黄淮白山羊瘤胃微生物Fosmid文库的构建与分析
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摘要
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 Xholand BamHI revealed 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 1 008 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.
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 Xholand BamHI revealed 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 1 008 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.
引文
[1]朱雅新,王加启,马润林,等.荷斯坦奶牛瘤胃微生物元基因组BAC文库的构建与分析[J].微生物学报,2007,47(2):213-216.
[2]Liu K,Wang J,Bu D,et al.Isolation and biochemical characterization of two lipases from a metagenomic library of China Holstein cow rumen[J].Biochemical and Biophysical Research Communications,2009,385:605-611.
[3]Shedova E N,Berezina O V,Lunina N A,et al.Cloning and characterisation of a large metagenomic DNA fragment containing glycosyl-hydrolase genes[J].Molecular Genetics,Microbiology and Virology,2009,24:12-16.
[4]郭鸿,封毅,莫新春,等.水牛瘤胃宏基因组的一个新的β-葡萄糖苷酶基因umcel3G的克隆、表达及其表达产物的酶学特性[J].生物工程学报,2008,24(2):232-238.
[5]Duan C J,Xian L,Zhao G C,et al.Isolation and partial characterization of novel genes encoding acidic cellulases from metagenomes of buffalo rumens[J].Journal of Applied Microbiology,2009,107:245-256.
[6]王佳堃,安培培,陈振明,等.湖羊瘤胃微生物Fosmid文库的构建和分析[J].动物营养学报,2010,22(2):341-345.
[7]Brulc J M,Antonopoulos D A,Miller M E B,et al.Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106:1948-1953.
[8]李秀婷,孙宝国,宋焕禄,等.一株高产木聚糖酶的枝链霉菌的分离鉴定及产酶[J].微生物学通报,2010,37(6):791-797.
[9]Morrison M,Adams S E,Nelson K E,et al.Metagenomic analysis of the microbiomes in ruminants and other herbivores[M].Dordrecht:Springer Netherlands,2005.
[10]王佳堃,安培培,刘建新.宏基因组学用于瘤胃微生物代谢的研究进展[J].动物营养学报,2010,22(3):527-535.
[11]冯国栋,许艳艳,吕丹青,等.一个新型湖羊瘤胃木聚糖酶基因的克隆和鉴定[J].科技通报,2010,26(3):345-357.
[12]Rees H C,Grant W D,Jones B E,et al.Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods[J].Extremophiles,2004,8:63-71.
[13]吴锡川,杨舒黎,苟潇,等.宏基因组学及其在瘤胃微生物中的应用研究进展[J].中国畜牧兽医,2011,38(12):106-110.
[14]Lee D G,Jeon J H,Jang M K,et al.Screening and characterization of a novel fibrinolytic metallprotease from a metagenomic library[J].Biotechnol Lett,2007,29(3):465-472.
[15]Béjào.To BAC or not to BAC:Marine ecogenomics[J].Current Opinion in Biotechnology,2004,15(3):187-190.
[16]Craig W M,Broderick G A,Ricker D B.Quantitation of microorganisms associated with the particulate phase of ruminal ingesta[J].Journal of Nutrition,1987,117:56-64.
[17]Forsberg C W,Lam R.Use of ade nosine-5-triphosphate as an indicator of the microbial biomass in rumen contents[J].Applied and Environmental Microbiology,1977,33:528-534.
[18]Williams A G,Strachan N H.Polysaccharide degrading enzymes in microbial populations from the liquid and solid fr actions of bovine rumen digesta[J].Canadian Journal of Animal Science,1984,64:58-59.
[19]李碧凤,朱雅新,苟潇,等.云南大额牛瘤胃宏基因组Fosmid文库的构建与分析[J].中国畜牧兽医,2013,40(12):61-65.
[20]林小洪,叶秀云,王国增,等.产木聚糖酶菌株的筛选、鉴定及酶学性质研究[J].中国食品学报,2016(1):115-122.
[21]郝涤非.木聚糖酶活性测定的实践与研究[J].中国酿造,2009(11):149-153.
[2]Liu K,Wang J,Bu D,et al.Isolation and biochemical characterization of two lipases from a metagenomic library of China Holstein cow rumen[J].Biochemical and Biophysical Research Communications,2009,385:605-611.
[3]Shedova E N,Berezina O V,Lunina N A,et al.Cloning and characterisation of a large metagenomic DNA fragment containing glycosyl-hydrolase genes[J].Molecular Genetics,Microbiology and Virology,2009,24:12-16.
[4]郭鸿,封毅,莫新春,等.水牛瘤胃宏基因组的一个新的β-葡萄糖苷酶基因umcel3G的克隆、表达及其表达产物的酶学特性[J].生物工程学报,2008,24(2):232-238.
[5]Duan C J,Xian L,Zhao G C,et al.Isolation and partial characterization of novel genes encoding acidic cellulases from metagenomes of buffalo rumens[J].Journal of Applied Microbiology,2009,107:245-256.
[6]王佳堃,安培培,陈振明,等.湖羊瘤胃微生物Fosmid文库的构建和分析[J].动物营养学报,2010,22(2):341-345.
[7]Brulc J M,Antonopoulos D A,Miller M E B,et al.Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106:1948-1953.
[8]李秀婷,孙宝国,宋焕禄,等.一株高产木聚糖酶的枝链霉菌的分离鉴定及产酶[J].微生物学通报,2010,37(6):791-797.
[9]Morrison M,Adams S E,Nelson K E,et al.Metagenomic analysis of the microbiomes in ruminants and other herbivores[M].Dordrecht:Springer Netherlands,2005.
[10]王佳堃,安培培,刘建新.宏基因组学用于瘤胃微生物代谢的研究进展[J].动物营养学报,2010,22(3):527-535.
[11]冯国栋,许艳艳,吕丹青,等.一个新型湖羊瘤胃木聚糖酶基因的克隆和鉴定[J].科技通报,2010,26(3):345-357.
[12]Rees H C,Grant W D,Jones B E,et al.Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods[J].Extremophiles,2004,8:63-71.
[13]吴锡川,杨舒黎,苟潇,等.宏基因组学及其在瘤胃微生物中的应用研究进展[J].中国畜牧兽医,2011,38(12):106-110.
[14]Lee D G,Jeon J H,Jang M K,et al.Screening and characterization of a novel fibrinolytic metallprotease from a metagenomic library[J].Biotechnol Lett,2007,29(3):465-472.
[15]Béjào.To BAC or not to BAC:Marine ecogenomics[J].Current Opinion in Biotechnology,2004,15(3):187-190.
[16]Craig W M,Broderick G A,Ricker D B.Quantitation of microorganisms associated with the particulate phase of ruminal ingesta[J].Journal of Nutrition,1987,117:56-64.
[17]Forsberg C W,Lam R.Use of ade nosine-5-triphosphate as an indicator of the microbial biomass in rumen contents[J].Applied and Environmental Microbiology,1977,33:528-534.
[18]Williams A G,Strachan N H.Polysaccharide degrading enzymes in microbial populations from the liquid and solid fr actions of bovine rumen digesta[J].Canadian Journal of Animal Science,1984,64:58-59.
[19]李碧凤,朱雅新,苟潇,等.云南大额牛瘤胃宏基因组Fosmid文库的构建与分析[J].中国畜牧兽医,2013,40(12):61-65.
[20]林小洪,叶秀云,王国增,等.产木聚糖酶菌株的筛选、鉴定及酶学性质研究[J].中国食品学报,2016(1):115-122.
[21]郝涤非.木聚糖酶活性测定的实践与研究[J].中国酿造,2009(11):149-153.