抗草甘膦牛筋草EPSPS基因的表达调控机理

To study the mechanisms of Eleusine indica resistance to glyphosate, we recently found that after been sprayed glyphosate, the expression of target enzyme gene EPSPS was significantly up-regulated in Glyphosate-resistance biotype of eleusine indica. But the EPSPS gene expression have no significant change in Glyphosate sensitive biotype of Eleusine indica. We speculated that up-regulation of EPSPS gene is one of reasons of Eleusine indica resistant to Glyphosate. Gene expression is co-regulated by some upstream regulatory factors after picked up the glyphosate signal. However, what factors regulate the expression of EPSPS gene have not been reported. Therefore, this project try to analysis the regulatory factors of EPSPS gene expression on the base of previous work. Cloning and analyses the regulatory sequence of EPSPS gene first, and then isolation and identification of the transcription factor regulation from glyphosate-resistance Eleusine indica . This study will help us to understand the key role factors and mechannism of EPSPS gene expression, and it could also enrich the theoretical basis of development of transgenic glyphosate-resistant crops which been transferred of exogenous EPSPS gene.

我们新近研究结果显示，在喷洒草甘膦之后，抗性牛筋草中草甘膦靶标酶基因EPSPS的mRNA表达量显著上调，而在敏感性牛筋草中该基因表达量变化不明显，推测这可能是牛筋草对草甘膦产生抗药性的原因之一。植物基因的表达是通过其上游调控序列中某些顺式作用元件和细胞核中的转录因子协同调控的，但具体是哪些作用元件和转录因子调控EPSPS基因表达，目前未见报道。因此，本项目拟在前期工作基础之上，通过对抗草甘膦牛筋草EPSPS基因调控序列的克隆、功能分析以及相关转录调控因子的分离与鉴定，探究调控牛筋草EPSPS基因表达的关键顺式元件及转录因子，阐明牛筋草EPSPS基因表达调控机理，并进一步充实转EPSPS基因抗草甘膦作物研究的理论基础。

草甘膦靶标酶EPSPS基因的表达量增加是杂草抵抗草甘膦胁迫的主要分子机制，但EPSPS基因转录水平的表达调控机理鲜有研究。本项目以敏感型和抗草甘膦牛筋草为材料，对EPSPS基因转录水平的表达调控机理进行了初步研究，较好的完成了项目预期内容。取得了重要的研究进展：1）通过HiTail-PCR的方法克隆了牛筋草EPSPS基因上游启动子序列，获得两条启动子序列Epro-S(862bp)和Epro-L(877bp)，生物信息学分析显示两者同源性为99%，主要序列差异存在于5’UTR Py-rich Strech 元件上，Epro-S和Epro-L序列上的5’UTR Py-rich Strech 元件序列分别命名为5ups-S和5ups-L。 2）启动子缺失实验表明， 5’UTR Py-rich Strech 元件的序列差异是导致Epro-L活性显著高于Epro-S活性得重要原因；3）Pull-down和EMSA结果显示，核酮糖-1、5-二磷酸羧化酶/加氧酶大亚基（rbcL）能与5ups-L元件相结合，却不能与5ups-S结合，编码rbcL蛋白的Ei-rbcl基因转录水平的表达量受草甘膦诱导显著上调。以上结果表明，核酮糖-1、5-二磷酸羧化酶/加氧酶大亚基（rbcL）可能具有转录因子的功能，能够与5ups-L元件结合调控EPSPS基因表达。研究结果为进一步深入了解牛筋草EPSPS基因的表达调控机理提供重要的实验证据。