Cloning And Characterization Of Endochitinase Gene In Trichoderma Hamatum On Inhibiting Pepper Southern Blight

Southern Blight is a widely spreading disease caused by a fungal pathogen fungus sclerotium rolfsli. Many vegetables and crops are infected with the pathogen, and which leads to the reduction of production. Interestingly, Trichoderma spp. fungi in the study was found to be an antagonist to the pathogen of Southern Blight through a series of complicated mycoparasitism. Therefore, Trichoderma spp. has been used a sustainable and effective way to protect the plant from the pathogen.In the early studies, we have isolated 23 strains of Trichoderma spp. in Liling which is a place severely suffered from pepper Southern Blight, and identified them with their ITS(Internal Transcribed Spacer) sequences. We found that 13 strains of Trichoderma spp.have the ability against sclerotium rolfsli. No.13 strain is the most efficient one named Th13. We selected Th13 as our research sample and other five strains with different inhibiting rate towards pathogen as control group. Firstly,we distracted, broke and isolated the cell wall of pathogen to prepare the medium for inducing the production of enzymes and genes expression in Th13 and its control group. Secondly, we detected the enzymatic activities of those three enzymes may play a significant role in the Mycoparasitism which is Chitinase, alkaline proteinase and ?-1,3-glucanse. Then we used SPSS software to analyze the data to find a remarkable correlations between the chitinase enzymatic activities and inhibition rates. Thirdly, We designed the primers according to the coding sequence region(CDS) of Trichoderma hamatum endochitinase gene(cgit42) which we searched on the NCBI website and then clone the gene by PCR. At the same time, the mRNA were extracted from the 6 strains of Trichoderma hamatum by using Trizol kit and we synthesis the cDNA first strand of Th13. Using the first strand of cDNA, the coding sequence of endochitinase were cloned and compared with the cloned DNA sequence, we obtained the information of the exon and intron. We also analyzed the protein molecular mass and predict the secondary and third structure. Simultaneously, all extracted mRNA were utilized in semi quantitative RT-PCR to analyze expression of endochitinase gene. We found out that the gene expression of Th 13 is evidently more in quality than that of other strains in the quatitative analysis of RT-PCR. At last, genome walking method was used in cloning regulatory sequence in the upstream which may influence the activity of the expression on endochitinase gene. Then we obtained the chit42 upstream sequence and compared to the sequences in the public database to analyze the transcription factors in the sequences. Finally we found several transcription factors binding on the sequences which may largely improve the transcription of endochitinase gene. In sum, we recognized endochitinase is one of the main factor of inhibition on sclerotium rolfsli, still the other pathways about how regulatory factors work and the effect of other proteinase in preventing and controlling the pathogen needs further study.