通过构建元基因组16S r RNA文库和Fosmid文库,对富含纤维素、半纤维素等大分子有机物的湖南沅江明星麻厂碱性沤麻废水处理池(p H9.35)微生物多样性进行了分析和新的工业用酶基因筛选,发现该碱性处理池原核微生物主要以细菌为主,包括厚...通过构建元基因组16S r RNA文库和Fosmid文库,对富含纤维素、半纤维素等大分子有机物的湖南沅江明星麻厂碱性沤麻废水处理池(p H9.35)微生物多样性进行了分析和新的工业用酶基因筛选,发现该碱性处理池原核微生物主要以细菌为主,包括厚壁菌门、变形菌门、拟杆菌门、绿菌门等四个细菌类群,其中变形菌门弯曲杆菌科硫磺单胞菌属细菌和腐败希瓦氏菌为优势菌群,其次为拟杆菌门和厚壁菌门。该环境古菌的生物量和丰度较少,以广古菌门马氏甲烷八叠球菌为优势类群。并且从该碱性池元基因组Fosmid文库中筛选出包括淀粉酶(36个)、脂肪酶(9个)、蛋白酶(4个)、纤维素酶(4个)在内的一系列食品和工业用酶活性克隆。为进一步开发和利用该碱性处理池微生物基因资源奠定了基础。展开更多
[Objective] The aim of this study was to compare three RNA extraction methods and thus find out the suitable one for isolating intact and high quality total RNA from Pinus bungeana.[Method] Employing three extraction ...[Objective] The aim of this study was to compare three RNA extraction methods and thus find out the suitable one for isolating intact and high quality total RNA from Pinus bungeana.[Method] Employing three extraction methods of Trizol,RNeasy Mini Kit,LiCl precipitation,total RNAs of P.bungeana were extracted from pine leaf samples,and their integrity and purity were detected via agarose gel electrophoresis and spectrophotometry for a comparative study.[Result] Among the three extraction methods,LiCl precipitation method demonstrated higher yield and better integrity of total extracted RNA,with OD260/OD280 ratio between 1.8-2.0 and clear 28 S and 18 S bands in electrophoresis pattern.[Conclusion] LiCl precipitation method could be used to extract highly pure and intact total RNA from P.bungeana.展开更多
A full-length cDNA library from the testis of dark-spotted frogs （ Rana nigromaculata ） was constructed with the SMART （switching mechanism at 5＇ end of RNA transcript） technique. Total RNA was extracted from the...A full-length cDNA library from the testis of dark-spotted frogs （ Rana nigromaculata ） was constructed with the SMART （switching mechanism at 5＇ end of RNA transcript） technique. Total RNA was extracted from the testis and reverse transcripted into full-length cDNA using PowerScript reverse transcriptase. The first-strand cDNA was amplified using long-distance PCR （LD-PCR）. After Sfi Ⅰ digestion and fractionation, cDNA （ 〉 500 bp） was ligated to λ TriplEx2 vector and packaged with GigapackⅢ Gold Packaging Extract. The titers of optimal primary libraries were 2.0×10^6 pfu/mL and 2.4 × 10^6 pfu/mL and the tlters of the amplified libraries were 0.48 × 10^9 pfu/mL and 3.0 × 10^9 pfu/ mL, respectively. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. The libraries were converted into pTriplEx2 plasmids in E. coli BM 25.8 strain. The insert sizes were measured by PCR which showed most fragments were over 500 bp and the average length was 1.0 kb approximately. A positive clone of 1 171 bp was sequenced and named RnUb based on sequence similarity with the known ubiquitin genes in Gen- Bank. This sequence was a full-length cDNA with complete coding sequences, which indicated that the library built a base for screening the full-length cDNA. These data showed that this library attained to the requirements of a standard cDNA library. This library provided a useful resource for the functional genomic research of Rana nigromaculata.展开更多
【目的】了解新疆艾比湖湿地国家级自然保护区非培养土壤细菌群落组成及多样性。【方法】采用非培养法直接从湿地土壤提取总DNA进行16S r RNA基因扩增,构建细菌16S r RNA基因克隆文库。使用MspⅠ和AfaⅠ限制性内切酶对阳性克隆进行16S r...【目的】了解新疆艾比湖湿地国家级自然保护区非培养土壤细菌群落组成及多样性。【方法】采用非培养法直接从湿地土壤提取总DNA进行16S r RNA基因扩增,构建细菌16S r RNA基因克隆文库。使用MspⅠ和AfaⅠ限制性内切酶对阳性克隆进行16S r RNA基因扩增片段的限制性酶切分析(Amplified r DNA restriction analysis,ARDRA),挑取具有不同双酶切图谱的克隆进行测序,序列比对并构建16S r RNA基因系统发育树。【结果】从土壤细菌的16S r RNA基因文库中随机挑取75个不同谱型的克隆子,共得到58个OTUs,系统发育归类为8个细菌类群:绿弯菌门(Chloroflexi)、蓝藻门(Cyanobacteria)、变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrob)和芽单胞菌门(Gemmatimonadetes)。其中,变形菌门为第一优势菌群,拟杆菌门为第二优势菌群,两者约占总克隆的65%。【结论】艾比湖湿地博乐河入口处土壤细菌多样性丰富,且存在一定数量的潜在微生物新种。展开更多
