贡嘎蝠蛾幼虫肠道细菌多样性分析
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摘要
贡嘎蝠蛾(Hepialus gonggaensis)幼虫是名贵中药材冬虫夏草(Cordyceps sinensis)的优势寄主之一。人工培植中幼虫抗逆力弱,易受微生物感染,是影响冬虫夏草资源保护和开发的重要制约因子。研究贡嘎蝠蛾幼虫肠道菌群多样性,对于研究其营养生理,进而改善和研制人工饲料,提高幼虫抗逆力和成活率有重要意义。
本研究以四川康定雅家埂山上采集的贡嘎蝠蛾为实验材料,按传统的培养分离方法对贡嘎蝠蛾幼虫的肠道细菌进行分离和鉴定。实验结果表明,贡嘎蝠蛾肠道细菌都属低温菌,最适生长温度15℃,在15℃培养2d 后才长出典型菌落。从肠道中分离纯化出12 个不同菌群,其中优势菌群为葡萄球菌(Staphylococcus ),菌群数量为2.05×109mL-1,检出率100%,应为幼虫肠道中的常住菌群,其它菌群检出率均低于50%,可能为肠道中的过路菌群。生化鉴定大多数菌群的符合值都较低,这与蝠蛾生活于高海拔高寒地区,其肠道微生物与常见微生物菌群有较大差异一致。
利用分子生物学方法研究了贡嘎蝠蛾幼虫肠道细菌多样性。研究用根据大肠杆菌16SrRNA 基因保守序列设计的引物对贡嘎蝠蛾幼虫肠道基因组DNA 进行PCR 扩增,再利用变性梯度凝胶电泳对扩增得到的16SrDNA 序列进行直观分离。采用变性剂浓度范围为10%-60%时,分离得到12 条带,进一步缩小变性剂范围为25%-48%,最终分离得到76 条带。因为每个条带很可能就代表一个不同的微生物物种,所以DGGE 带谱中条带的数量和亮度,即可相应反映出该环境中微生物种类的数量及群落中的优势菌群。从实验结果可以看出,贡嘎蝠蛾幼虫肠道中存在着大量细菌,它们的种类和数量各不相同,即贡嘎蝠蛾幼虫肠道中具有丰富的细菌多样性。多次重复地从健康贡嘎蝠蛾幼虫体内取得肠道富集后,应用DGGE 技术进行研究,都可以得出比较一致的结果。由此可见,以DGGE 技术分离16SrDNA条带研究昆虫肠道细菌多样性是可行的。
把DGGE 分离得到的最亮的8 条带进行纯化,克隆和测序,并对所得序列以分子信息技术进行BLAST 比对,结果表明:除3 条带外,其他条带的顺序与已知菌株的顺序都有较大的差异,暗示这些条带所代表的菌株可能尚未被人们获得并研究过,因此采用此方法对贡嘎蝠蛾幼虫肠道微生物进行研究将是可行的。
在DGGE 与纯培养的比较研究中,两种方法检测到的细菌多样性结果不完全一致,说明每种方法都有一定的选择性和局限性,得到纯培养的物种并不一定是肠道环境中的真正的优势类群,它们或许只占环境中细菌总数的一小部分。
The larvae of Hepialus gonggaensis is one of the predominant host of Cordyceps sinensis. The insect infected by C. sinensis can produce famous herb medicine Dongchongxiacao which has very high price in China and Asia area. The intestinal microbial flora played an important action in larvae’s development, nutrient and resistant to the diseases.
In the study, intestinal microbial flora of H. gonggaensis larvae, collected from Kangding, Sichuan Province, had been isolated and identified with classic methods. The results showed that the intestinal microbial flora of H. gonggaensis belongs to low-temperature bacteria. The bacteria grew very slowly and the typical colony appeared on cultural plate only after 2 days incubated under the most profitable temperature, 15℃. Totally 12 different isolates of bacteria were obtained from the larvae intestine with 5 different of cultural media. It showed that Staphylococcus. sp was the predominant community, whose numberable colony counted 2.05×109mL-1, and the detection rate as high as 100%. It revealed that Staphylococcus sp. could be the normal flora in the larva’s intestine. And the other isolates can only be detected occasionally; they were supposed to be the passer microbes. The biochemical identity showed that the identifying values are relative low for most bacteria, which are consistent with the larvae’s abnormal living environment.
Because of large sorts of bacteria could be uncultured, molecular technology were also applied to analyze the bacteria diversity in the larva gut of H. gonggaensis. The primers designed by conservative sequences of 16SrRNA gene of E.coli was used to amplificate intestinal bacterial gene group DNA from H. gonggaensis , and then amplificational products were separated directly by DGGE. Twelve bands were obtained with DGGE in the denaturant range of 10%~60%, and 76 bands were separated at last under the much narrow denaturant range of 25%~48%. Every band might present a different microbiological sort, so the number and the brightness of bands in DGGE patterns can reflect the number of bacterium sorts and the predominant bacterium in intestinal flora. All the study indicated that there were great number of bacterium sorts and quantity in the intestine of H. gonggaensis. Repeated experiment can get the same result. It showed that 16SrDNA sequence combined DGGE analysis was effective method for larvae’s intestinal bacterial diversity study.
The eight most bright bands separated by DGGE were purified, cloned and sequenced. The sequences were compared with the database in Gene Bank and aligned by the software BLAST. The results showed that the 5 of 8 sequences of isolators were different from that of bacteria which were already knew by biologists. This suggested that these bacteria had not be studied yet up to now. The different bacterial flora was obtained by classic culture and DGGE, especialy the predominant species. This inconsistant probably because of each method having limited use area and not perfect for all sorts of bacterial. It also indicated that the predominance bacterial strains got from purified culture were probably not the real predominant flora. They maybe only accounted a few part of flora in the insect intestine.
