玉米棉子糖合成酶(ZmRAFS)基因响应干旱胁迫的转录调控研究

Transcriptional regulation of maize raffinose synthase(ZmRAFS)gene in response to drought stress

玉米棉子糖合成酶基因ZmRAFS受干旱诱导表达,但其响应干旱的表达调控机理仍不清楚。本研究通过RNA-seq数据分析及相关实验,发现转录因子ZmHB7在玉米幼苗中响应干旱和ABA处理。通过PCR方法克隆了ZmHB7编码区片段,并构建了其原核表达载体和植物表达载体。体外诱导ZmHB7蛋白表达,纯化回收并制备相应抗体。Western blot实验证实了ZmHB7蛋白在玉米叶肉原生质体中正确表达。将ZmHB7和GFP植物表达载体分别与Zm RAFS基因全长启动子片段驱动的双荧光素酶报告载体转入玉米原生质体中,酶活检测结果显示,ZmHB7转录因子能够激活ZmRAFS启动子驱动的海参荧光素酶基因(Rluc)的表达。逐步截短Zm RAFS基因启动子,将Zm RAFS不同长度启动子片段驱动的双荧光素酶报告载体分别与GFP或ZmHB7植物表达载体转入玉米原生质体中,酶活检测结果发现,ZmHB7仍能上调ZmRAFS基因,这一结果说明ZmHB7可能间接调控ZmRAFS基因的表达。本文初步探索了ZmRAFS基因响应干旱的分子调控机制,为进一步深入研究棉子糖合成酶基因Zm RAFS的表达调控奠定了基础。

The expression of MAIZE RAFFINOSE SYNTHASE(ZmRAFS) gene is induced by drought stress,however, the transcription factors that regulate its expression in response to drought stress remains unknown.We found that transcription factor ZmHB7 was up-regulated by drought stress and ABA by analyzing RNAseq data and relevant experiments. The ZmHB7 coding sequence(CDS) was amplified by PCR, its prokaryotic and plant expression vectors were constructed. The ZmHB7 antibody was prepared by immunization of rabbit using the purified ZmHB7 protein. Western blot analysis showed that ZmHB7 protein was correctly expressed in maize mesophyll protoplast after being transformed with ZmHB7 plant expression vector. ZmHB7 or GFP plant expression vector and the dual luciferase reporter vector controlled by ZmRAFS full-length promoter were co-transformed into the maize protoplast. The luciferase activity assay showed that Renilla luciferase gene(Rluc) controlled by ZmRAFS full-length promoter was up-regulated by the ZmHB7. Either ZmHB7 or GFP plant expression vector was transformed into maize protoplast with the dual luciferase reporter which was controlled by the different length of ZmRAFS promoter. The luciferase activity assay showed that the Rluc gene was still up-regulated by the ZmHB7. These results indicate that ZmHB7 indirectly regulates the ZmRAFS expression under drought stress.