中国南海海绵共生微生物的宏基因组研究
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摘要
海绵动物是原始、低等滤食的后生动物，它的外形能随着生活环境的变化而改变，长期以来海绵的分类问题存在较大争议。传统的海绵分类是根据海绵的骨针或骨骼来鉴定的，骨针形状完全不同的海绵很容易分类。但是有些骨针的形状相似，但是弯曲程度和长度都有细微的变化，此时，单靠骨针来分类已不可靠。通过应用生物分子标签技术（Barcode），可以为海绵的的分类提供了可靠的依据。
利用16S rDNA克隆文库构建技术，克隆六种海绵共生微生物的16S基因片段，并将其六种海绵的细菌文库相应编号为：PBS2、PBS4、PBS5、PBS7、PBS8和PBS10，克隆数分别为240、203、288、288、288、288。用限制性内切酶MboI和BstUI对PBS2和PBS4片段进行RFLP分析，克隆文库相应的OTU为：29和21。挑选.代表性的克隆进行测序，并利用NCBI和RDP数据库进行细菌种属分析。PBS2文库主要由五大细菌门类，分别是：Actinobacteria（23个克隆，9.58%）Bacteroidetes（23个克隆，5.42%）、Firmicutes（1个克隆，0.42%）、Proteobacteria（62个克隆，25.83%）、 Cyanobacteria（137个克隆，57.08%）、未分类的细菌有4个克隆，占总克隆数的1.67%。PBS4文库共有203个克隆，主要由四大细菌门类，分别是：Actinobacteria（3个克隆，1.48%）Bacteroidetes（3个克隆，1.48%）、Proteobacteria（164个克隆，80.79%）、 Cyanobacteria（9个克隆，4.43%）、未能分类的细菌有24个克隆，占总克隆数的11.82%。
利用宏基因组和16S rDNA文库技术，对京一海绵进行研究。京一海绵的16SrDNA基因文库共得到244个克隆，15个OTU，主要由Actinobacteria、Planctomycetes、 Proteobacteria、 Cyanobacteria共四个细菌门类组成，其中Cyanobacteria的细菌门类多达91.39%，而Actinobacteria和Planctomycetes的门类低至0.41%，Proteobacteria门类为6.56%，没有明确分类的细菌门类为1.23%。京一海绵样品的宏基因组solexa测序得到原始数据约20G，拼接得到346749个contigs，约73.52M的非冗余数据，片段的平均长度为约212bp，最长的片段为27.73kb，最短的片段为100bp。其中，65.09%的congtigs片段分布在100-149bp之间，14.69%的片段分布在150-199bp，200bp以上的片段占20.22%。这346749个片段根据RDP16S classifier分析，共176个contigs属于Archaea，占总体contigs数目的0.05%；227021个片段属于Bacteria，占总体contigs数目的65.47%；没有明确分类的片段大约有119553个，占总体contigs数目的34.48%。在176个属于Archaea的contigs中，有32个contigs属于Crenarchaeota；剩余的没有明确分类；227021个Bacteria的contigs中，15个contigs属于Actinobacteria，1个属于Bacteroidetes，937个属于Firmicutes，5个属于Planctomycetes，10个属于Proteobacteria，9个属于Cyanobacteria。
Marine sponges， usually attached to the seabed or reef， are among the oldestmetazoans. Their bodies changed when environmental conditions are altered. Usingonly morphological features to classify marine sponges is controversial. Spongespicules are useful feature in traditional classification. It is easier to classify the kindsof marine sponge with their spicules unlikeness. However， some times spicules aresimilar in shape but different in spicule curve and length. In this case， theclassification of marine sponges becomes unreliable just only depending on the spiculeshapes. Application of the barcoding technology (DNA barcoding)， the classificationof marine sponges is beco分钟g more reliable.
