不同饲料饲育的家蚕幼虫肠道细菌的多样性分析
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摘要
【目的】为研究饲料对不同家蚕Bombyx mori品种肠道微生物菌群的影响。【方法】以筛选到的家蚕广食性品种GS和普通品种1015为研究对象,收集从收蚁开始分别饲育桑叶(GS. m和C1015. m组)和人工饲料(GS. b组)至4龄盛时期的家蚕肠道样本,采用高通量测序的方法对其肠道微生物16S r DNA的V3-V4区进行测序分析,比较它们之间肠道微生物的差异。【结果】在门水平上,所测家蚕肠道样本的优势菌为厚壁菌门(Firmicutes)和变形杆菌门(Proteobacteria);在科水平上,所测样本主要优势菌为明串珠菌科(Leuconostocaceae)、乳酸杆菌科(Lactobacillaceae)、肠杆菌科(Enterobacteriaceae)等;在属水平上,所测样本主要的优势菌为魏斯氏属Weissella、乳酸菌属Lactobacillus、布赫纳氏菌属Buchnera、甲基杆菌属Methylobacterium、叶瘤菌属Phyllobacterium、肠球菌属Enterococcus和脆弱拟杆菌属Bacteroides等。家蚕品种GS经桑叶和人工饲料饲育后,甲基杆菌属Methylobacterium、布赫纳氏菌属Buchnera等菌属仅在桑叶饲育的GS肠道内出现,而魏斯氏菌Weissella、短芽孢杆菌属Brevibacillus等菌属只在人工饲料饲育的GS肠道内出现。同是桑叶饲育的家蚕品种GS和1015,其肠道内相同的优势菌有叶瘤菌属Phyllobacterium、脆弱拟杆菌属Bacteroides、不动细菌属Acinetobacter等。相较于广食性蚕品种GS的肠道菌群,肠球菌属Enterococcus、草螺菌属Herbaspirillum、丝硫菌属Thiothrix等菌属仅在普通蚕品种1015肠道中被检测到。GS. b组家蚕肠道细菌的物种多样性低于GS. m和C1015. m。GS. m肠道中丰度差异显著性最高的菌群为变形菌门(Proteobacteria),GS. b肠道中丰度差异显著性最高的菌群为杆菌纲(Bacilli)和乳杆菌目(Lactobacillales),而C1015. m肠道中丰度差异显著性最高的菌群为粪肠球菌属Enterococcus和肠球菌科(Enterococcaceae)。【结论】经桑叶饲育的不同蚕品种(GS和1015)的肠道微生物比人工饲料饲育的家蚕肠道微生物更趋于一致;经桑叶饲育的广食性家蚕肠道微生物物种多样性较高于经人工饲料饲育的广食性家蚕。
【Aim】This study aims to investigate the effects of feed on intestinal microflora of different silkworm(Bombyx mori) varieties. 【Methods 】 The polyphagous silkworm strain GS and common silkworm strain 1015 were fed with mulberry leaves(groups GS. m and C1015. m) and artificial diet(group GS. b),respectively,and the intestinal samples of the 4 th instar larvae of B. mori were collected. The V3-V4 region of the 16 S rD NA of intestinal microorganisms was sequenced by using the high-throughput sequencing method,and the differences in intestinal microflora between the two strains were compared. 【Results】The results showed that the dominant bacteria in B. mori intestine at the phyla level were Firmicutes and Proteobacteria, those at the family level were Leuconostocaceae,Lactobacillaceae and Enterobacteriaceae,and those at the genus level were Weissella,Lactobacillus,Buchnera,Methylobacterium,Phyllobacterium,Enterococcus,Bacteroides,etc. After GS was fed with mulberry leaves and the artificial diet,Methylobacterium and Buchnera were present only in the intestine of GS fed with mulberry leaves,while Weissella and Brevibacillus appeared only in the intestine of GS fed with the artificial diet. The common dominant intestinal bacteria in silkworm strains GS and 1015 fed with mulberry leaves were Phyllobacterium,Bacteroides,Acinetobacter,etc. Compared with the intestinal microflora of the GS strain,Enterococcus,Herbaspirillum and Thiothrix were detected only in the strain1015. The species diversity of intestinal bacteria in group GS. b was lower than those of groups GS. m and C1015. m. The intestinal bacteria whose abundance showed the most significant difference was Proteobacteria in GS. m,Bacilli and Lactobacillales in GS. b,and Enterococcus and Enterococcacea in C1015. m. 【Conclusion】The intestinal microflora in different silkworm strains(GS and 1015) fed with mulberry leaves are more consistent as compared to that in silkworm strains fed with the artificial diet.The species diversity of intestinal microflora in the polyphagous silkworm fed with mulberry leaves is higher than that in the polyphagous silkworm fed with the artificial diet.
