Col生态型拟南芥AP3基因启动子克隆及植物表达载体构建

Molecular Cloning of Promoter of AP3 Gene from Arabidopsis thaliana (Ecotype Col) and Construction of Plant Expression Vector

隶属于MADS-Box基因家族的拟南芥花器官B类特征基因APETALA3（AP3）在花瓣和雄蕊中特异性地表达;AP3基因编码转录因子,与A类和C类特征基因协同作用控制双子叶植物花瓣和雄蕊的发育。研究表明AP3基因启动子为花特异表达启动子。因此,AP3基因启动子的克隆及功能鉴定对于园林植物与花相关的商业性状的定向改良具有重要作用。本文根据GenBank数据库报道的Ler生态型拟南芥（Arabidopsis thaliana）AP3基因启动子序列（U30729）设计了一对特异性扩增引物,基于PCR技术,用高保真的KOD-plusDNA聚合酶扩增了长度为1767bp的Col生态型拟南芥AP3基因启动子,并命名为pAtAP3,其Gen-Bank登录号为FJ619533。Bl2seq在线分析表明pAtAP3与U30729序列的相似性达98%,与Col生态型拟南芥BAC克隆T12E18（AL132971）9264~11030之间的碱基序列相似性达100%,且该段序列的下游基因编码AP3蛋白（CAB81799）,说明克隆序列为Col生态型拟南芥AP3基因的启动子。PLACE在线分析表明pAtAP3具有基本的启动子元件TATA-box和CAAT-box,还包含大量与花特异表达相关的顺式元件CArG1、CArG2、CArG3和anther-box等。本试验进一步构建了植物表达载体pAtAP3：：GUS,为该启动子的功能鉴定奠定了基础。

Belonging to MADS-Box gene family, Arabidopsis floral organ identity B gene APETALA3 （AP3） is expressed in petals and stamens specifically. AP3 gene which encodes a transcription factor controls the development of petals and stamens of dicotyledons, in combination with the class A and C genes, respectively. It indicated that the promoter of AP3 gene was a flower-specific expression promoter. Therefore, cloning and characterization of AP3 gene promoter from Arabidopsis thaliana will have significant effects on the oriented improvement of the commercial traits related to the flowers of ornamental plants. Here, a pair of specific PCR primers was designed according to promoter sequence of AP3 gene of Arabidopsis thaliana（ecotype Ler） reported in GenBank （U30729）. A 1 767 bp long sequence of AP3 gene promoter was isolated from Arabidopsis thaliana （ecotype Col） by using PCR technique with high-fidelity DNA polymerase KOD-plus. The GenBank accession of pAtAP3 is FJ619533. Online Bl2seq analysis indicated that the sequence similarity between pAtAP3 and U30729 was 98%. It also showed that the sequence similarity between pAtAP3 and the base from 9 264 to 11 030 of BAC clone T12E18 （AL132971） of Arabidopsis thaliana （ecotype Col） was up to 100%, and its downstream sequence encoded AP3 protein （CAB81799）, which approved that the cloned sequence was promoter of AP3 gene of Arabidopsis thaliana （ecotype Col）. Online PLACE analysis revealed that pAtAP3 contained basic cis-elements of promoter such as TATA-box and CAAT-box. It also comprised of several cis-elements like CArG1, CArG2, CArG3 and anther-box, which were all related to flower-specific expression. Moreover, plant expression vector of pAtAP3：：GUS was successfully constructed, laying the foundation for functional characterization of the AP3 promoter of Arabidopsis thaliana （ecotype Col）.