两种调控因子在嗜热毁丝霉纤维素酶基因表达调控中的功能研究

发布时间：2018-01-05 19:13

本文关键词：两种调控因子在嗜热毁丝霉纤维素酶基因表达调控中的功能研究　出处：《深圳大学》2017年硕士论文　论文类型：学位论文

【摘要】：木质纤维素是植物细胞壁的主要成分之一,是地球上最为丰富的可再生碳源。利用纤维素酶降解木质纤维素,在食品工业、造纸工业、畜牧业以及生物乙醇的研发等方面均有大量的研究应用。如何获得大量、高效、热稳定性好的纤维素酶已成为当今科学界重要的研究课题。许多微生物所产生的纤维素酶都能有效的降解木质纤维素,目前工业上主要用木霉、青霉等中温真菌来生产纤维素酶。与中温真菌相比,嗜热真菌可耐高温环境,培养过程中不易受中温真菌污染,所产纤维素酶具有更好的热稳定性,可加快降解速度,有广阔的应用前景。嗜热毁丝霉(Myceliophthora thermophile ATCC42464)是一种丝状子囊真菌,研究表明该菌能产生热稳定性良好的纤维素酶类,具有水解生物质大部分多糖的能力,是潜在的高效中高温酶库。本研究利用RNA-Seq技术,筛选到嗜热毁丝霉中两个潜在的与纤维素酶表达调控相关的转录因子MHR1和MHR2,并分别运用RNAi和同源过表达技术对上述两因子进行了研究。本论文的主要内容如下:(1)本实验设计了mhr1基因的RNA干扰序列,利用pUC19-M质粒构建mhr1基因的沉默表达盒,经原生质体转化、潮霉素平板筛选、PCR鉴定和测序,成功获得五株转化子,再经RT-qPCR选出干扰率最高的一株转化子MtR5,其mhr1基因的表达量仅为原菌WT中表达量的1%。分别在诱导条件和非诱导条件下,同时培养转化子MtR5和原菌WT,测定其胞外蛋白浓度、纤维素酶酶活及主要纤维素酶基因表达量。发现在非诱导培养144 h时,转化子MtR5的胞外蛋白浓度、滤纸酶活、内切葡聚糖酶酶活和木聚糖酶酶活分别是原菌的1.94倍、1.52倍、1.47倍和1.20倍;经qPCR检测发现,此时转化子MtR5的主要纤维素酶基因中cbh1、egl3和xyr1比原菌中的表达量高出6-10倍。在诱导培养72 h时,转化子MtR5的胞外蛋白浓度和纤维素酶酶活均比原菌有所提高;qPCR检测结果表明,此时转化子MtR5的主要纤维素酶基因中cbh2、egl3和调控因子xyr1的表达量比原菌中高出28-56倍。研究表明,干扰mhr1基因的表达,可提高嗜热毁丝霉中部分纤维素酶基因的表达量,增强纤维素酶活性。从上述结果可知,该转录因子MHR1是作为阻遏因子发挥作用的。(2)本实验通过查询NCBI数据库获得MHR2的基因全长序列,构建其过表达载体,经原生质体转化、潮霉素平板筛选、PCR鉴定获得五株阳性过表达转化子,再经RT-qPCR选出过表达量最高的一株转化子MtO24,研究了mhr2基因过表达对嗜热毁丝霉胞外蛋白含量、纤维素酶活性及纤维素酶基因表达的影响。实验发现,在诱导培养72h时,转化子MtO24和原菌WT的胞外蛋白浓度和纤维素酶酶活均达到峰值,且转化子MtO24的胞外蛋白浓度是原菌WT的1.58倍,滤纸酶活与内切葡聚糖酶酶活分别为原菌的1.30和1.24倍。qPCR检测发现,诱导培养72 h时,转化子MtO24的几种主要纤维素酶基因的表达量均比原菌WT提高了3-9倍不等,这与纤维素酶活性的提高基本一致。本研究结果表明,MHR2是一种与嗜热毁丝霉纤维素酶基因的表达调控相关的转录因子,且该因子对纤维素酶基因的表达具有激活作用。
[Abstract]:Lignocellulose is one of the main components of plant cell wall, is renewable carbon rich earth. The utilization of lignocellulose degradation of cellulase, in the food industry, paper industry, animal husbandry and the research and application of bio ethanol and other aspects of development. There are a lot of how to get a lot of high efficiency, good thermal stability, cellulase has become an important the research topic in the field of science. Generated by many microorganisms can degrade lignocellulose effectively, the industry is currently mainly used Trichoderma, Penicillium and other mesophilic fungi to produce cellulase. Compared with the mesophilic fungi and thermophilic fungi resistant to high temperature environment, the training process is not affected by the temperature of fungal contamination, thermal stability cellulase has better, can accelerate the degradation rate, and has a wide application prospect. Myceliophthora thermophila (Myceliophthora thermophile ATCC42464) is a filamentous The research shows that the fungi, bacteria can produce cellulases has good thermal stability, ability of hydrolysis of biomass polysaccharides is most efficient in high temperature, enzyme base potential. In this study, the use of RNA-Seq technology, screened by Myceliophthora thermophila in two potential cellulase and expression regulation of transcription factors related to MHR1 and MHR2, and then use RNAi and homologous overexpression was investigated based on the two factors mentioned above. The main contents of this thesis are as follows: (1) the experimental design of RNA interference