小龙虾肠道产木聚糖酶细菌的分离与鉴定
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摘要
【背景】小龙虾肠道微生物是小龙虾降解纤维素和半纤维素的主要驱动力。【目的】研究肠道内细菌的相对丰度,为揭示肠道微生物在小龙虾纤维素降解过程中的作用提供理论支撑。【方法】采用纯培养法从小龙虾肠道筛选产木聚糖酶细菌,并且对小龙虾肠道细菌进行16S高通量测序。【结果】形态学和16SrRNA基因分子鉴定表明,筛选到的4株产木聚糖酶细菌均属于芽孢杆菌科芽孢杆菌属;结合进一步的生理生化特征鉴定,结果为:菌株Z-3为枯草芽孢杆菌(Bacillus subtilis),菌株Z-4为贝莱斯芽孢杆菌(Bacillus velezensis),菌株Z-29为蜡状芽孢杆菌(Bacillus cereus),菌株Z-30为高地芽孢杆菌(Bacillus altitudinis);16S rRNA基因高通量测序结果表明:在属水平上,小龙虾肠道细菌主要是Candidatus Bacilloplasma、拟杆菌属、弧菌属、不动杆菌属、Dysgonomonas、Tyzzerella3、气单胞菌属和希瓦氏菌属细菌。【结论】小龙虾肠道内细菌资源丰富,且芽孢杆菌属细菌在木质纤维素降解过程中发挥一定功能。
[Background] The intestinal microbes are the main driving force of crayfish to degrade cellulose and hemicellulose. [Objective] The study of the relative abundance of bacteria in the intestine provides theoretical support for revealing the role of intestinal microorganisms in the degradation of lignocellulose. [Methods] The diversity of intestinal bacteria was studied by Illumina MiSeq high-throughput sequencing, and xylanase production bacteria were isolated from crayfish intestine by plate screening method. [Results] Morphological and 16 S rRNA gene molecular identification showed that all 4 strains belonged to Bacillus genus. The four strains were identified combining the physiological and biochemical characteristics. The results showed that the strain Z-3 was identified as Bacillus subtilis, strain Z-4 was Bacillus velezensis, strain Z-29 was Bacillus cereus, strain Z-30 was Bacillus altitudinis. The results of 16 S rRNA gene high-throughput sequencing showed that: at the genus level, the intestinal bacteria of crayfish were mainly Candidatus Bacilloplasma, Bacteroides, Vibrio, Acinetobacter,Dysgonomonas, Tyzzerella 3, Aeromonas and Shewanella. [Conclusion] Crayfish had abundant bacterial resources in the intestine, and Bacillus bacteria played an important role in the degradation of lignocellulose.
[Background] The intestinal microbes are the main driving force of crayfish to degrade cellulose and hemicellulose. [Objective] The study of the relative abundance of bacteria in the intestine provides theoretical support for revealing the role of intestinal microorganisms in the degradation of lignocellulose. [Methods] The diversity of intestinal bacteria was studied by Illumina MiSeq high-throughput sequencing, and xylanase production bacteria were isolated from crayfish intestine by plate screening method. [Results] Morphological and 16 S rRNA gene molecular identification showed that all 4 strains belonged to Bacillus genus. The four strains were identified combining the physiological and biochemical characteristics. The results showed that the strain Z-3 was identified as Bacillus subtilis, strain Z-4 was Bacillus velezensis, strain Z-29 was Bacillus cereus, strain Z-30 was Bacillus altitudinis. The results of 16 S rRNA gene high-throughput sequencing showed that: at the genus level, the intestinal bacteria of crayfish were mainly Candidatus Bacilloplasma, Bacteroides, Vibrio, Acinetobacter,Dysgonomonas, Tyzzerella 3, Aeromonas and Shewanella. [Conclusion] Crayfish had abundant bacterial resources in the intestine, and Bacillus bacteria played an important role in the degradation of lignocellulose.
