The Effect Of Hfq Protein For Giant Panda Bacillus Subtilis’ Adaptive Ability In High Fibre Environment

Bacillus subtilis is a typical Gram-positive bacteria,as one of the most famous animal intestinal probiotics candidates,not only because of its excellent cellulolytic activity,but also it can help the animal to maintain the balance of intestinal flora by producding antimicrobial peptide and creating an anaerobic environment of the intestine.The high fiber environment is a stressful environment for Bacillus subtilis,so adapting to the giant panda’ intestinal high fiber environment is the premise of its various probiotic functions.We want to find the key regulatory factor in the process that Bacillus subtilis adapt to giant panda’ intestinal high fiber environment.,and then we can transform bacteria by genetic engineering technolog to get Bacillus subtilis which have better ability to adapt giant panda’ intestinal high fiber environment and be able to function better.Hfq protein is widely present in prokaryotes and serves as a key factor in post-transcriptional regulation and plays an important role in the regulation of multiple life activities of bacteria.However,it is not clear whether Hfq protein plays a role in regulating the process of Bacillus subtilis to adapt to the process of giant panda’s high intestinal fibrous environment.In order to clarify this,This study knocked out the hfq gene of Bacillus subtilis HH2 and studied the ability of isolates and deletion strains to adapt to high fiber environment.The main research work is as follows:1.Based on the homologous recombination principle,we constructed hfq gene mutant by suicide vector system and we named it B.subtilis HH2△hfq.2.Cultivating B.subtilis HH2 and B.subtilis HH2△hfq in the glucose medium(C),mixed medium Ⅰ(C2F1),mixed medium Ⅱ(C1F2)and cellulose medium(F)and mapping growth curve.3.Determination the Cellulose Decomposition Ability of B.subtilis HH2 and B.subtilis HH2△hfq by Congo Red Cellulose Tablet Decomposition Test and Filter Paper Disintegration Test.4.Cultivating B.subtilis HH2 and B.subtilis HH2△hfq in the glucose medium(C),mixed medium Ⅰ(C2F1),mixed medium Ⅱ(C1F2)and cellulose medium(F).The relative expression of cellulase gene was measured by quantitative real-time PCR which based on the cellulase synthesis gene,Cel042.The results are as follows:1.Successfully constructed B.subtilis HH2△hfq,the hfq gene deletion strain of B.subtilis HH2.2.The growth curve shows that the growth of B.subtilis HH2 and B.subtilis HH2△hfq is not very different in all-glucose medium.In each group of cellulose medium,compared to B.subtilis HH2,the adjustment period of B.subtilis HH2△hfq is prolonged,the logarithmic slope is decreased and the number of bacteria in the stable period is decreased,which reflects the ability of B.subtilis HH2△hfq to adapt to high fiber environments deterioration.3.The results of the cellulose decomposition test shows that the Dp value and he disintegration rate of the filter paper were significantly decreased after the hfq gene deletion.4.Fluorescence quantitative PCR results shows that the expression of endoglucanase gene Cel042 in B.subtilis HH2△hfq was down-regulated in each group of cellulose medium.Comparison between groups shows thatthe expression levels of endoglucanase gene Cel042 were the lowest for both B.subtilis HH2 and B.subtilis HH2△hfq In the whole glucose medium.increased with the increase of cellulose concentration in the medium,but the increase of Cel042 gene in B.subtilis HH2△hfq was less than that of B.subtilis HH2.The results show that we Successfully constructed B.subtilis HH2△hfq,the hfq gene deletion strain of B.subtilis HH2.And we found that the deletion of hfq gene had no obvious effect on B.subtilis HH2’ ability to adapt to glucose environment,but had a significant effect on its ability to adapt to high fiber environment.It is presumed that the Hfq protein can increase the cellulolytic capacity of B.subtilis HH2△hfq by increasing the expression of endoglucanase gene Cel042 in high fiber environment,so as to improve its ability to adapt to high fiber environment.After hfq gene knockout,HH2 strain could still increase the expression of Cel042 gene to some extent when the concentration of environmental cellulose increased,which may reflect the other regulatory mechanisms in the process of B.subtilis HH2 adaptation to high fiber environment and this is for further study.