本文選題：果膠酶基因　切入點：纖維素酶基因　出處：《華南農(nóng)業(yè)大學》2016年碩士論文　論文類型：學位論文

【摘要】：非淀粉多糖(Non-starch polysaccharides,NSP)是廣泛存在于植物細胞壁中的抗營養(yǎng)因子,包括纖維素、半纖維素(阿拉伯木聚糖、β-葡聚糖、甘露聚糖等)、果膠等物質(zhì)。豬、雞等單胃動物的胃和小腸中缺乏降解這類物質(zhì)的酶,無法充分吸收細胞內(nèi)的營養(yǎng)物質(zhì),不僅造成了飼料的浪費,還造成了環(huán)境污染。飼料企業(yè)往往通過添加NSP酶制劑的方式來緩解或消除NSP產(chǎn)生的抗營養(yǎng)作用,而酶制劑在飼料制粒、貯藏等過程中易失活,影響了其實際利用價值。此外,在飼料中添加NSP酶制劑還增加了飼料成本。轉(zhuǎn)基因技術(shù)為上述問題的解決提供了新的思路。本研究從微生物和低等真核生物中篩選了大量NSP酶基因,通過豬密碼子優(yōu)化及信號肽替換后克隆到pCDNA3.1(+)真核表達載體上,瞬時轉(zhuǎn)染豬PK15細胞后通過收集細胞分泌上清分析酶學性質(zhì)(最適pH、p H穩(wěn)定性、胃/胰蛋白酶耐受性),以篩選可在豬細胞中高效表達的NSP酶基因。經(jīng)基因連接方式的篩選和優(yōu)化及體外細胞表達驗證,構(gòu)建了一條多個NSP酶基因共表達的多順反子,并構(gòu)建了一個唾液腺特異共表達多個NSP酶的載體,為新型環(huán)保轉(zhuǎn)基因豬的制備奠定基礎(chǔ),對我國養(yǎng)豬業(yè)的可持續(xù)發(fā)展具有重要意義。本研究主要結(jié)果如下:(1)通過對微生物和低等真核生物來源的多個纖維素酶基因的表達活性及酶學性質(zhì)分析,篩選到兩個可在豬細胞中高效表達出具有較高活性纖維素酶的基因:egII和TeEGI,二者兼具葡聚糖酶活性。在PK15細胞中,以上兩個基因表達的纖維素酶和葡聚糖酶活性分別為0.20 U/mL(egII)、0.30 U/mL(TeEGI)(CMC-Na,pH4.00)和0.61 U/mL(egII)、0.66 U/mL(Te EGI)(β-葡聚糖,pH 4.62)。(2)通過對微生物源的三個果膠酶基因的表達活性及酶學性質(zhì)分析,篩選到兩個可在豬細胞中高效表達出具有較高活性果膠酶的基因:PG7fn和PG。二者在PK15細胞中表達的果膠酶最高酶活分別為1.15 U/mL(多聚半乳糖醛酸、pH4.0)和1 U/mL(多聚半乳糖醛酸、pH5.50)。(3)對比分析了三種微生物源木聚糖酶基因(asp-xyn、Xynl11、Penxyl)的表達活性及酶學性質(zhì),篩選到可在豬細胞中高效表達木聚糖酶的基因:asp-xyn。以上三種木聚糖酶基因表達的最高酶活力分別為:1.88 U/mL、1.65 U/mL、0.72 U/mL(木聚糖,pH5.0)。(4)通過基因重組技術(shù),構(gòu)建了四條共表達木聚糖酶和纖維素酶的雙順反子:xyn-flag-egII-T2A,xyn-flag-egII,xyn-egII-T2A,xyn-egII。其中xyn-egII融合效果最好,其木聚糖酶活性為asp-xyn表達活性的57.35%(pH 5.00);β-葡聚糖酶活性egII表達活性的46.90%(pH 4.50);CMC的活性為egII表達活性的67.97%(pH 6.00)。(5)成功構(gòu)建pCD-EsAPPA-T2A和pCD-TeEGI-T2A表達載體,其在PK15細胞中表達的酶活性低于單基因表達活性。(6)優(yōu)化多基因共表達的連接方案,確定了四個基因(pg7fn、asp-xyn、EsAPPA、TeEGI)的最佳連接方案,成功構(gòu)建了PG7fn-asp-xyn-EsAPPA-TeEGI多順反子,在PK15細胞中成功共表達果膠酶-木聚糖酶-植酸酶-纖維素酶。(7)成功構(gòu)建唾液腺特異表達四種基因(pg7fn、asp-xyn、EsAPPA、TeEGI)的轉(zhuǎn)基因載體pPB-mPSP-PXAT-neoGFP。
[Abstract]:Non starch polysaccharides (Non-starch, polysaccharides, NSP) is the anti nutritional factors, widely exists in the cell wall of plant including cellulose, hemicellulose (Arabia xylan, beta glucan, mannan, pectin and so on). The lack of this kind of material degradation of pig, chicken and other enzymes in monogastric animal stomach and small intestine, unable to fully absorb the nutrients in the cell, not only cause feed waste, but also caused environmental pollution. Feed enterprises often by adding NSP enzyme to alleviate or eliminate the anti nutritional effects of NSP, and enzyme preparation in feed granulating, storage in the process of deactivation, its actual influence use value. In addition, adding NSP enzyme also increased the cost of feed in the feed. The transgenic technology provides a new way to solve the above problems. This study selected a large number of NSP genes from bacteria and lower eukaryotes,. Pig codon optimization and signal peptide replacement was cloned into pCDNA3.1 (+) eukaryotic expression vector, transfection of porcine PK15 cells after the cells were collected