本研究以四川康定雅家埂山上采集的贡嘎蝠蛾为实验材料,按传统的培养分离方法对贡嘎蝠蛾幼虫的肠道细菌进行分离和鉴定。实验结果表明,贡嘎蝠蛾肠道细菌都属低温菌,最适生长温度15℃,在15℃培养2d 后才长出典型菌落。从肠道中分离纯化出12 个不同菌群,其中优势菌群为葡萄球菌(Staphylococcus ),菌群数量为2.05×109mL-1,检出率100%,应为幼虫肠道中的常住菌群,其它菌群检出率均低于50%,可能为肠道中的过路菌群。生化鉴定大多数菌群的符合值都较低,这与蝠蛾生活于高海拔高寒地区,其肠道微生物与常见微生物菌群有较大差异一致。
利用分子生物学方法研究了贡嘎蝠蛾幼虫肠道细菌多样性。研究用根据大肠杆菌16SrRNA 基因保守序列设计的引物对贡嘎蝠蛾幼虫肠道基因组DNA 进行PCR 扩增,再利用变性梯度凝胶电泳对扩增得到的16SrDNA 序列进行直观分离。采用变性剂浓度范围为10%-60%时,分离得到12 条带,进一步缩小变性剂范围为25%-48%,最终分离得到76 条带。因为每个条带很可能就代表一个不同的微生物物种,所以DGGE 带谱中条带的数量和亮度,即可相应反映出该环境中微生物种类的数量及群落中的优势菌群。从实验结果可以看出,贡嘎蝠蛾幼虫肠道中存在着大量细菌,它们的种类和数量各不相同,即贡嘎蝠蛾幼虫肠道中具有丰富的细菌多样性。多次重复地从健康贡嘎蝠蛾幼虫体内取得肠道富集后,应用DGGE 技术进行研究,都可以得出比较一致的结果。由此可见,以DGGE 技术分离16SrDNA条带研究昆虫肠道细菌多样性是可行的。
把DGGE 分离得到的最亮的8 条带进行纯化,克隆和测序,并对所得序列以分子信息技术进行BLAST 比对,结果表明:除3 条带外,其他条带的顺序与已知菌株的顺序都有较大的差异,暗示这些条带所代表的菌株可能尚未被人们获得并研究过,因此采用此方法对贡嘎蝠蛾幼虫肠道微生物进行研究将是可行的。
在DGGE 与纯培养的比较研究中,两种方法检测到的细菌多样性结果不完全一致,说明每种方法都有一定的选择性和局限性,得到纯培养的物种并不一定是肠道环境中的真正的优势类群,它们或许只占环境中细菌总数的一小部分。
The larvae of Hepialus gonggaensis is one of the predominant host of Cordyceps sinensis. The insect infected by C. sinensis can produce famous herb medicine Dongchongxiacao which has very high price in China and Asia area. The intestinal microbial flora played an important action in larvae’s development, nutrient and resistant to the diseases.
In the study, intestinal microbial flora of H. gonggaensis larvae, collected from Kangding, Sichuan Province, had been isolated and identified with classic methods. The results showed that the intestinal microbial flora of H. gonggaensis belongs to low-temperature bacteria. The bacteria grew very slowly and the typical colony appeared on cultural plate only after 2 days incubated under the most profitable temperature, 15℃. Totally 12 different isolates of bacteria were obtained from the larvae intestine with 5 different of cultural media. It showed that Staphylococcus. sp was the predominant community, whose numberable colony counted 2.05×109mL-1, and the detection rate as high as 100%. It revealed that Staphylococcus sp. could be the normal flora in the larva’s intestine. And the other isolates can only be detected occasionally; they were supposed to be the passer microbes. The biochemical identity showed that the identifying values are relative low for most bacteria, which are consistent with the larvae’s abnormal living environment.
Because of large sorts of bacteria could be uncultured, molecular technology were also applied to analyze the bacteria diversity in the larva gut of H. gonggaensis. The primers designed by conservative sequences of 16SrRNA gene of E.coli was used to amplificate intestinal bacterial gene group DNA from H. gonggaensis , and then amplificational products were separated directly by DGGE. Twelve bands were obtained with DGGE in the denaturant range of 10%~60%, and 76 bands were separated at last under the much narrow denaturant range of 25%~48%. Every band might present a different microbiological sort, so the number and the brightness of bands in DGGE patterns can reflect the number of bacterium sorts and the predominant bacterium in intestinal flora. All the study indicated that there were great number of bacterium sorts and quantity in the intestine of H. gonggaensis. Repeated experiment can get the same result. It showed that 16SrDNA sequence combined DGGE analysis was effective method for larvae’s intestinal bacterial diversity study.
The eight most bright bands separated by DGGE were purified, cloned and sequenced. The sequences were compared with the database in Gene Bank and aligned by the software BLAST. The results showed that the 5 of 8 sequences of isolators were different from that of bacteria which were already knew by biologists. This suggested that these bacteria had not be studied yet up to now. The different bacterial flora was obtained by classic culture and DGGE, especialy the predominant species. This inconsistant probably because of each method having limited use area and not perfect for all sorts of bacterial. It also indicated that the predominance bacterial strains got from purified culture were probably not the real predominant flora. They maybe only accounted a few part of flora in the insect intestine.
引文
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