列与已报道序列同源性为98.55%。应用pcgene软件进行序列分析,结果表明试验克隆的启动子含有启动子所必须的所有调控元件,这与报道的序列基本一致。[结论]试验成功地克隆了CHSA启动子并将其与"F3′5′H"构建成能够在植物中进行遗传转化的表达载体,为培育新型花色新品种奠定了基础。The special expression vector- F3′5′H, which was new type of vector, a combination of blue color gene vector-F3′5′H and PchsA cloned, for other flowers in Petunia hybrida could be amused. [ Method] According to the sequence of flower tissue-specific expression gene CHSA promoter in Petunia hybrida reported by GenBank, a pair of primers was synthesized. With total DNA of Petunia hybrida used as template, a DNA fragment about 550 bp was amplified by PCR procedure. [ Results] The DNA fragrment was linked in pGM-T vector and the recombinant was confirmed after its transformation and filtration. Then the recombinant was identified with restriction enzymes and subjected to sequence analysis. The result showed that this fiagment＇s length was 553/919. These sequences were proved to share 98.55% horaology with the sequence reported by BLAST program and DNAMAN analysis. We cloned The special promoter tested with PchsA indicated that it contained the necessary adjustment and control factor of a promoter and its sequence was similar with the reported result. [Conclusion] The promoter- CHSA was successfully cloned and the expression vector, which could be used in gene transformation, was constructed after it was combined with F3′5′H so that the basis of new colooring variety-brecdhlg was laid.

关 键 词：矮牵牛 PchsA启动子 克隆 蓝色基因F3′5′H 表达载体 

分 类 号：S188[农业科学—农业基础科学]