through analysis of the enzymatic properties of secretion supernatant (optimum pH, P stability H, gastric / trypsin tolerance), NSP gene to screen highly expressed in pig cells the connection way screening and optimization and in vitro expression verified by gene, a co expression of multiple NSP gene polycistron construct, and the construction of the carrier of multiple NSP enzyme co expression of a salivary gland specific, as the new environmental protection lay the foundation for the preparation of transgenic pigs, has important the meaning of sustainable development of pig industry in China. The main results of this study are as follows: (1) the expression and characterization of a cellulase gene on microorganisms and lower eukaryotes source analysis, identified two highly expressed in pig cells in issue High activity cellulase genes: egII and TeEGI, two. Both endoglucanase activity in PK15 cells, the expression of more than two cellulase genes and glucanase activity were 0.20 U/mL (egII), 0.30 U/mL (TeEGI) (CMC-Na, pH4.00) and 0.61 U/mL (egII), 0.66 U/mL (Te EGI (pH) beta glucan, 4.62). (2) the expression and characterization of three pectinase gene on microbial source analysis, screened two in pig cells highly expressed with high activity of pectinase genes: PG7fn and PG. two expression in PK15 cells the highest enzyme activity of pectinase were 1.15 U/mL (poly galacturonic acid, pH4.0) and 1 U/mL (poly galacturonic acid, pH5.50). (3) compared the three kinds of microbial xylanase gene (asp-xyn, Xynl11, Penxyl) expression and enzymatic activity properties, screened high expression of xylan in pig cells in Gene: the highest enzyme activity asp-xyn. more than three kinds of xylanase gene expression were 1.88 U/mL, 1.65 U/mL, 0.72 U/mL (xylan, pH5.0). (4) by gene recombination technology, constructed four co expression of xylanase and cellulase bicistron: xyn-flag-egII-T2A, xyn-flag-egII. Xyn-egII-T2A xyn-egII. xyn-egII, the fusion effect is the best, the xylanase activity was the expression of asp-xyn 57.35% (pH 5); beta glucan enzyme activity expression of egII 46.90% (pH 4.50); the activity of CMC and expression of egII activity by 67.97% (pH 6). (5) pCD-EsAPPA-T2A and pCD-TeEGI-T2A expression vector successfully construction activity of its expression in PK15 cells than single gene expression. (6) the connection scheme optimization of co expression of multiple genes, identified four genes (pg7fn, asp-xyn, EsAPPA, TeEGI) the best connection scheme, the successful construction of PG7fn-asp-xyn-E SAPPA-TeEGI polycis trans son, CO expressed pectinase xylanase phytase cellulase in PK15 cells. (7) successfully constructed salivary gland specific expression vectors of four genes (pg7fn, asp-xyn, EsAPPA, TeEGI).

【學位授予單位】：華南農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S816;S828

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