文摘通过构建元基因组16S r RNA文库和Fosmid文库,对富含纤维素、半纤维素等大分子有机物的湖南沅江明星麻厂碱性沤麻废水处理池(p H9.35)微生物多样性进行了分析和新的工业用酶基因筛选,发现该碱性处理池原核微生物主要以细菌为主,包括厚壁菌门、变形菌门、拟杆菌门、绿菌门等四个细菌类群,其中变形菌门弯曲杆菌科硫磺单胞菌属细菌和腐败希瓦氏菌为优势菌群,其次为拟杆菌门和厚壁菌门。该环境古菌的生物量和丰度较少,以广古菌门马氏甲烷八叠球菌为优势类群。并且从该碱性池元基因组Fosmid文库中筛选出包括淀粉酶(36个)、脂肪酶(9个)、蛋白酶(4个)、纤维素酶(4个)在内的一系列食品和工业用酶活性克隆。为进一步开发和利用该碱性处理池微生物基因资源奠定了基础。
基金Supported by National Program on Key Basic Research Project(2009CB119204)~~
文摘[Objective] The aim of this study was to compare three RNA extraction methods and thus find out the suitable one for isolating intact and high quality total RNA from Pinus bungeana.[Method] Employing three extraction methods of Trizol,RNeasy Mini Kit,LiCl precipitation,total RNAs of P.bungeana were extracted from pine leaf samples,and their integrity and purity were detected via agarose gel electrophoresis and spectrophotometry for a comparative study.[Result] Among the three extraction methods,LiCl precipitation method demonstrated higher yield and better integrity of total extracted RNA,with OD260/OD280 ratio between 1.8-2.0 and clear 28 S and 18 S bands in electrophoresis pattern.[Conclusion] LiCl precipitation method could be used to extract highly pure and intact total RNA from P.bungeana.
基金The National Natural Science Foundation of China(30640048)the Natural Science Foundation in Anhui Province(01043202)
文摘A full-length cDNA library from the testis of dark-spotted frogs （ Rana nigromaculata ） was constructed with the SMART （switching mechanism at 5＇ end of RNA transcript） technique. Total RNA was extracted from the testis and reverse transcripted into full-length cDNA using PowerScript reverse transcriptase. The first-strand cDNA was amplified using long-distance PCR （LD-PCR）. After Sfi Ⅰ digestion and fractionation, cDNA （ 〉 500 bp） was ligated to λ TriplEx2 vector and packaged with GigapackⅢ Gold Packaging Extract. The titers of optimal primary libraries were 2.0×10^6 pfu/mL and 2.4 × 10^6 pfu/mL and the tlters of the amplified libraries were 0.48 × 10^9 pfu/mL and 3.0 × 10^9 pfu/ mL, respectively. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. The libraries were converted into pTriplEx2 plasmids in E. coli BM 25.8 strain. The insert sizes were measured by PCR which showed most fragments were over 500 bp and the average length was 1.0 kb approximately. A positive clone of 1 171 bp was sequenced and named RnUb based on sequence similarity with the known ubiquitin genes in Gen- Bank. This sequence was a full-length cDNA with complete coding sequences, which indicated that the library built a base for screening the full-length cDNA. These data showed that this library attained to the requirements of a standard cDNA library. This library provided a useful resource for the functional genomic research of Rana nigromaculata.
文摘【目的】了解新疆艾比湖湿地国家级自然保护区非培养土壤细菌群落组成及多样性。【方法】采用非培养法直接从湿地土壤提取总DNA进行16S r RNA基因扩增,构建细菌16S r RNA基因克隆文库。使用MspⅠ和AfaⅠ限制性内切酶对阳性克隆进行16S r RNA基因扩增片段的限制性酶切分析(Amplified r DNA restriction analysis,ARDRA),挑取具有不同双酶切图谱的克隆进行测序,序列比对并构建16S r RNA基因系统发育树。【结果】从土壤细菌的16S r RNA基因文库中随机挑取75个不同谱型的克隆子,共得到58个OTUs,系统发育归类为8个细菌类群:绿弯菌门(Chloroflexi)、蓝藻门(Cyanobacteria)、变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrob)和芽单胞菌门(Gemmatimonadetes)。其中,变形菌门为第一优势菌群,拟杆菌门为第二优势菌群,两者约占总克隆的65%。【结论】艾比湖湿地博乐河入口处土壤细菌多样性丰富,且存在一定数量的潜在微生物新种。