引文
[1]Liu CC,Liu B,Lan JL,et al.Screening of xylanase-producing Bacillus sp.and optimization of fermentation process[J].Fujian Journal of Agricultural Sciences,2014,29(8):757-767(in Chinese)刘程程,刘波,蓝江林,等.产木聚糖酶芽胞杆菌的筛选及产酶条件优化[J].福建农业学报,2014,29(8):757-767
[2]Hu FJ,Yu JH,Zhang QH,et al.Cloning,expression and optimization of xylanase Xyn2441 from Clostridium clariflavum DSM19732[J].Food and Fermentation Industries,2016,42(7):16-22(in Chinese)胡方觊,余嘉慧,张庆华,等.Clostridium clariflavum DSM19732木聚糖酶Xyn2441的克隆表达及条件优化[J].食品与发酵工业,2016,42(7):16-22
[3]Meng DD,Ying Y,Chen XH,et al.Distinct roles for carbohydrate-binding modules of glycoside hydrolase 10(GH10)and GH11 xylanases from Caldicellulosiruptor sp.strain F32 in thermostability and catalytic efficiency[J].Applied and Environmental Microbiology,2015,81(6):2006-2014
[4]Mattéotti C,Bauwens J,Brasseur C,et al.Identification and characterization of a new xylanase from Gram-positive bacteria isolated from termite gut(Reticulitermes santonensis)[J].Protein Expression and Purification,2012,83(2):117-127
[5]He YD,Hua XM,Zhao CY,et al.Fat requirement and optimal dietary fat to carbohydrate ratio for red swamp crayfish(Procambarus clarkii)[J].Chinese Journal of Animal Nutrition,2013,25(5):1017-1024(in Chinese)何亚丁,华雪铭,赵朝阳,等.克氏原螯虾的脂肪需求量及饲料中脂肪与糖类适宜比例研究[J].动物营养学报,2013,25(5):1017-1024
[6]James J,Davidson KE,Richardson G,et al.Reduced aggression and foraging efficiency of invasive signal crayfish(Pacifastacus leniusculus)infested with non-native branchiobdellidans(Annelida:Clitellata)[J].Parasites&Vectors,2015,8(1):596
[7]Nishijima S,Nishikawa C,Miyashita T.Habitat modification by invasive crayfish can facilitate its growth through enhanced food accessibility[J].BMC Ecology,2017,17(1):37
[8]Stern DB,Breinholt J,Pedraza-Lara C,et al.Phylogenetic evidence from freshwater crayfishes that cave adaptation is not an evolutionary dead-end[J].Evolution,2017,71(10):2522-2532
[9]Cai W,Ma Z,Yang C,et al.Using eDNA to detect the distribution and density of invasive crayfish in the Honghe-Hani rice terrace World Heritage site[J].PLoS One,2017,12(5):e0177724
[10]Huang SW,Yang WC,Zhu H,et al.Cloning and expression of red swamp crayfish major allergen tropomyosin gene and identification of its immunoglogic activity[J].Chinese Journal of Immunology,2011,27(7):642-647(in Chinese)黄素文,杨文潮,朱海,等.淡水小龙虾主要过敏原原肌球蛋白基因的克隆、表达及其免疫活性鉴定[J].中国免疫学杂志,2011,27(7):642-647
[11]Lan JF,Li XC,Sun JJ,et al.Prohibitin interacts with envelope proteins of white spot syndrome virus and prevents infection in the red swamp crayfish,Procambarus clarkii[J].Journal of Virology,2013,87(23):12756-12765
[12]Song JH,Dai F,Bai X,et al.Recent incidence of Paragonimus westermani metacercariae in freshwater crayfish,Cambaroides similis,from two enzootic sites in Jeollanam-do,Korea[J].Korean Journal of Parasitology,2017,55(3):347-350
[13]Li Q,Xie LL,Lin YC,et al.Dietary phosphorus requirement of juvenile red swamp crayfish,Procambarus clarkii[J].Journal of Huazhong Agricultural University,2013,32(2):109-115(in Chinese)李强,谢玲玲,林郁葱,等.克氏原螯虾对饲料中磷的需求量[J].华中农业大学学报,2013,32(2):109-115