The16S-rRNA genes are cloned through polymerase chain reaction (PCR) viaprimer27F and1492R. The symbiotic microbial libraries of six different marinesponges are constructed with16S-rDNA clone library technology via plasmidPMD-19T. The name of the six sponges clone library is as follow: PBS2， PBS4，PBS5， PBS7， PBS8and PBS10. And analyzed clones are240，203，288，288，288and288respectively. After constructing the clone library， the inserts whichcontain the target16S rRNA gene fragment by universal primer of plasmid (M13F andRV-47) is amplified to produce enough copies. Then the restriction enzyme Mbo I andBstU I are used to digest the fragments and different operational taxonomic units (OTU)are identified. The OTUs of PBS2and PBS4are29and21. The representative clonesof different OTUs are sequenced and the types of bacteria are identified by comparingthe sequences with NCBI and RDP database. PBS2library comprises five categories ofbacteria: Actinobacteria (23clones，9.58%)， Bacteroidetes (23clones，5.42%)， Firmicutes (1clones，0.42%)，Proteobacteria (62clones，25.83%)，Cyanobacteria(137clones，57.08%)， and unclassified bacteria (4clones，1.67%). PBS4librarycontain203clones， and bacteria categories are: Actinobacteria (3clones，1.48%)Bacteroidetes (3clones，1.48%)， Proteobacteria (164clones，80.79%)，Cyanobacteria (9clone，4.43%)， unclassified bacteria(24clones，11.82%).Metagenomic approach based on next generation sequence and16S-rDNA clonedlibrary have been applied in a marine sponge sample analyses. The16S-rDNA libraryof sponge Jing1was analysed and244clones were classified into fifteen types OTU.Actinobacteria(1clones，0.41%)， Planctomycetes(1clones，0.41%)，Proteobacteria(16clones，6.56%)， Cyanobacteria(222clones，91.39%)， andunclassified bacteria(3clones，1.23%) were composed of the categories of bacteria ofjing1. The percentage of bacteria Cyanobacteria covers up to91.39%whilePlanctomycetes and Actinobacteria only occupy0.41%. Proteobacteria bacteria is6.56%， and unclassified bacteria is1.23%.
From the sponge sample of Jing1about20GB data were collected through Illu分钟asequencer，GAⅡx. After using software to assemble，about346，749contigs，73.52MB non-redundant data were acquired. The average fragment length is212bp whilethe longest fragment is27.73kb， the shortest is100bp. Among the data，65.09%ofcontigs were distribute in the length100-149bp，14.69%of the contigs in150-199bp，over200bp length contigs is20.22%. Those contigs accord to RDP16S classifieranalysis， a total of176contigs belong to Archaea， accounting for0.05%of allcontigs;227，021contigs belong to Bacteria，accounting for65.47%; the contigs withno clear classification are about119，553， accounting for34.48%. The176contigsbelonging to Archaea， there are32contigs are Crenarchaeota; the remaining has noclear classification.227021bacteria contigs，15contigs belong to Actinobacteria，1fall within the category Bacteroidetes，937fall within the category Firmicutes，5fallwithin the category Planctomycetes，10belong to Proteobacteria，9fall in thecategory Cyanobacteria.
利用16S rDNA克隆文库构建技术，克隆六种海绵共生微生物的16S基因片段，并将其六种海绵的细菌文库相应编号为：PBS2、PBS4、PBS5、PBS7、PBS8和PBS10，克隆数分别为240、203、288、288、288、288。用限制性内切酶MboI和BstUI对PBS2和PBS4片段进行RFLP分析，克隆文库相应的OTU为：29和21。挑选.代表性的克隆进行测序，并利用NCBI和RDP数据库进行细菌种属分析。PBS2文库主要由五大细菌门类，分别是：Actinobacteria（23个克隆，9.58%）Bacteroidetes（23个克隆，5.42%）、Firmicutes（1个克隆，0.42%）、Proteobacteria（62个克隆，25.83%）、 Cyanobacteria（137个克隆，57.08%）、未分类的细菌有4个克隆，占总克隆数的1.67%。PBS4文库共有203个克隆，主要由四大细菌门类，分别是：Actinobacteria（3个克隆，1.48%）Bacteroidetes（3个克隆，1.48%）、Proteobacteria（164个克隆，80.79%）、 Cyanobacteria（9个克隆，4.43%）、未能分类的细菌有24个克隆，占总克隆数的11.82%。
利用宏基因组和16S rDNA文库技术，对京一海绵进行研究。京一海绵的16SrDNA基因文库共得到244个克隆，15个OTU，主要由Actinobacteria、Planctomycetes、 Proteobacteria、 Cyanobacteria共四个细菌门类组成，其中Cyanobacteria的细菌门类多达91.39%，而Actinobacteria和Planctomycetes的门类低至0.41%，Proteobacteria门类为6.56%，没有明确分类的细菌门类为1.23%。京一海绵样品的宏基因组solexa测序得到原始数据约20G，拼接得到346749个contigs，约73.52M的非冗余数据，片段的平均长度为约212bp，最长的片段为27.73kb，最短的片段为100bp。其中，65.09%的congtigs片段分布在100-149bp之间，14.69%的片段分布在150-199bp，200bp以上的片段占20.22%。这346749个片段根据RDP16S classifier分析，共176个contigs属于Archaea，占总体contigs数目的0.05%；227021个片段属于Bacteria，占总体contigs数目的65.47%；没有明确分类的片段大约有119553个，占总体contigs数目的34.48%。在176个属于Archaea的contigs中，有32个contigs属于Crenarchaeota；剩余的没有明确分类；227021个Bacteria的contigs中，15个contigs属于Actinobacteria，1个属于Bacteroidetes，937个属于Firmicutes，5个属于Planctomycetes，10个属于Proteobacteria，9个属于Cyanobacteria。
Marine sponges， usually attached to the seabed or reef， are among the oldestmetazoans. Their bodies changed when environmental conditions are altered. Usingonly morphological features to classify marine sponges is controversial. Spongespicules are useful feature in traditional classification. It is easier to classify the kindsof marine sponge with their spicules unlikeness. However， some times spicules aresimilar in shape but different in spicule curve and length. In this case， theclassification of marine sponges becomes unreliable just only depending on the spiculeshapes. Application of the barcoding technology (DNA barcoding)， the classificationof marine sponges is beco分钟g more reliable.
