Pib启动子中茉莉酸和乙烯响应元件的转基因分析

Analysis of the molecular motif for inducing response to jasmonic acid and ethylene in Pib promoter via rice transformation

水稻Pib基因的表达受茉莉酸、乙烯等激素诱导,为了确定该基因启动子响应茉莉酸和乙烯诱导的必需区域,进一步阐明茉莉酸和乙烯响应分子元件,文章用PCR制备了Pib全长启动子-3572～2bp及3个5′端有不同长度缺失的Pib启动子片段-2692～2bp、-1335～2bp、-761～2bp。4个不同长度Pib启动子分别置换掉双元质粒中gus基因上游的35S构建为重组质粒,经农杆菌介导转入水稻获得转基因植株。转基因水稻中gus活性的蛋白质水平和mRNA水平的定性和定量分析结果表明,全长Pib启动子(-3572～2bp,pNAR901)启动活性最强,茉莉酸或乙烯诱导6h后,其驱动gus基因在转基因植株各部组织中的表达量明显上升。而-3572～-2692bp区段序列缺失后不但Pib启动子启动活性显著降低而且也丧失了对茉莉酸和乙烯的诱导活性。pNAR902(-2692～2bp),pNAR903(-1335～2bp)和pNAR904(-761～2bp)中的Pib启动子序列的缺失长度相差达2倍和3倍以上,但其对茉莉酸和乙烯的诱导响应没有区别。这些结果显示3个Pib启动子缺失体构建中,其共同缺失序列即-3572～-2692bp区域是Pib启动子茉莉酸和乙烯诱导响应的必需区域。软件检索证实,Pib启动子序列中只在上述共同缺失区段之内的-2722bp处有一个GCCGCC基序。文章报道的转基因实验表明GCCGCC基序可能是Pib基因中有关茉莉酸和乙烯诱导响应的顺式分子元件。

The expression of Pib gene in rice was induced by hormone, such as jasmonic acid and ethylene. In order to determine the necessary regions of sequence or motifs for response to jasmonic acid and ethylene in Pib promoter, the full length promoter of Pib （-3 572-2 bp） and three different 5＇ deletion fragments of Pib promoter （-2 692-2 bp, -1 335-2 bp, -761-2 bp） were synthesized by PCR and then were substituted for 35S upstream gus in a binary plasmid to construct recombined plasmids of Pib promoter-gus fusions. Transgenic rice plants of the four recombined plasmids were produced by Agrobacterium-mediated transformation. Quality and quantum analysis of gus activities in transgenie plants at both protein and mRNA levels were conducted. The promotion activity of the full length promoter of Pib （-3 572-2 bp, pNAR901） was the highest in the four recombinants and the gus activities in its transgenic plant organs were enhanced obviously at 6 h after treatment with jasmonic acid or ethylene. The promotion activity of the deleted Pib promoters was significantly decreased and the response to jasmonic acid or ethylene treatment was not present when the -3 572- -2 692 bp sequence was knocked out from the Pib promoter. Although the disparity in the lengths of the deleted Pib promoter of pNAR902 （-2 692-2 bp）, pNAR903 （-1 335-2 bp）, and pNAR904 （-761-2 bp） was more than 2 or 3 times, the response to jasmonic acid or ethylene treatment was not different among their transgenic plants. All these results indicated that the common deleted sequences （-3 572 - -2 692 bp） in the three deleted Pib promoter constructs were the essential region to the response to jasmonic acid and ethylene treatment. The result ofpib promoter sequence searching indicated that there was only one GCCGCC motif at -2 722 bp of this common deleted segment in the Pib promoter sequence. Our rice transgenic results showed that the GCCGCC may be a cis-motif for Pib gene conferring response to jasmonic acid and ethylene for Pib gene.