| The GGDEF domain,as the core site of the important synthase of c-di-GMP signaling pathway in bacteria,plays an important role in regulating the life activities of bacteria.In this experiment,four genes containing GGDEF domain（MS82GL0000189,MS82GL002587,MS82GL005092,MS82GL004701 genes,hereinafter referred to as 0189,2587,5092,4701 genes）were selected from the whole genome sequence of Pseudomonas fluorescens MS82 for knockout.In addition,four GGDEF domain-containing genes deletion mutants were obtained.Furthermore,to explore the biocontrol bacteria-Pseudomonas fluorescens,the wild strains and mutant strains（four mutant strains and the previously obtained random mutant strain MT19）were studied on the antifungal activity,biofilm formation ability,polysaccharide content,and movement behavior and so on.The effect of GGDEF domain-containing genes on c-di-GMP signaling pathway to regulate bacterial life activities,complementing the working mechanism and signaling network of c-di-GMP.At the same time,in terms of application research,it provides theoretical basis for the development and application of Pseudomonas fluorescens as a biological pesticide.The specific research results are as follows:1)Based on the gene sequence information containing the GGDEF domain in the MS82 whole genome sequence,the results show that the lengths of the 0189,2587,5092 and 4701 gene sequences are 1932 bp,2082bp,2124 bp and 3822 bp,respectively.The upstream and downstream fragments of the four target genes were amplified respectively,and recombinant plasmids PEX18-0189-S17λ,PEX18-2587-S17λ,PEX18-5092-S17λ and PEX18-4701-S17λwere constructed through vectors.The corresponding GGDEF domain gene del etion mutant strains MT0189,MT2587,MT5092 and MT4701 were successfully constructed by means of protoplast homologous recombination.After successfully constructing the recombinant cloning vector and transfer vector corresponding to each target gene,PCR and double digestion were verified to be correct,and the sequence comparison was carried out in NCBI.After multiple generations of culture,mutant strains can be stably inherited.2)Compared with the transparent inhibition zone produced by the wild strain MS82 using the inhibition zone method,the bacteriostatic activities of the mutant strains MT0189,MT2587,MT5092 and MT4701 against Trichoderma viriens were all decreased.The antibacterial activity of the wild strain MS82 was significantly different from the mutant strains MT19 and MT4701,which was constructed by different knockout methods for the same gene（4701 gene）,and they had no significant difference in the antibacterial activity.The antifungal activity of each strain from high to low was MS82> MT0189> MT5092> MT2587>MT4701> MT19.3)At the same time,the plate confrontation method was used to detect whether the wild-type strain and the mutant strain had influence on the mycelial growth of different varieties of edible fungi.We selected 5 common edible fungi varieties that are easily infected by pathogenic bacteria-Trichoderma virens: Pleurotus ostreatus,Flammulina velutipes,Pleurotus eryngii,Auricularia auricula,and Hypsizygus marmoreus.Certain inhibitory effect,the effect was very weak,no obvious effect on the growth of mycelium.The results indicated that the wild strain MS82 and the mutant strains were safe for the above five kinds of edible fungi.4)From the results of the microplate quantitative detection method,it can be seen that the mutant strains MT0189,MT5092 and MT4701 after knocking out the GGDEF domain have significantly lower biofilm formation ability within 48 h than the wild strain MS82,while the mutant strain MT2587 has higher biofilm formation ability in each period.The amount of film formation was consistent with the degree of color difference of the purple ring on the wall of the test tube obtained by the small test tube method at 48 h.At the same time,synthesizing the biofilm formation rate of each strain formed by the data measured every 12 hours,it was found that there was a difference in the formation rate of biofilm among the strains.5)Simultaneously detect the corresponding polysaccharide content in each time period of determining the biofilm formation amount of each strain.The data measured at 12 h showed that there was a difference between the wild strain and the mutant strain（p<0.05）;there was a very significant difference between the wild strain and the mutant strain at 24h（p<0.01）;There was no significant difference between the wild strain and the mutant strain at 36h（p>0.05）;at 48 h,there was no significant difference between the wild strain MS82 and the mutant strain MT2587（p>0.05）,and there was a difference between the mutant strains MT4701 and MT19（p<0.05）,and there was a very significant differences between mutant strains MT0189 and MT5092（p<0.01）.Comparing the polysaccharides between MT4701 and MT19,it was found that the two showed extremely significant difference（p<0.01）only at24 h.6)Through the exercise ability test,it was found that the swimming movement,swaming movement and twitching movement of mutant strains MT0189,MT2587,MT5092,MT4701 were all reduced to varying degrees compared with MS82.Among them,the difference between swimming movement and swarming movement is more significant than twitching movement.The diffusion diameters of the three types of movements of the mutant strain were smaller than those of the wild strain,and the trajectory was lighter,while the outermost circle of the individual strains was irregular like a circle,which was significantly different from that of the MS82 strain（p<0.01）.There was also a very significant difference between MT19（p<0.01）.Based on the above experimental results,it is concluded that the gene containing the GGDEF domain plays a key role in the second signal in MS82 of Pseudomonas fluorescens MS82 in mediating the various life activities of bacteria which was regulated by c-di-GMP,and it can be genetically modified as a safe and effective biocontrol bacteria for edible fungus Trichoderma disease. |
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