The16S-rRNA genes are cloned through polymerase chain reaction (PCR) viaprimer27F and1492R. The symbiotic microbial libraries of six different marinesponges are constructed with16S-rDNA clone library technology via plasmidPMD-19T. The name of the six sponges clone library is as follow: PBS2， PBS4，PBS5， PBS7， PBS8and PBS10. And analyzed clones are240，203，288，288，288and288respectively. After constructing the clone library， the inserts whichcontain the target16S rRNA gene fragment by universal primer of plasmid (M13F andRV-47) is amplified to produce enough copies. Then the restriction enzyme Mbo I andBstU I are used to digest the fragments and different operational taxonomic units (OTU)are identified. The OTUs of PBS2and PBS4are29and21. The representative clonesof different OTUs are sequenced and the types of bacteria are identified by comparingthe sequences with NCBI and RDP database. PBS2library comprises five categories ofbacteria: Actinobacteria (23clones，9.58%)， Bacteroidetes (23clones，5.42%)， Firmicutes (1clones，0.42%)，Proteobacteria (62clones，25.83%)，Cyanobacteria(137clones，57.08%)， and unclassified bacteria (4clones，1.67%). PBS4librarycontain203clones， and bacteria categories are: Actinobacteria (3clones，1.48%)Bacteroidetes (3clones，1.48%)， Proteobacteria (164clones，80.79%)，Cyanobacteria (9clone，4.43%)， unclassified bacteria(24clones，11.82%).Metagenomic approach based on next generation sequence and16S-rDNA clonedlibrary have been applied in a marine sponge sample analyses. The16S-rDNA libraryof sponge Jing1was analysed and244clones were classified into fifteen types OTU.Actinobacteria(1clones，0.41%)， Planctomycetes(1clones，0.41%)，Proteobacteria(16clones，6.56%)， Cyanobacteria(222clones，91.39%)， andunclassified bacteria(3clones，1.23%) were composed of the categories of bacteria ofjing1. The percentage of bacteria Cyanobacteria covers up to91.39%whilePlanctomycetes and Actinobacteria only occupy0.41%. Proteobacteria bacteria is6.56%， and unclassified bacteria is1.23%.
From the sponge sample of Jing1about20GB data were collected through Illu分钟asequencer，GAⅡx. After using software to assemble，about346，749contigs，73.52MB non-redundant data were acquired. The average fragment length is212bp whilethe longest fragment is27.73kb， the shortest is100bp. Among the data，65.09%ofcontigs were distribute in the length100-149bp，14.69%of the contigs in150-199bp，over200bp length contigs is20.22%. Those contigs accord to RDP16S classifieranalysis， a total of176contigs belong to Archaea， accounting for0.05%of allcontigs;227，021contigs belong to Bacteria，accounting for65.47%; the contigs withno clear classification are about119，553， accounting for34.48%. The176contigsbelonging to Archaea， there are32contigs are Crenarchaeota; the remaining has noclear classification.227021bacteria contigs，15contigs belong to Actinobacteria，1fall within the category Bacteroidetes，937fall within the category Firmicutes，5fallwithin the category Planctomycetes，10belong to Proteobacteria，9fall in thecategory Cyanobacteria.
引文
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