【Aim】This study aims to investigate the effects of feed on intestinal microflora of different silkworm(Bombyx mori) varieties. 【Methods 】 The polyphagous silkworm strain GS and common silkworm strain 1015 were fed with mulberry leaves(groups GS. m and C1015. m) and artificial diet(group GS. b),respectively,and the intestinal samples of the 4 th instar larvae of B. mori were collected. The V3-V4 region of the 16 S rD NA of intestinal microorganisms was sequenced by using the high-throughput sequencing method,and the differences in intestinal microflora between the two strains were compared. 【Results】The results showed that the dominant bacteria in B. mori intestine at the phyla level were Firmicutes and Proteobacteria, those at the family level were Leuconostocaceae,Lactobacillaceae and Enterobacteriaceae,and those at the genus level were Weissella,Lactobacillus,Buchnera,Methylobacterium,Phyllobacterium,Enterococcus,Bacteroides,etc. After GS was fed with mulberry leaves and the artificial diet,Methylobacterium and Buchnera were present only in the intestine of GS fed with mulberry leaves,while Weissella and Brevibacillus appeared only in the intestine of GS fed with the artificial diet. The common dominant intestinal bacteria in silkworm strains GS and 1015 fed with mulberry leaves were Phyllobacterium,Bacteroides,Acinetobacter,etc. Compared with the intestinal microflora of the GS strain,Enterococcus,Herbaspirillum and Thiothrix were detected only in the strain1015. The species diversity of intestinal bacteria in group GS. b was lower than those of groups GS. m and C1015. m. The intestinal bacteria whose abundance showed the most significant difference was Proteobacteria in GS. m,Bacilli and Lactobacillales in GS. b,and Enterococcus and Enterococcacea in C1015. m. 【Conclusion】The intestinal microflora in different silkworm strains(GS and 1015) fed with mulberry leaves are more consistent as compared to that in silkworm strains fed with the artificial diet.The species diversity of intestinal microflora in the polyphagous silkworm fed with mulberry leaves is higher than that in the polyphagous silkworm fed with the artificial diet.
引文
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Martin M,2011.Cutadapt removes adapter sequences from highthroughput sequencing reads.EMBnet.J.,17(1):10-12.
Pechal JL,Crippen TL,Benbow ME,Tarone AM,Dowd S,Tomberlin JK,2014.The potential use of bacterial community succession in forensics as described by high throughput metagenomic sequencing.Inter.J.Legal Med.,128(1):193-205.
Quast C,Pruesse E,Yilmaz P,Gerken J,Schweer T,Yarza P,Peplies J,Glckner FO,2013.The SILVA ribosomal RNA gene database project:improved data processing and web-based tools.Nucleic Acids Res.,41(Database issue):590-596.
Shao MX,2016.High-throughput Sequencing Analysis of Intestinal Normal Flora of Small Tail Han Sheep and Development of Sheepderived Microecological Preparation.MSc Thesis,Shandong Agricultural University,Tai'an,Shandong.[邵明旭,2016.小尾寒羊肠道正常菌群高通量测序分析及羊源微生态制剂的研制.山东泰安:山东农业大学硕士学位论文]
Sun ZL,2016.Effect of Fluoride on Intestinal Microbial Community in Silkworm.MSc Thesis,Suzhou University,Suzhou,Jiangsu.[孙振丽,2016.病毒感染和高温处理对家蚕肠道菌群结构的影响.江苏苏州:苏州大学硕士学位论文]
Takizawa Y,Iizuka T,1968.The aerobic bacterial flora in the gut of larvae of the silkworm,Bombyx mori L.(Ⅰ).The relation between media and the numbers of living cells.J.Sericul.Sci.Jpn.,37(4):295-305.
Tian ZH,Hui FL,Ke T,Han YC,Wen ZZ,2007.Analysis of intestinal bacterial population structure in silkworm.Sci.Sericul.,33(4):592-595.[田贞华,惠丰立,柯涛,阚云超,文桢中,2007,家蚕肠道细菌种群结构分析.蚕业科学,33(4):592-595]
Wang Q,Garrity GM,Tiedje JM,Cole JR,2007.Nave Bayesian classifier for rapid assignment of r RNA sequences into the new bacterial taxonomy.Appl.Environ.Microbiol.,73(16):5261-5267.
