扣囊复膜孢酵母菌MIG1基因的调控功能及其信号传导通路研究

In recent years, more and more people studied Saccharomycopsis fibuligera that was widely used in fermentation industry. In the previous research, we cloned the MIG1 gene that was the most important gene in glucose repression from Saccharomycopsis fibuligera; in S. fibuligera, we has proved that Mig1, which was the product of MIG1 gene expression, was involved in the regulation of the α-amylase, glucoamylse, acid protease and β-glucosidase gene expression; we found that Mig1 also participated in the regulation of cell wall synthesis, colony morphology, size of colony, and so on. This project intends to characterize the MIG1 gene regulation and signal transduction pathway at the mRNA and protein levels by comparing the correlation analysis of transcriptome and proteome of S. fibuligera wild-type and MIG1 knockout strains. We verificate Mig1 binging the DNA of the regulated gene by yeast one-hybrid system and DNaseⅠ foot-printing assay, and then we identify the interaction between the proteins in the signal transduction pathway by yeast two-hybrid system and biomolecular fluorescence complementation assay. The result of this project not only provides the important theoretical basis for genetic improvement of S. fibuligera and the technical reference for the study on the function of MIG1 gene of other species, and is also conducive to the comprehensive and systematic understanding of the function of MIG1 gene for us.

扣囊复膜孢酵母在发酵工业中应用广泛。前期研究中，从扣囊复膜孢酵母中克隆到了直接参与葡萄糖阻遏的MIG1基因，是已知参与葡萄糖阻遏基因中最重要的一个，所表达的Mig1被证明参与了α-淀粉酶等多种酶的基因调控；还发现MIG1基因参与细胞壁合成、菌落大小形态等多种生物学过程的调控。本项目拟对扣囊复膜孢酵母野生型菌株和MIG1基因敲除菌株的转录组和蛋白质组进行比较关联分析，从mRNA和蛋白质两个水平来综合研究Mig1在扣囊复膜孢酵母中所参与调控的生物学过程及其信号传导通路；采用酵母单杂交系统和DNaseⅠ足迹实验验证Mig1与其调控基因DNA的结合情况，采用酵母双杂交系统和双分子荧光互补技术验证信号传导通路中蛋白质间的相互作用。本研究结果不仅为扣囊复膜孢酵母菌的遗传改良提供重要理论依据，还有利于人们对MIG1基因功能的全面系统的认识，为研究其它菌种的MIG1基因功能提供理论基础和技术参考。

扣囊复膜孢酵母在发酵工业中应用广泛。前期研究中，从扣囊复膜孢酵母中克隆到了直接参与葡萄糖阻遏的MIG1基因，是已知参与葡萄糖阻遏基因中最重要的一个，所表达的Mig1被证明参与了α-淀粉酶等多种酶的基因调控。本项目构建了扣囊复膜孢酵母的cDNA文库、完成了对扣囊复膜孢酵母全基因组的测序，数据上传到NCBI网站（登录号：STUDY: PRJNA433274），这是研究扣囊复膜孢酵母基因功能的基础工作，具有重要的意义；完成了扣囊复膜孢酵母野生型菌株和MIG1基因敲除菌株的转录组测序工作，数据上传传NCBI网站（登录号：STUDY: PRJNA432463），并对差异表达基因做了细致的分析，尤其是对MIG1基因功能信号传导通路的预测；实验验证MIG1基因信号传导通路的结点，采用酵母双杂交系统来证明Mig1蛋白在Tup1参与下与Ssn6结合，采用DMS (Dimethylsulfate)足迹实验来验证Mig1蛋白结合差异表达基因（α-淀粉酶基因）上游顺式作用元件，Mig1-Ssn6-Tup1复合体与CCGAATATAA ATAGAGACACA ATCCACCTCTGGAC序列绑定，从而阻止了α-淀粉酶基因的表达。通过本项目研究，主要取得以下几个成果：（1）扣囊复膜孢酵母的MIG1基因的信号传导通路，阐明了MIG1基因的基因功能，可以广泛应用到扣囊复膜孢酵母发酵以及水解淀粉上；（2）在本项目的研究中还发现，扣囊复膜孢酵母的淀粉酶信号肽和性蛋白酶信号肽可以应用到抗菌肽表达上，能够很好地将表达的抗菌肽引导分泌到细胞外部，目前已经申请了专利，这2个信号肽还可以应用到其他分泌型白蛋的表达上；（3）此外，扣囊复膜孢酵母全基因组的测序、cDNA文库的构建、转录组的测定，都为以后研究扣囊复膜孢酵母其他基因功能起着重要作用。