藏猪肠道微生物多样性的研究
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摘要
本研究以藏猪和长白猪新鲜粪便为试验材料，利用PCR/DGGE技术和16S rNDA序列分析方法研究了藏猪的肠道微生物多样性。试验直接从藏猪和长白猪粪便中提取微生物的总DNA，并根据微生物的16S rRNA基因V6-V8区保守序列通用引物对总DNA进行PCR扩增，将扩增后的纯化产物直接利用变性剂浓度范围为38%-60%的变性梯度凝胶电泳分离。扫描拍照后将DGGE图谱中清晰可辨的条带割胶回收，经过菌落PCR和EcoRⅠ和SaLⅠ双酶切鉴定后挑取阳性克隆进行测序。对两种猪肠道内的微生物进行DGGE指纹图谱的多样性分析，并用Quantity One软件进行UPGMA聚类分析。将测序结果进行BLAST对比后发现从DGGE图谱中回收的优势菌的27条克隆中有20个克隆与基因库相关序列的相似性介于90%-96%之间，仅有7条克隆的相似性达到了97%以上，未报道的优势微生物占总微生物的74.1%。且有14条序列被鉴定为与纤维素分解菌或半纤维素分解菌相似。试验主要结果如下：
1.藏猪肠道内优势微生物约30多种，比长白猪肠道内优势微生物（约20种）的更具多样性。
2.藏猪与长白猪之间肠道微生物相似性仅为0.44，种内相似性大于种间相似性。
3.藏猪与长白猪肠道微生物组成有差异，藏猪体内含长白猪体内没有的（溶糊精琥珀酸弧菌、毛螺旋菌科、梭菌目、梭状芽孢杆菌属等）肠道优势微生物。
4.藏猪体内优势微生物的种类与长白猪相比，藏猪体内含有与溶糊精琥珀酸弧菌、直肠真杆菌、挑剔真杆菌、产琥珀酸厌氧螺菌、梭菌目、毛螺旋菌科及乳杆菌目等相似的微生物，这些微生物是具有降解纤维素或半纤维素功能的微生物。
Taken fresh feces of Landrace and Tibetan swine as materials, PCR/DGGE combinedwith16S rNDA sequence analysis were applied for biodiversity analysis of thegastrointestinal tract of Tibetan pig. DNA was directly extracted from feces of two swinespecies, and then Polymerase Chain Reaction (PCR) amplification was conducted to amplifyV6-V8region of16S rDNA gene. All the PCR products were separated by38%-60%Denaturing Gradient Gel Electrophoresis (DGGE) to compare the predominant bacterialcommunity structure. Then the advantaged marked bands of DGGE patterns were recoveredand subcloned into E. coli respectively. Colony PCR and double enzyme (EcoRⅠand SalⅠ)digestion analysis were applied to select the positive clones. Then all the positive clones weresequenced and compared to the existed ones in NCBI and Quantity One software was appliedfor UPGMA analysis. The resultant27clone sequences in16S rDNA DGGE librariesrevealed that7clones show similarity over97%with that of database sequences, while therest present similarity in a range of90%-96%. The content of the new microbes in intestinalmicroflora of the Tibetan pigs is74.1%.14clones were similar to cellulose-decomposingmicroorganisms or hemicellulose-decomposing microorganisms. The main conclusions of thestudy were as follows:
1. The dominant microbes of the Tibetan pigs are (more than30species)moremultiformity than The dominant microbes of the Landrace(about20species).
2. The bacteria community structure of different species of pigs was similar to each otheronly0.44. The similarities within species were noticeably higher than that between species.
3. Intestinal microflora of the Tibetan pigs include larger amount of unknown unculturedmicroorganisms(Succinivibrio dextrinosolvens、Lachnospiraceae bacterium、Clostridialesbact-erium、Clostridium sp.) than intestinal microflora of Landrace.
4. The dominant microbes of the Tibetan pigs (Succinivibrio dextrinosolvens、Eubacterium rectale、 Eubacterium eligens、 Anaerobiospirillum succiniciproducens、Clostridiales bacterium、Lachnospiraceae bacterium and Lactobacillales bacterium) weresimilar to cellulose-decomposing microorganisms or hemicellulose-decomposingmicroorganisms.
1.藏猪肠道内优势微生物约30多种，比长白猪肠道内优势微生物（约20种）的更具多样性。
2.藏猪与长白猪之间肠道微生物相似性仅为0.44，种内相似性大于种间相似性。
3.藏猪与长白猪肠道微生物组成有差异，藏猪体内含长白猪体内没有的（溶糊精琥珀酸弧菌、毛螺旋菌科、梭菌目、梭状芽孢杆菌属等）肠道优势微生物。
4.藏猪体内优势微生物的种类与长白猪相比，藏猪体内含有与溶糊精琥珀酸弧菌、直肠真杆菌、挑剔真杆菌、产琥珀酸厌氧螺菌、梭菌目、毛螺旋菌科及乳杆菌目等相似的微生物，这些微生物是具有降解纤维素或半纤维素功能的微生物。
Taken fresh feces of Landrace and Tibetan swine as materials, PCR/DGGE combinedwith16S rNDA sequence analysis were applied for biodiversity analysis of thegastrointestinal tract of Tibetan pig. DNA was directly extracted from feces of two swinespecies, and then Polymerase Chain Reaction (PCR) amplification was conducted to amplifyV6-V8region of16S rDNA gene. All the PCR products were separated by38%-60%Denaturing Gradient Gel Electrophoresis (DGGE) to compare the predominant bacterialcommunity structure. Then the advantaged marked bands of DGGE patterns were recoveredand subcloned into E. coli respectively. Colony PCR and double enzyme (EcoRⅠand SalⅠ)digestion analysis were applied to select the positive clones. Then all the positive clones weresequenced and compared to the existed ones in NCBI and Quantity One software was appliedfor UPGMA analysis. The resultant27clone sequences in16S rDNA DGGE librariesrevealed that7clones show similarity over97%with that of database sequences, while therest present similarity in a range of90%-96%. The content of the new microbes in intestinalmicroflora of the Tibetan pigs is74.1%.14clones were similar to cellulose-decomposingmicroorganisms or hemicellulose-decomposing microorganisms. The main conclusions of thestudy were as follows:
1. The dominant microbes of the Tibetan pigs are (more than30species)moremultiformity than The dominant microbes of the Landrace(about20species).
2. The bacteria community structure of different species of pigs was similar to each otheronly0.44. The similarities within species were noticeably higher than that between species.
3. Intestinal microflora of the Tibetan pigs include larger amount of unknown unculturedmicroorganisms(Succinivibrio dextrinosolvens、Lachnospiraceae bacterium、Clostridialesbact-erium、Clostridium sp.) than intestinal microflora of Landrace.
4. The dominant microbes of the Tibetan pigs (Succinivibrio dextrinosolvens、Eubacterium rectale、 Eubacterium eligens、 Anaerobiospirillum succiniciproducens、Clostridiales bacterium、Lachnospiraceae bacterium and Lactobacillales bacterium) weresimilar to cellulose-decomposing microorganisms or hemicellulose-decomposingmicroorganisms.
引文
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