Xiang H,Li MW,Zhao Y,Zhao LP,Zhang YH,Huang YP,2007.Bacterial community in midguts of the silkworm larvae estimated by PCR/DGGE and 16S r DNA gene library analysis.Acta Entomol.Sin.,50(3):222-233.[相辉,李木旺,赵勇,赵立平,张月华,黄勇平,2007.家蚕幼虫中肠细菌群落多样性的PCR/DGGE和16S r DNA文库序列分析.昆虫学报,50(3):222-233]
Xiang YQ,2010.Studies on the Type Composition and Differences of Intestinal Microecologically Predominant Bacteria in Bombyx mori Different Feeds.MSc Thesis,Southwest University,Chongqing.[向芸庆,2010.不同饲料饲养家蚕其肠道微生态优势菌群的类型组成及差异性研究.重庆:西南大学硕士学位论文]
Xu G,Sun ZL,Hu XL,Xue RY,Cao GL,Gong CL,2015.Analysis on intestinal bacterial diversity in silkworm(Bombyx mori)based on16S r RNA gene sequences.Sci.Sericul.,41(4):641-649.[许刚,孙振丽,胡小龙,薛仁宇,曹广力,贡成良,2015.基于16S r RNA基因序列分析家蚕肠道细菌的多样性.蚕业科学,41(4):641-649]
Yang X,Chen L,Wang CQ,2008.Advances in application of 16Sr RNA gene sequence analysis technology in bacterial classification.J.Northwest A&F Univ.(Nat.Sci.Ed.),36(2):55-60.[杨霞,陈陆,王川庆,2008.16S r RNA基因序列分析技术在细菌分类中应用的研究进展.西北农林科技大学学报(自然科学版),36(2):55-60]
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Chen H,Wan J,Chen K,Luo G,Fan J,Clark J,Zhang S,2016.Biogas production from hydrothermal liquefaction wastewater(HTLWW):focusing on the microbial communities as revealed by high-throughput sequencing of full-length 16S r RNA genes.Water Res.,106:98-107.
Cui WZ,2003.The physiology and genetics of Bombyx mori feeding on artificial feed.Sci.Sericul.,29(2):107-113.[崔为正,2003.家蚕对人工饲料摄食性的生理遗传学研究与展望.蚕业科学,29(2):107-113]
Cui WZ,Zhang SH,Liu QX,Wang YW,Wang HL,Liu XL,Mou ZM,2016.The general situation of research on the artificial feed for silkworm in our country and the progress of practical production.Sci.Sericul.,42(1):3-15.[崔为正,张升祥,刘庆信,王彦文,王洪利,刘训理,牟志美,2016.我国家蚕人工饲料的研究概况及生产实用化进展.蚕业科学,42(1):3-15]
Edgar R C,Haas BJ,Clemente JC,Quince C,Knight R,2011.Uchime improves sensitivity and speed of chimera detection.Bioinformatics,27(16):2194-2200.
Edgar RC,2004.MUSCLE:multiple sequence alignment with high accuracy and high throughput.Nucleic Acids Res.,32(5):1792-1797.
Edgar RC,2013.UPARSE:highly accurate OTU sequences from microbial amplicon reads.Nat.Methods,10:996-998.
Haas BJ,Gevers D,Earl AM,Feldgarden M,Ward DV,Giannoukos G,Ciulla D,Tabbaa D,Highlander SK,Sodergren E,MethéB,De Santis TZ,Petrosino JF,Knight R,Birren BW,2011.Chimeric16S r RNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons.Genome Res.,21(3):494-504.
Hao ZH,Zhu JL,Huang ZJ,2016.Research progress of intestinal microorganism in silkworm.Guangdong Seric.,50(5):1-6.[郝志华,朱佳林,黄志君,2016.家蚕肠道微生物的研究进展.广东蚕业,50(5):1-6]
Huang K,Zhang XX,Shi P,Wu B,Ren H,2014.A comprehensive insight into bacterial virulence in drinking water using 454pyrosequencing and Illumina high-throughput sequencing.Ecotox.Environ.Safe.,109:15-21.
Ji WZ,Zhu Y,Kan PC,Cai Y,Wang ZD,Wu ZJ,Yang P,2017.Analysis of intestinal microbial communities of cerebral infarction and ischemia patients based on high throughput sequencing technology and glucose and lipid metabolism.Mol.Med.Rep.,16(4):5413-5417.
Li DP,Guo MZ,Xu WT,2015.Advances and applications on methodology of 16S r RNA sequencing in gut microbiota analysis.Biotech.Bull.,31(2):71-77.[李东萍,郭明璋,许文涛,2015.16S r RNA测序技术在肠道微生物中的应用研究进展.生物技术通报,31(2):71-77]
Li GN,Xia XJ,Tang WC,Zhu Y,2016.Intestinal microecology associated with fluoride resistance capability of the silkworm(Bombyx mori L.).Appl.Microb.Biotechnol.,100(15):6715-6724.
