独龙牛瘤胃细菌纤维素酶基因克隆

发布时间：2018-03-10 17:48

  本文选题：独龙牛瘤胃　切入点：基因文库　出处：《南方农业学报》2017年05期 　论文类型：期刊论文

【摘要】：【目的】从分子生物学水平对独龙牛的瘤胃纤维素酶基因资源进行筛选及酶学特性研究,为后续开发利用新的纤维素酶提供参考依据,也为揭示瘤胃微生物降解纤维素的作用机理打下基础。【方法】提取独龙牛瘤胃微生物中的大片段基因组DNA,构建瘤胃微生物基因组文库,并进行纤维素酶活性筛选,筛选获得的高活性基因经测序后进行生物信息学分析与酶学性质研究。【结果】从独龙牛瘤胃中共获得20352个阳性克隆,白斑率达92%,构建的瘤胃微生物基因组文库容量899.6 Mb,空载率1.82%。从瘤胃微生物基因组文库筛选获得2个具有纤维素酶活性的阳性克隆(B1和B2),其中,B1基因序列长1230 bp,编码409个氨基酸,基因编码产物与来自Ruminococcus albus纤维素酶基因编码产物(β-1,4-内切葡聚糖酶,Gen Bank登录号P23661.1)的覆盖率高达99%,其同源性高达97%;B2基因序列长1002bp,编码333个氨基酸,基因编码产物与Uncultured microorganism纤维素酶基因编码产物(纤维糊精酶,Gen Bank登录号ADB80112.1)的覆盖率高达99%,其同源性为83%。B1和B2基因可在Rosetta原核表达宿主菌中成功诱导表达,B1纤维素酶的最适pH为6.0,最适温度40℃;B2纤维素酶的最适pH为6.0,最适温度40~50℃。【结论】从构建的独龙牛瘤胃微生物基因文库中筛选获得2株具有较高活力的纤维素酶(B1和B2),其中,B1为β-1,4-内切葡聚糖酶,而B2为新的纤维糊精酶,可为纤维素的体外降解提供新型材料。
[Abstract]:[objective] to screen the rumen cellulase gene resources and study the characteristics of cellulase from Dulong cattle at molecular biological level, so as to provide a reference for the further development and utilization of new cellulase. It also laid a foundation for revealing the mechanism of degradation of cellulose by rumen microbes. [methods] A large fragment of genomic DNA was extracted from rumen microorganisms of Dulong cattle, a genomic library of rumen microorganisms was constructed, and cellulase activity was screened. The highly active genes were sequenced and analyzed by bioinformatics and enzymatic properties. [results] A total of 20352 positive clones were obtained from the rumen of Dulong cattle. The rumen microbial genomic library was constructed with a capacity of 899.6 Mb and a no-load rate of 1.82.Two positive clones with cellulase activity were obtained from the rumen microbial genomic library, in which the sequence of B 1 gene was 1230 BP, encoding 409 amino acids. The coverage of the gene encoding product from Ruminococcus albus cellulase gene (尾 -1O4-endoglucanase genase accession number P23661.1) is as high as 99.1%, and the homology of the gene sequence is as high as 97bp1, encoding 333 amino acids, the length of which is 1002bp. The gene encoding product and the Uncultured microorganism cellulase gene encoding product (cellulose dextrinase GenGen Bank accession number ADB80112.1) have a high coverage of 99%. The homology is 83.B1 and B2 genes can be successfully induced to express B1-cellulase in Rosetta prokaryotic expression host bacteria. The optimum pH was 6.0, the optimum pH of cellulase was 6.0 at 40 鈩，

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