本文選題：里氏木霉 + milRNA�。� 參考：《深圳大學(xué)》2017年碩士論文

【摘要】：里氏木霉(Trichoderma reesei)是重要的纖維素酶產(chǎn)生菌,也是用于研究絲狀真菌纖維素酶合成調(diào)控機(jī)制的常用菌株,其中的纖維素酶合成調(diào)控機(jī)理已獲得了較充分的認(rèn)識。Micro RNA(miRNA)廣泛存在于各種生物中,能夠與靶mRNA特異性結(jié)合對基因表達(dá)進(jìn)行調(diào)控。近年來,與miRNA結(jié)構(gòu)相似的小分子RNA(micro RNA-like RNA,milRNA),在多種真菌中通過高通量測序分析后被鑒定,如粗糙脈胞菌、核盤菌、黑僵菌以及里氏木酶等。然而,關(guān)于里氏木酶miRNA或milRNAs研究的報(bào)道較少。本研究以課題組前期對里氏木酶高通量測序得到的13個(gè)milRNAs為基礎(chǔ),根據(jù)milRNAs在誘導(dǎo)和非誘導(dǎo)條件下的表達(dá)水平,選取了4個(gè)里氏木酶QM9414的milRN A進(jìn)行功能研究。根據(jù)已獲得的milRNA序列信息,設(shè)計(jì)milRNA的Tough Decoy(TuD)序列以及PCR擴(kuò)增過表達(dá)序列,進(jìn)行PCR鑒定及DNA測序比對分析;并利用里氏木酶強(qiáng)組成型啟動(dòng)子Ppdc構(gòu)建milRNA抑制表達(dá)載體和過表達(dá)載體,將載體轉(zhuǎn)入里氏木酶進(jìn)行原生質(zhì)體轉(zhuǎn)化后表達(dá)。篩選構(gòu)建成功的重組菌培養(yǎng)于纖維素酶誘導(dǎo)培養(yǎng)基,利用熒光定量PCR檢測milRNA、酶基因cbh1、egl1的表達(dá)水平,測定濾紙酶活(FPA)以及羧甲基纖維素酶活(CMCA),探討milRNA是否調(diào)控纖維素酶的表達(dá),分析milRNA與纖維素酶表達(dá)調(diào)控的相關(guān)功能關(guān)系。再將調(diào)控效果明顯的milRNA重組菌進(jìn)行培養(yǎng)基碳氮源組分的優(yōu)化培養(yǎng),測定纖維素酶調(diào)控因子cre1、xyr1的表達(dá)量以及濾紙酶活,進(jìn)一步提高重組菌株的纖維素酶活。研究結(jié)果表明,milR4、milR9參與了纖維素酶的表達(dá)調(diào)控。熒光定量PCR數(shù)據(jù)顯示,抑制表達(dá)的菌株milRNA表達(dá)水平降低,說明TuD序列發(fā)揮了抑制作用;過表達(dá)菌株milRNA表達(dá)水平提高,說明成功擴(kuò)增了milRNA的前體序列。酶活測定結(jié)果顯示,各個(gè)菌株在誘導(dǎo)培養(yǎng)120 h后達(dá)到最高值。其中,過表達(dá)菌株P(guān)-milR4酶活是原始菌株的0.919倍,并有下降的趨勢;抑制表達(dá)菌株TuD4酶活有一定提高,是原始菌株的1.228倍,milR4參與了纖維素酶表達(dá)的正調(diào)控。抑制表達(dá)菌株TuD9酶活是原始菌株的0.946倍,過表達(dá)菌株P(guān)-milR9酶活提高明顯,是原始菌株的1.349倍,milR9參與了纖維素酶表達(dá)的負(fù)調(diào)控。對纖維素酶產(chǎn)酶培養(yǎng)基進(jìn)行優(yōu)化后,得到一株產(chǎn)酶量相對較高的重組菌P-milR9,其酶活為原始菌株的1.454倍。本工作通過構(gòu)建TuD抑制表達(dá)載體和過表達(dá)載體得到的小分子RNA:milR4和milR9能顯著改變T.reesei纖維素酶酶活;并通過優(yōu)化培養(yǎng)基進(jìn)一步提高了重組菌株P(guān)-milR9的纖維素酶活。本工作為研究里氏木霉中的小分子RNA調(diào)控機(jī)制提供了新認(rèn)識,并為進(jìn)一步提高T.reesei纖維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.
【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q55;Q78

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 彭可可;歐陽伶宣;鄧朝謙;蔣元;徐素萍;李曉寧;韋顯凱;羅廷榮;;MicroRNA研究概述[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2016年04期

2 黃俊駿;王華華;梁衛(wèi)紅;;非編碼小RNA的多樣性[J];生物技術(shù)通報(bào);2014年11期

3 王方忠;蔣藝;劉奎美;姜寶杰;王明鈺;方詡;;絲狀真菌中纖維素酶與半纖維素酶的合成調(diào)控[J];生物加工過程;2014年01期

4 辛琪;徐金濤;汪天虹;劉巍峰;陳冠軍;;絲狀真菌紅褐肉座菌(Hypocrea jecorina)纖維素酶基因的轉(zhuǎn)錄調(diào)控研究進(jìn)展[J];微生物學(xué)報(bào);2010年11期

5 吳丹,仇華吉,童光志;幾種表達(dá)系統(tǒng)的比較[J];生物技術(shù)通報(bào);2002年02期

，

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