笋瓜韧皮部特异性启动子的克隆分析及新型植物表达载体构建

Cloning and Analysis of the Phloem Specific Promoter from Cucurbita maxima and Construction of New Plant Expression Vector

根据已报道具韧皮部组织特异性的笋瓜韧皮部蛋白2(PP2)基因序列设计引物,以山西本地种笋瓜叶片基因组DNA为模板,用PCR法扩增得到了长度为966bp的PP2基因启动子片段,克隆入pUCm-T载体后,经鉴定获得了新的重组质粒pUCm-PSP,测序和序列分析表明与两个已报道的片段分别有95%和99%的同源性,推测具有相似的启动子功能。分别用限制性内切酶PstI和BglII双酶切重组质粒pUCm-PSP和由CaMV35S组成型启动子驱动GFP报告基因的双元植物表达载体pCAMBIA1302,分别回收pUCm-PSP重组质粒中的PSP小片段和pCAMBIA1302植物表达载体中去掉GFP报告基因上游CaMV35S组成型启动子的大片段,经连接、转化和鉴定后,构建了由PSP驱动报告基因GFP的新型植物表达载体pHZ03。利用细胞感受态法将新植物表达载体分别导入根癌农杆菌EHA105、GV3101、LBA4404和发根农杆菌Ri15834中,为进一步研究其表达功能奠定了基础。

A pair of primers was designed according to the sequence of phloem protein 2 （PP2） , which shows phloem - specificity from Cucubita maxima, and a 966bp fragment of PP2 gene in the promoter region was amplified by polymerase chain reaction （PCR） using the genomie DNA of a local C. maxima ev. variety as the template. The fragment was cloned into pUCm - T vector, and a new reeombined vector named pUCm - PSP was obtained after analyzed with PCR and restriction digestion. The results of sequence analysis indicated that this fragment had 95% and 98% homology compared with two reported promoters, respectively. This result suggested that it might have the same function as other PSP. A new plant expression vector named pHZ03 in which the reporter gene GFP is droved by PSP was constructed after cutting two vectors pUCm - PSP and pCAMBIA1302 with two restriction enzymes Pst Ⅰ and Bgl Ⅱ, subsequently recovering the small PSP frag- ment from pUCm - PSP reeombined vector and the long fragment excised CaMV35S promoter in the upper region of GFP reporter gene from pCAMBIA1302 plant expression vector, and then ligation, transformation and identification. The recombined plant expression vector was transferred into Agrobacterium tumefaciens strains of LBA4404, GV3101, EHA105 and A. rhizogenes strain of 15834 by using cell competent method, and the foundation has been set up for further research work in expression and function of this promoter.