sequence of mhr1 gene, mhr1 gene was constructed by pUC19-M gene silencing expression cassette by protoplast transformation, hygromycin screening, identification and PCR sequencing, successfully obtained five transformants were selected by RT-qPCR, then interference transformants were the highest rate of MtR5, the expression of mhr1 gene expression is only original bacteria amount of WT 1%. in induced and non induced conditions, At the same time culturing a transformant MtR5 and original strain WT, the determination of protein concentration, the expression of cellulase activity and cellulase gene. Found in the non induced 144 h, the concentration of extracellular protein MtR5 transformants, filter paper enzyme activity, enzyme activity of endoglucanase and xylanase activity were 1.94 times the original, bacteria 1.52 times, 1.47 times and 1.20 times; the results of qPCR showed that the cbh1 mainly cellulase transformant MtR5 genes, egl3 and xyr1 is 6-10 times higher than the original strain. The amount of expression in the induction of 72 h transformants, MtR5 protein concentration and cellulose enzyme the activity was compared with the original strain increased; the results of qPCR showed that the main cbh2 cellulase transformant MtR5 gene, expression and regulation of egl3 factor xyr1 is 28-56 times higher than the original strain. The research shows that interfering the expression of mhr1 gene can improve the thermophilic cellulase destroyed part of silk in the mould The amount of gene expression, enhanced cellulase activity. It can be seen from the above results, the transcription factor MHR1 is play a role as a repressor. (2) obtained the full-length sequence of MHR2 gene in this experiment by querying the NCBI database, the construction of its expression vector, by protoplast transformation, hygromycin screening, identification of five strains of PCR the positive expression of transformants by RT-qPCR had the highest expression level of selected transformants were MtO24, the overexpression of mhr2 gene on Myceliophthora thermophila extracellular protein content, the effect of cellulase activity and cellulase gene expression. The experimental results showed that, in the induction of 72h, extracellular protein concentration and cellulase enzyme conversion MtO24 and the original strain WT activities reached peak, the concentration of extracellular proteins and the transformant MtO24 was 1.58 times higher than the original strain WT, and endoglucanase enzyme activity were 1.30 and 1.24 times of.QPCR detection of pathogenic bacteria of filter paper activity Found that induction of 72 h, the expression of several major cellulase genes transformed MtO24 were compared with the original strain WT was increased by 3-9 times. This increase and cellulase activity are basically the same. The results of this study show that MHR2 is a Myceliophthora thermophila cellulase gene expression regulation of related transcription factors activate the expression, and the factor of cellulase gene.

【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q55

【参考文献】