甘薯耐盐基因IbNFU1特异启动子的克隆及功能分析

Environmental stresses such as drought, high salt and low temperature affect plant growth and decrease sweet potato productivity extremely. Numerous stress-relative genes have been introduced into crops to improve stress tolerance. One strategy for enhancing expression of exogenous genes is to search proper promoter, which drives and activates the expression of stress genes.In the present strudy, enzyme digested and adapter-ligated genomic DNA from sweet potato were used to isolate the promoter of sweet potato IbNFU1 gene with specific primers corresponding to the 5' sequence of IbNFU1,termed IbNFU1P1. To characterize the expression pattern of the promoter, the IbNFU1P1 sequence was fused to the GUS reporter gene and introduced into tobacco and sweet potato by Agrobacterium-mediated transformation. GUS expression levels in various organs of the transgenic plants were detected in response to salt treatment. Moreover, we designed a series of indel mutation to define the promoter core sequence. We constructed a vector expressing IbNFU1 gene driven by the IbNFU1P1 core promoter for evaluation of salt tolerance. This strategy not only provide self-owned cis-regulatory elements for sweet potato transgenic engineering, but also provide new insights to delineate the mechanism of salt tolerance, and favor the utilization of saline-alkali soil.

植物非生物胁迫，例如高盐、干旱、低温等会影响甘薯生长发育，严重降低其产量。将抗逆基因转入抗逆性差的作物可显著提高作物对非生物逆境的抵抗能力。选择合适的启动子启动和增强相应的抗性基因表达具有非常重要的意义。本研究利用反向PCR技术，扩增甘薯IbNFU1基因上游的启动子序列，命名为IbNFU1P1。将IbNFU1P1连接在GUS报告基因的上游构建植物表达载体连接，经农杆菌介导获得转基因烟草和甘薯植株。结合组织化学染色法，检测GUS报告基因在不同组织中的表达特性，并采用不同的处理检测其在胁迫情况下的响应模式，明确IbNFU1P1的表达特性。构建一系列的缺失突变体，确定该启动子的核心序列。将启动子核心元件与IbNFU1基因连接转入烟草和甘薯，验证其能否提高耐盐性。这不仅能够为甘薯基因工程提供具有自主知识产权的调控元件，而且为植物耐盐机理的深入研究提供了新的资料，可望为我国盐碱地的利用提供支持。

甘薯是一种世界上重要的粮食作物，其产量经常受到盐胁迫影响。培育耐盐的优良甘薯品种，是甘薯育种工作者的一项非常重要的任务。基因的有效表达需要有合适的启动子，目前在转基因研究中主要使用花椰菜花叶病毒CaMV 35S启动子，该启动子为组成型表达启动子。因此通过开发诱导型启动子，使耐盐基因在胁迫条件下启动表达，是开展甘薯耐盐性转基因研究的基础。本研究利用反向PCR技术，扩增出甘薯IbNFU1基因上游的启动子序列，将其命名为IbNFU1P1，该启动子全长为1436bp。采用启动子分析软件分析并预测IbNFU1基因启动子的功能，同时构建了8个的缺失突变体。将IbNFU1P1-8连接在GUS报告基因的上游，经农杆菌介导获得转基因烟草植株，通过GUS酶活试验分析启动子的活性，确定了该启动子的核心序列。同时以耐盐突变体和野生型为实验材料，对包括该启动子在内的IbNFU1基因开展了甲基化分析。在IbNFU1基因的启动子区域有2个位点产生了甲基化差异，其中1个位点在野生型中发生了10%的甲基化，而在突变体中为0%；另一个位点在野生型中发生了90%甲基化，而在突变体中仅为50%，由此推断，突变体耐盐性增强可能是由于启动子区域的去甲基化，从而提高了基因表达水平，进而增强了耐盐性。