黄淮白山羊瘤胃微生物Fosmid文库的构建与分析

Construction and analysis of Fosmid library of rumen microbiota of Huanghuai white goat

采用CTAB法和蛋白酶K法提取黄淮白山羊瘤胃微生物总DNA,酶切获得DNA片段,大小为23～50 kb,以E.coli EPI300为宿主细胞,构建瘤胃微生物宏基因Fosmid文库。研究发现该文库共获得克隆28 800个,插入片段大小约35 kb,空载率小于2%,容量1 008 Mb。通过羧甲基纤维素酶和木聚糖酶(Xylanase)活性筛选,分别获得具有两种酶活性的阳性克隆60和32个,木聚糖酶和羧甲基纤维素酶(CMC,Carboxymethyl cellulase)共同作用阳性克隆15个。通过已测序MF3木聚糖酶阳性克隆酶学活性分析,该木聚糖酶在最适应pH 4.5, 40℃条件下,酶活力为79.287μ·mL^(-1)。为进一步研究黄淮白山羊瘤胃内纤维素酶(Cellulase)基因奠定基础。

A Fosmid library of uncultured microorganism from Huanghai white goat rumen was constructed to explore the potential of the ruminants'gut ecosystem.The high molecular weight DNA was extracted an purified from rumen contents of Huanghai white goat by CTAB method and Proteinase K treatment.Restriction analysis by XhoⅠand BamHⅠrevealed a high level of diversity of the cloned DNA fragments.The insert size of clone ranged from 23 to 50 kb,and transformed into E.coli EPI300.A total of 28 800 clones were acquired from the Fosmid library,with the average insert size of 35 kb,the capacity of this Fosmid library was 1008 Mb,and the empty rate was lower than 2%.The screening of the partial metagenomic library identified 32 clones with xylanase activity,60 clones with carboxymethyl cellulase,and 15 clones with xylanase activity and carboxymethyl cellulase.The xylanase activity of clone MF3(which was sequenced)was 79.287 μ·mL^-1 under the conditions of pH 4.5 and 40 ℃.These results provided the foundation to further research on cellulase gene in Huanghai white goat rumen.