Construction Of SRNA-C12P Mutant Of B.subtilis HH2 Isolated From Giant Panda And Cellulose Decomposition Ability Study Of HH2 And HH2?C12P Strains

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.