非淀粉多糖酶基因筛选及其与植酸酶基因共表达载体构建

发布时间：2018-03-13 15:34

本文选题：果胶酶基因　切入点：纤维素酶基因　出处：《华南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：非淀粉多糖(Non-starch polysaccharides,NSP)是广泛存在于植物细胞壁中的抗营养因子,包括纤维素、半纤维素(阿拉伯木聚糖、β-葡聚糖、甘露聚糖等)、果胶等物质。猪、鸡等单胃动物的胃和小肠中缺乏降解这类物质的酶,无法充分吸收细胞内的营养物质,不仅造成了饲料的浪费,还造成了环境污染。饲料企业往往通过添加NSP酶制剂的方式来缓解或消除NSP产生的抗营养作用,而酶制剂在饲料制粒、贮藏等过程中易失活,影响了其实际利用价值。此外,在饲料中添加NSP酶制剂还增加了饲料成本。转基因技术为上述问题的解决提供了新的思路。本研究从微生物和低等真核生物中筛选了大量NSP酶基因,通过猪密码子优化及信号肽替换后克隆到pCDNA3.1(+)真核表达载体上,瞬时转染猪PK15细胞后通过收集细胞分泌上清分析酶学性质(最适pH、p H稳定性、胃/胰蛋白酶耐受性),以筛选可在猪细胞中高效表达的NSP酶基因。经基因连接方式的筛选和优化及体外细胞表达验证,构建了一条多个NSP酶基因共表达的多顺反子,并构建了一个唾液腺特异共表达多个NSP酶的载体,为新型环保转基因猪的制备奠定基础,对我国养猪业的可持续发展具有重要意义。本研究主要结果如下:(1)通过对微生物和低等真核生物来源的多个纤维素酶基因的表达活性及酶学性质分析,筛选到两个可在猪细胞中高效表达出具有较高活性纤维素酶的基因:egII和TeEGI,二者兼具葡聚糖酶活性。在PK15细胞中,以上两个基因表达的纤维素酶和葡聚糖酶活性分别为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)通过对微生物源的三个果胶酶基因的表达活性及酶学性质分析,筛选到两个可在猪细胞中高效表达出具有较高活性果胶酶的基因:PG7fn和PG。二者在PK15细胞中表达的果胶酶最高酶活分别为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)对比分析了三种微生物源木聚糖酶基因(asp-xyn、Xynl11、Penxyl)的表达活性及酶学性质,筛选到可在猪细胞中高效表达木聚糖酶的基因:asp-xyn。以上三种木聚糖酶基因表达的最高酶活力分别为:1.88 U/mL、1.65 U/mL、0.72 U/mL(木聚糖,pH5.0)。(4)通过基因重组技术,构建了四条共表达木聚糖酶和纤维素酶的双顺反子:xyn-flag-egII-T2A,xyn-flag-egII,xyn-egII-T2A,xyn-egII。其中xyn-egII融合效果最好,其木聚糖酶活性为asp-xyn表达活性的57.35%(pH 5.00);β-葡聚糖酶活性egII表达活性的46.90%(pH 4.50);CMC的活性为egII表达活性的67.97%(pH 6.00)。(5)成功构建pCD-EsAPPA-T2A和pCD-TeEGI-T2A表达载体,其在PK15细胞中表达的酶活性低于单基因表达活性。(6)优化多基因共表达的连接方案,确定了四个基因(pg7fn、asp-xyn、EsAPPA、TeEGI)的最佳连接方案,成功构建了PG7fn-asp-xyn-EsAPPA-TeEGI多顺反子,在PK15细胞中成功共表达果胶酶-木聚糖酶-植酸酶-纤维素酶。(7)成功构建唾液腺特异表达四种基因(pg7fn、asp-xyn、EsAPPA、TeEGI)的转基因载体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).

【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S816;S828

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