[14]Buchanan RE,Gibbons NE.Bergey?s Manual of Determinative Bacteriology[M].Translation Group of Berger Bacterial Identification Manual,Institute of Microbiology,Chinese Academy of Sciences,trans.Beijing:Science Press,1984:729-758(in Chinese)布坎南RE,吉本斯NE.伯杰细菌鉴定手册[M].中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组,译.北京:科学出版社,1984:729-758
[15]Dong XZ,Cai MY.Manual of Systematic Determinative General Bacteriology[M].Beijing:Science Press,2001:349-412(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:349-412
[16]Weisburg WG,Barns SM,Pelletier DA,et al.16S ribosomal DNA amplification for phylogenetic study[J].Journal of Bacteriology,1991,173(2):697-703
[17]Zhang QF,Xu J,Yang XL,et al.Screening of a xylanase-producing strain and optimization in fermentation conditions[J].Chemistry&Bioengineering,2017,34(10):20-24,31(in Chinese)张庆芳,许晶,杨旭立,等.一株产木聚糖酶菌株的筛选及发酵产酶条件优化[J].化学与生物工程,2017,34(10):20-24,31
[18]Sun MZ,Zheng HC,Sun JS,et al.Screening of thermostable xylanase producing strains and its condition optimization for xylanase production[J].China Brewing,2013,32(8):15-20(in Chinese)孙明哲,郑宏臣,孙君社,等.产耐高温木聚糖酶菌株的筛选及其产酶条件优化[J].中国酿造,2013,32(8):15-20
[19]Gao ZQ,Li X,Xu YS,et al.Isolation and identification of a xylanase-producing bacterium and its enzymatic characteristics[J].Chinese Journal of Applied and Environmental Biology,2017,23(3):443-447(in Chinese)高志强,李茜,徐岳松,等.高产木聚糖酶细菌的筛选、鉴定及其部分酶学特性[J].应用与环境生物学报,2017,23(3):443-447
[20]Chen ZB,Li B,Wang DK,et al.Diversity of endophytic bacteria in walnut analyzed by Illumina MiSeq high-throughput sequencing technology[J].Jiangsu Journal of Agricultural Sciences,2015,31(5):1129-1133(in Chinese)陈泽斌,李冰,王定康,等.Illumina MiSeq高通量测序分析核桃内生细菌多样性[J].江苏农业学报,2015,31(5):1129-1133
[21]Liu GH,Liu B,Lin NQ,et al.Phyletic evolution and taxonomic characteristics of Bacillus[J].Fujian Journal of Agricultural Sciences,2008,23(4):436-449(in Chinese)刘国红,刘波,林乃铨,等.芽孢杆菌的系统进化及其属分类学特征[J].福建农业学报,2008,23(4):436-449
[22]Wang L,Qu Q,Li L.Review on microbiologically influenced corrosion by Bacillus genus[J].Total Corrosion Control,2013,27(10):55-60(in Chinese)王蕾,屈庆,李蕾.芽抱杆菌属微生物腐蚀研究进展[J].全面腐蚀控制,2013,27(10):55-60
[23]Huang QC,Tan BP,Dong XH,et al.Effect of chitosan oligosaccharide and mycotoxin adsorbents on intestinal mucosa structure and gut flora of Litopenaeus vannamei[J].Journal of Fishery Sciences of China,2018,25(2):373-383(in Chinese)黄钦成,谭北平,董晓慧,等.饲料中添加壳寡糖和霉菌毒素吸附剂对凡纳滨对虾肠道黏膜形态及菌群结构的影响[J].中国水产科学,2018,25(2):373-383
[24]Liu QQ,Zheng JT,Liu HC.Isolation and characterization of a Bacteroides strain from Tideland of East China Sea[J].China Biogas,2013,31(6):3-8(in Chinese)刘全全,郑经堂,刘海昌.一株拟杆菌的分离鉴定及代谢特征分析[J].中国沼气,2013,31(6):3-8
[25]Xue B,Li ST,Peng XC.Effect of aqueous extracts of six flavoring mterials on the construction of two rats’colonic bacteria[J].Modern Food Science and Technology,2013,29(7):1501-1504,1490(in Chinese)薛斌,李少艇,彭喜春.六种香辛料水提取物对大鼠中两类主要肠道菌分布的影响[J].现代食品科技,2013,29(7):1501-1504,1490
[26]Sun Y,Liu Y,Wang HL,et al.Designing of vibrio 16S rDNAspecific primers and analysis of seawater vibrio community structure[J].Periodical of Ocean University of China(Natural Science Edition),2010,40(S1):122-130(in Chinese)孙颖,刘泳,王华磊,等.弧菌16S rRNA基因特异引物设计及海洋弧菌菌群结构分析[J].中国海洋大学学报:自然科学版,2010,40(S1):122-130
