玉米大斑病菌蛋白激酶C基因的克隆及表达规律分析

摘要

[Objective]The aim of this research was to clone the gene encoding the protein kinase C (PKC) and its promoter in Setosphaeria turcica, analyze the gene copy number in the genome of S.turcica and study the effects of different conditions on expression of PKC.[Method]The homologous fragment of PKCwas isolated by degenerate PCR, the 5’ and 3’ flanking sequences were cloned using genome walking, and the full length cDNA was obtained by RT-PCR.Gene structure and putative cis-acting elements were analyzed by bioinformatics methods.Then the gene's expression was analyzed by semi-quantitative RT-PCR, when Setosphaeria turcica was cultured on different carbon/nitrogen media, and different abiological stresses.[Result]PKC, encoding a protein of 1 171 amino acid residues, including 7 extons and 6 introns, was 3 837 bp of DNA and 3 516 bp of cDNA.There were CAAT-box and TATA-box in its upstream sequence, while the transcriptional initiator such as HSF, AP1 and Sp1-binding site, was observed.Southern blotting analysis indicated that there was a single copy of the PKC in the genome of S.turcica.By semi-quantitative RT-PCR analysis, it was found that the expression of PKC was the highest on saccharose as the carbon source,while it was distinctly inhibited by NH4+ and heavy metal ions such as Mn2+, Cu2+, and Zn2+.After addition of different concentrations of sorbitol, it has a positive correlation with the inhibition and concentrations.However, the highest expression was observed in response to isotonic NaCl.[Conclusion]Cloning of PKC and its promoter region in S.turcica have enriched the biological information resource of filamentous fungi, and thus laying a foundation for the functional analysis of the signal transduction pathway in phytopathogenic fungi.%[目的]克隆蛋白激酶C基因(PKC)及其启动子,验证该基因拷贝数,同时对PKC的表达规律进行研究,了解该基因在信号转导途径中的激活条件,为进一步研究该基因的功能奠定基础.[方法]首先通过简并引物PCR法获得PKC的同源片段,再利用Genome walking技术克隆片段的3'端和5'端侧翼序列,采用RT-PCR法扩增基因全长,并对基因结构和上游调控元件进行生物信息学分析.利用Southern blotting验证基因拷贝数.最后,利用半定量RT-PCR技术研究玉米大斑病菌在不同碳源、氮源培养以及非生物胁迫条件下,PKC的表达量的变化规律.[结果]获得PKC全长序列及其上游部分启动子区,生物信息学分析表明,PKC全长DNA序列为3837bp.cDNA序列为3516bp.由7个外显子和6个内含子组成,编码产物包含1171个氨基酸残基.在1380bp的上游侧翼序列中包含CAAT-box及TATA-box,并且存在热激转录因子(HSF)结合元件和SP1.AP1等结合元件.PKC在玉米大斑病菌基因组中以单拷贝形式存在.半定量RT-PCR结果表明:在以蔗糖为碳源时PKC,表达量高于其它碳源,铵态氮为氮源时PKC的表达明显受到抑制.重金属离子Mn(2+)、Cu(2+)、Zn(2+)显著抑制PKC表达.在高渗胁迫环境中下,山梨醉抑制PKC表达,且抑制程度与浓度成正相关;而高浓度NaCl造成高渗胁迫时,PKC由低浓度NaCl时的低丰度表达状态中被激活,表达量迅速增加.[结论]玉米大斑病菌PKC及其启动子的成功克隆与该基因的表达规律丰富了植物病原真菌的生物信息学资源,为深入了解植物病原真菌信号转导途径奠定了基础.