罗伦隐球酵母rho1基因的克隆及其在酿酒酵母中的转化和表达

发布时间：2018-03-22 05:39

本文选题：rho1基因　切入点：罗伦隐球酵母　出处：《浙江大学》2016年硕士论文　论文类型：学位论文

【摘要】：细胞壁完整性(Cell wall integrity, CWI)信号通路是真菌中普遍存在一条保守通路,在生长、繁殖、对逆境胁迫的感受和调节中均具有重要作用。Rho1蛋白作为CWI通路的主要调节因子能够与膜传感器信号整合后激活下游MAPK信号通路,参与细胞壁合成等多种生理调控。罗伦隐球酵母是目前广泛报道的一种用于果实采后病害的生防酵母,但因其遗传背景尚不明确,导致其在基因水平上研究进展缓慢。本课题通过转录组技术获得罗伦隐球酵母的rhol基因序列,在序列分析的基础上通过酿酒酵母表达系统异源表达罗伦隐球酵母rhol基因,探究rho1基因转化在提高重组酵母抗逆性和生防效力方面的可行性,研究结果对于明确rho1在生防酵母逆境适应和抑制果实病害中的作用等具有重要意义。主要研究结果如下：罗伦隐球酵母rho1基因序列和所编码的蛋白结构分析。通过对罗伦隐球酵母转录组高通量测序分析获得rho1基因的cDNA序列为597 bp,运用PCR扩增得到rho1基因全长966 bp,包含7个外显子和6个内含子,在基因结构上与新型隐球酵母及其相似；对罗伦隐球酵母Rho1蛋白结构预测结果显示：Rho1蛋白由199个氨基酸组成,存在典型小GTP结合蛋白保守结构域,并有6个α1-螺旋结构、6个p-折叠和5个Loop结构,与已知的Rho家族蛋白空间结构相似,因此推断罗伦隐球酵母Rho1蛋白与已知Rho家族蛋白可能具有相似的生物学功能。罗伦隐球酵母rho1基因克隆及在酿酒酵母中表达。用特异性引物扩增得到rho1基因cDNA全长序列,利用Clonexpress高效的DNA定向克隆技术将rho1基因连接到载体上,并将重组载体通过电转化导入酿酒酵母感受态细胞,在含有150μg/ml的杀稻瘟菌素的培养基筛选得到转化子,测序鉴定后构建重组酿酒酵母SC-rho1。重组酿酒酵母SC-rho1抗逆性和生防效力评价。重组酿酒酵母对温度和渗透压的耐受性明显高于野生型酿酒酵母,同时在37℃和渗透压胁迫下重组酿酒酵母能更加有效抑制梨果实病害,与野生型酿酒酵母相比分别将发病率降低了33%和25%,其机理可能与rho1基因过表达后提高CWI信号通路的转录效率有关。
[Abstract]:Cell wall integrity (Cell wall integrity, CWI) signaling pathway is a conserved pathway exists in fungi growth, reproduction, stress perception and regulation plays an important role as a major regulator of.Rho1 protein CWI pathway with membrane sensor signal integration after activation of downstream MAPK signaling pathway is involved in the cell. The physiological regulation of wall synthesis. C.laurentii is used for postharvest diseases of biocontrol yeast widely reported, but because of its genetic background is not clear, the slow progress of the study on the gene level. In this paper obtains the rhol gene sequence of c.laurentii by transcriptome technology, through the yeast expression system for heterologous expression of c.laurentii rhol gene on the basis of sequence analysis, explore the transformation of Rho1 gene in improving recombinant yeast resistance and biocontrol effect of the can For, the results is of great significance for the biocontrol yeast Rho1 in adversity adaptation and suppression of fruit diseases in action. The main results are as follows: the analysis of protein structure c.laurentii Rho1 gene sequence and encoding. The c.laurentii transcriptome high-throughput sequencing analysis of cDNA sequence of Rho1 gene for 597 BP, using PCR amplified Rho1 gene was 966 BP, contains 7 exons and 6 introns in the gene structure and Cryptococcus and similar; display of c.laurentii Rho1 protein structure prediction results: the Rho1 protein consists of 199 amino acids, are typical of small GTP binding protein conserved domain, and have 6 alpha helix structure of 1-, 6 p- and 5 Loop folding structure, similar to the Rho protein family known spatial structure, it is deduced that c.laurentii Rho1 protein with known Rho family proteins May have similar biological functions. Cloning of yeast Rho1 gene profile of ball Luo and expression in Saccharomyces cerevisiae. The full-length sequence of Rho1 gene cDNA with specific primers, using DNA Clonexpress efficient directional cloning the Rho1 gene was cloned into the vector, and the recombinant vector was transformed by electroporation into yeast competent cells. In the presence of 150 g/ml blasticidin medium screened transformants, sequencing the recombinant Saccharomyces cerevisiae SC-rho1. recombinant Saccharomyces cerevisiae SC-rho1 resistance and biocontrol efficacy evaluation of a recombinant Saccharomyces cerevisiae tolerance to temperature and osmotic pressure was significantly higher than that of wild type yeast, also at 37 degrees C and osmotic pressure under the stress of recombinant Saccharomyces cerevisiae can effectively inhibit pear fruit disease, compared with wild type yeast respectively incidence rate decreased by 33% and 25%, and its mechanism may be related to Rho1 gene The expression increases the transcriptional efficiency of the CWI signaling pathway.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TS261.11

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