[27]Liu YP,Huang GJ,Liu TQ,et al.Establishment and application of PCR assay for dection of Vibrio spp.[J].Freshwater Fisheries,2015,45(3):88-92(in Chinese)刘衍鹏,黄冠军,刘天强,等.弧菌属PCR检测方法的建立与应用[J].淡水渔业,2015,45(3):88-92
[2]Hu FJ,Yu JH,Zhang QH,et al.Cloning,expression and optimization of xylanase Xyn2441 from Clostridium clariflavum DSM19732[J].Food and Fermentation Industries,2016,42(7):16-22(in Chinese)胡方觊,余嘉慧,张庆华,等.Clostridium clariflavum DSM19732木聚糖酶Xyn2441的克隆表达及条件优化[J].食品与发酵工业,2016,42(7):16-22
[3]Meng DD,Ying Y,Chen XH,et al.Distinct roles for carbohydrate-binding modules of glycoside hydrolase 10(GH10)and GH11 xylanases from Caldicellulosiruptor sp.strain F32 in thermostability and catalytic efficiency[J].Applied and Environmental Microbiology,2015,81(6):2006-2014
[4]Mattéotti C,Bauwens J,Brasseur C,et al.Identification and characterization of a new xylanase from Gram-positive bacteria isolated from termite gut(Reticulitermes santonensis)[J].Protein Expression and Purification,2012,83(2):117-127
[5]He YD,Hua XM,Zhao CY,et al.Fat requirement and optimal dietary fat to carbohydrate ratio for red swamp crayfish(Procambarus clarkii)[J].Chinese Journal of Animal Nutrition,2013,25(5):1017-1024(in Chinese)何亚丁,华雪铭,赵朝阳,等.克氏原螯虾的脂肪需求量及饲料中脂肪与糖类适宜比例研究[J].动物营养学报,2013,25(5):1017-1024
[6]James J,Davidson KE,Richardson G,et al.Reduced aggression and foraging efficiency of invasive signal crayfish(Pacifastacus leniusculus)infested with non-native branchiobdellidans(Annelida:Clitellata)[J].Parasites&Vectors,2015,8(1):596
[7]Nishijima S,Nishikawa C,Miyashita T.Habitat modification by invasive crayfish can facilitate its growth through enhanced food accessibility[J].BMC Ecology,2017,17(1):37
[8]Stern DB,Breinholt J,Pedraza-Lara C,et al.Phylogenetic evidence from freshwater crayfishes that cave adaptation is not an evolutionary dead-end[J].Evolution,2017,71(10):2522-2532
[9]Cai W,Ma Z,Yang C,et al.Using eDNA to detect the distribution and density of invasive crayfish in the Honghe-Hani rice terrace World Heritage site[J].PLoS One,2017,12(5):e0177724
[10]Huang SW,Yang WC,Zhu H,et al.Cloning and expression of red swamp crayfish major allergen tropomyosin gene and identification of its immunoglogic activity[J].Chinese Journal of Immunology,2011,27(7):642-647(in Chinese)黄素文,杨文潮,朱海,等.淡水小龙虾主要过敏原原肌球蛋白基因的克隆、表达及其免疫活性鉴定[J].中国免疫学杂志,2011,27(7):642-647
[11]Lan JF,Li XC,Sun JJ,et al.Prohibitin interacts with envelope proteins of white spot syndrome virus and prevents infection in the red swamp crayfish,Procambarus clarkii[J].Journal of Virology,2013,87(23):12756-12765
[12]Song JH,Dai F,Bai X,et al.Recent incidence of Paragonimus westermani metacercariae in freshwater crayfish,Cambaroides similis,from two enzootic sites in Jeollanam-do,Korea[J].Korean Journal of Parasitology,2017,55(3):347-350
[13]Li Q,Xie LL,Lin YC,et al.Dietary phosphorus requirement of juvenile red swamp crayfish,Procambarus clarkii[J].Journal of Huazhong Agricultural University,2013,32(2):109-115(in Chinese)李强,谢玲玲,林郁葱,等.克氏原螯虾对饲料中磷的需求量[J].华中农业大学学报,2013,32(2):109-115
[14]Buchanan RE,Gibbons NE.Bergey?s Manual of Determinative Bacteriology[M].Translation Group of Berger Bacterial Identification Manual,Institute of Microbiology,Chinese Academy of Sciences,trans.Beijing:Science Press,1984:729-758(in Chinese)布坎南RE,吉本斯NE.伯杰细菌鉴定手册[M].中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组,译.北京:科学出版社,1984:729-758