Li MY,Xu HQ,Yu XH,Wang YR,2000.Studies on intestinal flora in silkworm.Jiangsu Seric.,(2):5-7.[李蒙英,许宏庆,虞晓华,王懿仁,2000.家蚕肠道菌群的研究.江苏蚕业,(2):5-7]
Li QY,Fang F,Du GC,Chen J,2017.Application of Weissella in fermented food.Food Ferm.Ind.,43(10):241-247.[李巧玉,方芳,堵国成,陈坚,2017.魏斯氏菌在发酵食品中的应用.食品与发酵工业,43(10):241-247]
Liu C,Li JB,Rui JP,An JX,Li XZ,2015.The application of 16Sr RNA gene in microbial ecology.Acta Ecol.Sin.,35(9):2769-2788.[刘驰,李家宝,芮俊鹏,安家兴,李香真,2015.16Sr RNA基因在微生物生态学中的应用.生态学报,35(9):2769-2788]
Luo HX,Yang Y,Xie XL,2017.Analysis of intestinal microbial diversity in diarrhea and healthy lambs.Prog.Anim.Med.,38(12):86-90.[罗海霞,杨易,谢秀兰,2017.腹泻羔羊与健康羔羊肠道微生物群落分析.动物医学进展,38(12):86-90]
Martin M,2011.Cutadapt removes adapter sequences from highthroughput sequencing reads.EMBnet.J.,17(1):10-12.
Pechal JL,Crippen TL,Benbow ME,Tarone AM,Dowd S,Tomberlin JK,2014.The potential use of bacterial community succession in forensics as described by high throughput metagenomic sequencing.Inter.J.Legal Med.,128(1):193-205.
Quast C,Pruesse E,Yilmaz P,Gerken J,Schweer T,Yarza P,Peplies J,Glckner FO,2013.The SILVA ribosomal RNA gene database project:improved data processing and web-based tools.Nucleic Acids Res.,41(Database issue):590-596.
Shao MX,2016.High-throughput Sequencing Analysis of Intestinal Normal Flora of Small Tail Han Sheep and Development of Sheepderived Microecological Preparation.MSc Thesis,Shandong Agricultural University,Tai'an,Shandong.[邵明旭,2016.小尾寒羊肠道正常菌群高通量测序分析及羊源微生态制剂的研制.山东泰安:山东农业大学硕士学位论文]
Sun ZL,2016.Effect of Fluoride on Intestinal Microbial Community in Silkworm.MSc Thesis,Suzhou University,Suzhou,Jiangsu.[孙振丽,2016.病毒感染和高温处理对家蚕肠道菌群结构的影响.江苏苏州:苏州大学硕士学位论文]
Takizawa Y,Iizuka T,1968.The aerobic bacterial flora in the gut of larvae of the silkworm,Bombyx mori L.(Ⅰ).The relation between media and the numbers of living cells.J.Sericul.Sci.Jpn.,37(4):295-305.
Tian ZH,Hui FL,Ke T,Han YC,Wen ZZ,2007.Analysis of intestinal bacterial population structure in silkworm.Sci.Sericul.,33(4):592-595.[田贞华,惠丰立,柯涛,阚云超,文桢中,2007,家蚕肠道细菌种群结构分析.蚕业科学,33(4):592-595]
Wang Q,Garrity GM,Tiedje JM,Cole JR,2007.Nave Bayesian classifier for rapid assignment of r RNA sequences into the new bacterial taxonomy.Appl.Environ.Microbiol.,73(16):5261-5267.
Xiang H,Li MW,Zhao Y,Zhao LP,Zhang YH,Huang YP,2007.Bacterial community in midguts of the silkworm larvae estimated by PCR/DGGE and 16S r DNA gene library analysis.Acta Entomol.Sin.,50(3):222-233.[相辉,李木旺,赵勇,赵立平,张月华,黄勇平,2007.家蚕幼虫中肠细菌群落多样性的PCR/DGGE和16S r DNA文库序列分析.昆虫学报,50(3):222-233]
Xiang YQ,2010.Studies on the Type Composition and Differences of Intestinal Microecologically Predominant Bacteria in Bombyx mori Different Feeds.MSc Thesis,Southwest University,Chongqing.[向芸庆,2010.不同饲料饲养家蚕其肠道微生态优势菌群的类型组成及差异性研究.重庆:西南大学硕士学位论文]
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