【作者】 李进；

【导师】 彭广能；

【作者基本信息】 四川农业大学 ， 临床兽医学， 2016， 硕士

【摘要】 大熊猫是世界生物多样性保护的旗舰物种,每天食入约12.5 kg竹子。大熊猫肠道结构短不能消化其中的大部分纤维素,依赖于肠道菌群。枯草芽孢杆菌能形成芽孢,分解纤维素,协助大熊猫消化竹子,在面对环境压力胁迫时具有很强的环境适应能力。在原核生物中有一种非编码小RNA(sRNA),在细菌的物质代谢、环境适应、群体感应和细菌毒力等方面发挥了重要的转录后调节作用。sRNA可以在转录后水平与靶基因互补配对促进/抑制mRNA的翻译,sRNA在基因转录后水平发挥十分重要的调控作用。实验室从大熊猫肠道中分离得到枯草芽孢杆菌HH2菌株,将其分别培养于葡萄糖和纤维素培养基,提取RNA反转录建立cDNA文库进行高通量测序,分析转录组表达差异,选取有效序列,发现两个样本绝大部分序列由sRNA组成。在预测到的这些sRNA中,sRNA-C12P在纤维素和葡萄糖培养基两个组别中表达差异非常大,可能是一个新发现的与高纤维环境适应相关的关键sRNA。为了验证sRNA-C12P是否存在,及其是否对枯草芽孢杆菌适应高纤维环境有调控作用,本研究进行了以下试验:1.通过RNA体外反转录扩增C12P片段,荧光定量PCR检测HH2分离株sRNA-C12P在不同碳源培养环境中的表达差异;2.通过基因敲除构建sRNA-C12P缺失型菌株HH2ΔC12P;3.对比HH2分离株与HH2ΔC12P纤维素分解能力差异。结果:1.HH2菌株存在sRNA-C12P,且随着培养基中纤维素浓度升高sRNA-C12P表达量呈上升趋势;2.同源置换构建HH2ΔC12P成功;3.对比C12P基因敲除前后菌株生长情况和纤维素分解能力,发现HH2ΔC12P在全纤维素培养基中适应期时间长于葡萄糖培养基,在葡萄糖培养基中生长速度大于纤维素培养基;原菌株纤维素水解圈直径为28.04±0.22mm,C12P基因敲除后,菌株纤维素分解能力降低,纤维素分解圈直径为10.96±0.12mm。综上所述,大熊猫肠道枯草芽孢杆菌HH2分离株中含有sRNA-C12P,并在纤维素培养基中大量表达,敲除sRNA-C12P基因后,菌株在高纤维素环境胁迫下的适应能力降低。刚果红纤维素钠试验显示HH2ΔC12P纤维素分解能力被抑制,但没有彻底消失,说明C12P对HH2株适应高纤维环境具有一定的调控作用,但其具体调控机制仍需要进一步研究。更多还原

【Abstract】 The giant panda is a flagship species for the biological diversity protection.It only feeds on bamboo and it eats 12.5 kg bamboo every day.The giant panda owns a relatively short intestinal tract with a single stomach and without cecum result in poor digestibility.B.subtilis can form a tough,protective endospore,allowing it to tolerate extreme environmental conditions.And it is able to secret various kinds of enzymes such as protease,lipase,cellulose to help the digestion.There exists a certain RNA named sRNA which usually smaller than 300 nucleotides and coded by intergenic region but can’t translate into proteins.sRNA plays post-transcriptionally significant roles in substance metabolism,environmental adaptation,quorum sensing,bacterial virulence and so on.sRNA can regulate the expression of gene by combination of target gene with complementary base pairing,hence promote/inhibit the translation of mRNA.We had isolated the B.subtilis HH2 strain from the feces of giant pandas,then cultivated them under different carbon mediums,as the growth of bacteria reached the logarithmic phase then extracted the genome RNA followed with RNA-seq,with bioinformatics method we predicted that the difference of sRNA-Cl2P expression between the cellulose medium and glucose medium is obvious,based on the results we doubt that whether the sRNA-C12P plays a role in the regulation mechanism of adaption under high fiber environment or not,in order to verify the existence of sRNA-C12P and the role it may play in the adaption we conduct the following trials.1.To amplify the C12P gene fragment with the reverse transcription of RNA,to detect the expression difference of sRNA-C12P in different carbon medium by qPCR;2.To construct the sRNA-C12P gene mutant strain by homologous recombination;3.Comparing the cellulose decomposition ability of the strain before/after the knockout of C12P.Results:1.There exsists a certain sRNA-C12P in HH2 strain,and the qPCR shows that sRNA-C12P possesses the highest expression quantity in the cellulose medium,and the higher the cellulose concentration in medium,the higher expression of the C12P;2.Have successfully constructed the targeting vector,and then transform the targeting vector into HH2 competent cell to gain the sRNA-C12P gene mutant strain by homologous recombination;3.Detecte the cellulose decomposing ability of HH2 strain suggests that it owns a high cellulose decomposing ability with a 28.04±0.22 mm Cellulose hydrolysis circle averagely while 10.96±0.12 mm of the mutant strain;the smaller cellulose hydrolysis circle shows that its cellulose decomposing ability obviously reduced compared with the HH2 strain,and the adaptive capacity in high cellulose culture declined dramatically.Compared with the HH2 strain,the mutant strain’s growth is in a much slower pace,besides,the mutant strain forms spores much earlier.It took much more time to live through the adaptive phase in cellulose medium and the slope of growth curve in logarithmic phase in glucose medium is greatest while in cellulose is smallest.In summary,there exists sRNA-C12P in HH2 strain iolated from the Giant Panda Intestines,and with the highest expression quantity in the cellulose medium.The mutant strain shows a reduction of the ability to adapt the high concentration of cellulose after the deletion of sRNA-Cl2P.The cellulose decomposing ability of mutant strain also reduced by the Congo red cellulose test,suggests that the sRNA-C12P owns a role to adapt the high fibre environment,but the specific regulatory mechanism still needs further research.更多还原