[15]Dong XZ,Cai MY.Manual of Systematic Determinative General Bacteriology[M].Beijing:Science Press,2001:349-412(in Chinese)东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:349-412
[16]Weisburg WG,Barns SM,Pelletier DA,et al.16S ribosomal DNA amplification for phylogenetic study[J].Journal of Bacteriology,1991,173(2):697-703
[17]Zhang QF,Xu J,Yang XL,et al.Screening of a xylanase-producing strain and optimization in fermentation conditions[J].Chemistry&Bioengineering,2017,34(10):20-24,31(in Chinese)张庆芳,许晶,杨旭立,等.一株产木聚糖酶菌株的筛选及发酵产酶条件优化[J].化学与生物工程,2017,34(10):20-24,31
[18]Sun MZ,Zheng HC,Sun JS,et al.Screening of thermostable xylanase producing strains and its condition optimization for xylanase production[J].China Brewing,2013,32(8):15-20(in Chinese)孙明哲,郑宏臣,孙君社,等.产耐高温木聚糖酶菌株的筛选及其产酶条件优化[J].中国酿造,2013,32(8):15-20
[19]Gao ZQ,Li X,Xu YS,et al.Isolation and identification of a xylanase-producing bacterium and its enzymatic characteristics[J].Chinese Journal of Applied and Environmental Biology,2017,23(3):443-447(in Chinese)高志强,李茜,徐岳松,等.高产木聚糖酶细菌的筛选、鉴定及其部分酶学特性[J].应用与环境生物学报,2017,23(3):443-447
[20]Chen ZB,Li B,Wang DK,et al.Diversity of endophytic bacteria in walnut analyzed by Illumina MiSeq high-throughput sequencing technology[J].Jiangsu Journal of Agricultural Sciences,2015,31(5):1129-1133(in Chinese)陈泽斌,李冰,王定康,等.Illumina MiSeq高通量测序分析核桃内生细菌多样性[J].江苏农业学报,2015,31(5):1129-1133
[21]Liu GH,Liu B,Lin NQ,et al.Phyletic evolution and taxonomic characteristics of Bacillus[J].Fujian Journal of Agricultural Sciences,2008,23(4):436-449(in Chinese)刘国红,刘波,林乃铨,等.芽孢杆菌的系统进化及其属分类学特征[J].福建农业学报,2008,23(4):436-449
[22]Wang L,Qu Q,Li L.Review on microbiologically influenced corrosion by Bacillus genus[J].Total Corrosion Control,2013,27(10):55-60(in Chinese)王蕾,屈庆,李蕾.芽抱杆菌属微生物腐蚀研究进展[J].全面腐蚀控制,2013,27(10):55-60
[23]Huang QC,Tan BP,Dong XH,et al.Effect of chitosan oligosaccharide and mycotoxin adsorbents on intestinal mucosa structure and gut flora of Litopenaeus vannamei[J].Journal of Fishery Sciences of China,2018,25(2):373-383(in Chinese)黄钦成,谭北平,董晓慧,等.饲料中添加壳寡糖和霉菌毒素吸附剂对凡纳滨对虾肠道黏膜形态及菌群结构的影响[J].中国水产科学,2018,25(2):373-383
[24]Liu QQ,Zheng JT,Liu HC.Isolation and characterization of a Bacteroides strain from Tideland of East China Sea[J].China Biogas,2013,31(6):3-8(in Chinese)刘全全,郑经堂,刘海昌.一株拟杆菌的分离鉴定及代谢特征分析[J].中国沼气,2013,31(6):3-8
[25]Xue B,Li ST,Peng XC.Effect of aqueous extracts of six flavoring mterials on the construction of two rats’colonic bacteria[J].Modern Food Science and Technology,2013,29(7):1501-1504,1490(in Chinese)薛斌,李少艇,彭喜春.六种香辛料水提取物对大鼠中两类主要肠道菌分布的影响[J].现代食品科技,2013,29(7):1501-1504,1490
[26]Sun Y,Liu Y,Wang HL,et al.Designing of vibrio 16S rDNAspecific primers and analysis of seawater vibrio community structure[J].Periodical of Ocean University of China(Natural Science Edition),2010,40(S1):122-130(in Chinese)孙颖,刘泳,王华磊,等.弧菌16S rRNA基因特异引物设计及海洋弧菌菌群结构分析[J].中国海洋大学学报:自然科学版,2010,40(S1):122-130
[27]Liu YP,Huang GJ,Liu TQ,et al.Establishment and application of PCR assay for dection of Vibrio spp.[J].Freshwater Fisheries,2015,45(3):88-92(in Chinese)刘衍鹏,黄冠军,刘天强,等.弧菌属PCR检测方法的建立与应用[J].淡水渔业,2015,45(3):88-92