發(fā)布時(shí)間：2018-03-10 17:48

  本文選題：獨(dú)龍牛瘤胃　切入點(diǎn)：基因文庫　出處：《南方農(nóng)業(yè)學(xué)報(bào)》2017年05期 　論文類型：期刊論文

【摘要】：【目的】從分子生物學(xué)水平對獨(dú)龍牛的瘤胃纖維素酶基因資源進(jìn)行篩選及酶學(xué)特性研究,為后續(xù)開發(fā)利用新的纖維素酶提供參考依據(jù),也為揭示瘤胃微生物降解纖維素的作用機(jī)理打下基礎(chǔ)�！痉椒ā刻崛—�(dú)龍牛瘤胃微生物中的大片段基因組DNA,構(gòu)建瘤胃微生物基因組文庫,并進(jìn)行纖維素酶活性篩選,篩選獲得的高活性基因經(jīng)測序后進(jìn)行生物信息學(xué)分析與酶學(xué)性質(zhì)研究�！窘Y(jié)果】從獨(dú)龍牛瘤胃中共獲得20352個(gè)陽性克隆,白斑率達(dá)92%,構(gòu)建的瘤胃微生物基因組文庫容量899.6 Mb,空載率1.82%。從瘤胃微生物基因組文庫篩選獲得2個(gè)具有纖維素酶活性的陽性克隆(B1和B2),其中,B1基因序列長1230 bp,編碼409個(gè)氨基酸,基因編碼產(chǎn)物與來自Ruminococcus albus纖維素酶基因編碼產(chǎn)物(β-1,4-內(nèi)切葡聚糖酶,Gen Bank登錄號P23661.1)的覆蓋率高達(dá)99%,其同源性高達(dá)97%;B2基因序列長1002bp,編碼333個(gè)氨基酸,基因編碼產(chǎn)物與Uncultured microorganism纖維素酶基因編碼產(chǎn)物(纖維糊精酶,Gen Bank登錄號ADB80112.1)的覆蓋率高達(dá)99%,其同源性為83%。B1和B2基因可在Rosetta原核表達(dá)宿主菌中成功誘導(dǎo)表達(dá),B1纖維素酶的最適pH為6.0,最適溫度40℃;B2纖維素酶的最適pH為6.0,最適溫度40~50℃。【結(jié)論】從構(gòu)建的獨(dú)龍牛瘤胃微生物基因文庫中篩選獲得2株具有較高活力的纖維素酶(B1和B2),其中,B1為β-1,4-內(nèi)切葡聚糖酶,而B2為新的纖維糊精酶,可為纖維素的體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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