Cloning And Preliminary Functional Analysis Of The Transcription Factor Sl Cmr1 From Stemphylium Lycopersici

Stemphylium Wallroth is an important group of dematiaceous hyphomycetes.Many of its members are common weak parasitic pathogens on plants.They can produce phytotoxins to kill host cells and obtain nutrients from dead tissues.S.lycopersici is one of the members of Stemphylium with strong pathogenicity and wide host range caused stemphylium blight on tomato,pepper,lettuce and other vegetables in China.It was found previously that the virulence of the S.lycopersici strains isolated from lettuce were related to their orange red pigment production.Transcriptome analysis by RNA-Seq showed that the expression of the gene homologous to Cmr1(Colletotrichum melanin regulation)was down-regulated in the strains with low pigment production.In order to explore the function of Cmr1,gene knockout vector and gene complement vector were constructed and transformed in S.lycopersici by PEG-mediated protoplast transformation.The main results are as follows:1.SlCmr1 gene cloning and sequence analysis: Primers were designed according to the S.lycopersici genomic sequence in the NCBI database to PCR amplification the full length and CDS of the SlCmr1 gene from CS12 strain with high pigment production.There are 3 intron of SlCmr1,the full length of the encoded protein is1,005 aa.The SlCMR1 contains two adjacent Zn F＿C2H2 domains and a GAL4 domain,which is a typical transcription factor widespread in fungi.2.The establishment of PEG-mediated protoplast transformation of S.lycopersici: The effects of different enzyme combinations,enzyme concentration,enzymolysis time,enzymolysis temperature,hyphae age and the type and concentration of osmotic stabilizer on protoplast preparation were analyzed.The optimal conditions finally obtained: 10 mg/L kitalase treat the 36 hours old hyphae3.5 h in 0.7 M Na Cl at 28°C can produce good quality and sufficient protoplasts for transformation.10-15 μg plasmids added into 200 μL protoplasts and transformed in PTC solution containing PEG8000,then regenerated in RM liquid medium could obtained tramsformed strains.3.SlCmr1 gene knockout and complementation: The upstream and downstream sequence of the SlCmr1 gene were inserted into p CX62 vector by enzyme digestion and ligation respectively to construct the gene knockout vector.The SlCmr1 gene with its upstream promoter were inserted into the KSTNP vector by restriction digestion and ligation to construct complement vector.Sequencing and restriction enzyme digestion confirmed that both the knockout vector and the complement vector of SlCmr1 were successfully constructed,named p CX62-SlCmr1 and KSTNPSlCmr1,respectively.The plasmid p CX62-SlCmr1 was transformed into the wild-type strain CS12,and the transformants were screened on Hyg plates.The transformants#2-ΔSlCmr1,#20-ΔSlCmr1,#33-ΔSlCmr1 were btained by PCR and Southern blot confirmation.The complement vector KSTNP-SlCmr1 was transformed into gene knockout strain #2-ΔSlCmr1,and the transformants were screened on G418 plates.The complement transformants O-2-SlCmr1,O-4-SlCmr1,GO-2-SlCmr1 were obtained by PCR and Southern blot confirmation.The differences of colony morphology,growth rate and pathogenicity were compared between wild-type,gene knockout strains and gene complement strains.The results showed that the orange red pigment and the pathogenicity of gene knockout strains decreased significantly,however the growth rate increased.The synthesis of orange red pigment,growth rate and pathogenicity of the complement strain were the same as the wild-type CS12.The results showed that SlCmr1 gene affected the pathogenicity of S.lycopersici by affecting the synthesis of orange red pigment.