里氏木霉中milRNA的表达干预及其对纤维素酶表达的调控研究

发布时间：2018-06-18 10:38

  本文选题：里氏木霉 + milRNA　； 参考：《深圳大学》2017年硕士论文

【摘要】：里氏木霉(Trichoderma reesei)是重要的纤维素酶产生菌,也是用于研究丝状真菌纤维素酶合成调控机制的常用菌株,其中的纤维素酶合成调控机理已获得了较充分的认识。Micro RNA(miRNA)广泛存在于各种生物中,能够与靶mRNA特异性结合对基因表达进行调控。近年来,与miRNA结构相似的小分子RNA(micro RNA-like RNA,milRNA),在多种真菌中通过高通量测序分析后被鉴定,如粗糙脉胞菌、核盘菌、黑僵菌以及里氏木酶等。然而,关于里氏木酶miRNA或milRNAs研究的报道较少。本研究以课题组前期对里氏木酶高通量测序得到的13个milRNAs为基础,根据milRNAs在诱导和非诱导条件下的表达水平,选取了4个里氏木酶QM9414的milRN A进行功能研究。根据已获得的milRNA序列信息,设计milRNA的Tough Decoy(TuD)序列以及PCR扩增过表达序列,进行PCR鉴定及DNA测序比对分析;并利用里氏木酶强组成型启动子Ppdc构建milRNA抑制表达载体和过表达载体,将载体转入里氏木酶进行原生质体转化后表达。筛选构建成功的重组菌培养于纤维素酶诱导培养基,利用荧光定量PCR检测milRNA、酶基因cbh1、egl1的表达水平,测定滤纸酶活(FPA)以及羧甲基纤维素酶活(CMCA),探讨milRNA是否调控纤维素酶的表达,分析milRNA与纤维素酶表达调控的相关功能关系。再将调控效果明显的milRNA重组菌进行培养基碳氮源组分的优化培养,测定纤维素酶调控因子cre1、xyr1的表达量以及滤纸酶活,进一步提高重组菌株的纤维素酶活。研究结果表明,milR4、milR9参与了纤维素酶的表达调控。荧光定量PCR数据显示,抑制表达的菌株milRNA表达水平降低,说明TuD序列发挥了抑制作用;过表达菌株milRNA表达水平提高,说明成功扩增了milRNA的前体序列。酶活测定结果显示,各个菌株在诱导培养120 h后达到最高值。其中,过表达菌株P-milR4酶活是原始菌株的0.919倍,并有下降的趋势;抑制表达菌株TuD4酶活有一定提高,是原始菌株的1.228倍,milR4参与了纤维素酶表达的正调控。抑制表达菌株TuD9酶活是原始菌株的0.946倍,过表达菌株P-milR9酶活提高明显,是原始菌株的1.349倍,milR9参与了纤维素酶表达的负调控。对纤维素酶产酶培养基进行优化后,得到一株产酶量相对较高的重组菌P-milR9,其酶活为原始菌株的1.454倍。本工作通过构建TuD抑制表达载体和过表达载体得到的小分子RNA:milR4和milR9能显著改变T.reesei纤维素酶酶活;并通过优化培养基进一步提高了重组菌株P-milR9的纤维素酶活。本工作为研究里氏木霉中的小分子RNA调控机制提供了新认识,并为进一步提高T.reesei纤维素酶酶活提供新的方法。
[Abstract]:Trichoderma reeseii (Trichoderma reeseii) is an important cellulase-producing strain and a common strain used to study the mechanism of cellulase synthesis in filamentous fungi. The regulation mechanism of cellulase synthesis has been fully recognized. Micro RNA-miRNAs exist widely in various organisms and can specifically bind to target mRNA to regulate gene expression. In recent years, small RNA-microRNA-like RNA-milRNAs, similar to miRNA structures, have been identified by high-throughput sequencing analysis in a variety of fungi, such as C. crassica, Sclerotinia sclerotiorum, Metarhizium anisopliae, and Lymphozyme. However, there are few reports on the study of Ribsbeck enzyme miRNA or milRNAs. On the basis of 13 milRNAs obtained by high-throughput sequencing, four milRNAs of QM9414 were selected for functional study according to the expression levels of milRNAs in induced and non-induced conditions. According to the obtained milRNA sequence information, we designed the Tough Decoyo TuD sequence of milRNA and amplified the overexpression sequence of milRNA, identified by PCR and compared with DNA sequencing, and constructed the expression vector and overexpression vector of milRNA by using Ppdc, a strong promoter of the enzyme. The vector was transferred to Lycrase for protoplast transformation and expressed. The recombinant bacteria were screened and cultured in cellulase induction medium. The expression levels of milRNAs, cbh1egl1 gene, FPAA and CMCAA were detected by fluorescence quantitative PCR, and the effect of milRNA on the expression of cellulase was investigated. To analyze the functional relationship between milRNA and cellulase expression regulation. The recombinant milRNA strain with obvious regulation effect was cultured to optimize the composition of carbon and nitrogen source in culture medium. The expression of cellulase regulation factor cre1xyr1 and the activity of filter paper enzyme were determined, and the cellulase activity of the recombinant strain was further improved. The results showed that Mil R4 Mil R9 was involved in the regulation of cellulase expression. Fluorescence quantitative PCR data showed that the expression level of milRNA decreased, indicating that TuD sequence played an inhibitory role, and the overexpression strain milRNA expression level increased, which indicated that the precursor sequence of milRNA was successfully amplified. The results of enzyme activity test showed that each strain reached the highest value after 120 hours of induction and culture. The enzyme activity of the overexpression strain P-milR4 was 0.919 times of that of the original strain, and the enzyme activity of the inhibiting strain TuD4 was increased to some extent, which was 1.228 times of the original strain, which was involved in the positive regulation of cellulase expression. The enzyme activity of inhibiting expression strain TuD9 was 0.946 times of that of original strain, and the enzyme activity of over-expression strain P-milR9 was obviously increased. It was 1.349 times of original strain that MilR9 participated in the negative regulation of cellulase expression. After optimization of cellulase production medium, a recombinant strain P-milR9 with relatively high enzyme production was obtained, and its enzyme activity was 1.454 times of that of the original strain. In this work, the small molecule RNAmilR4 and milR9 obtained by constructing TuD inhibitory expression vector and over-expressing vector could significantly change the activity of T. reesei cellulase, and the cellulase activity of the recombinant strain P-milR9 was further improved by optimizing the medium. This work provides a new understanding of the regulation mechanism of small RNA in Trichoderma leuci and provides a new method for further enhancing the activity of T. reesei cellulase.
【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】：Q55;Q78

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