Gene Cloning And Function Analysis Of Aspartate Aminotransferase From Race 1 And Race 4 Of Fusarium Oxysporum F.sp.cubense

Based on the comparative transcriptomic analysis of race 1(Foc1)and race 4(Foc4)of banana wilt(Fusarium oxysporum f.sp.cubense)induced with three carbon sources completed by our laboratory,five target genes with low expression in Foc1 and high expression in Foc4 were screened from Foc transcriptomic induced with cell wall in this study,BLAST the transcriptomic data with NCBI database,by which we found comp6613,comp6626 and comp22765 coding hypothetical protein,comp23886 coding aspartate aminotransferase,comp25382 coding manganese transporter.The real-time fluorescence quantitative PCR were used to show the expression changes of the 5 genes after Foc treated banana roots,comp23886 up-regulated obvious and the expression of comp23886 at 48 h was 350-fold increase compared with the expression at 0 h,it increased to 600-fold at 96 h.Although the expression of comp6613,comp22765,comp25382 were up at 48 h and 96 h,the maximum can reach nearly 400 times,but the ratio of up-regulated was far less than comp23886,and after the Foc treatment of banana root,the expression of comp6626 shown downward trend,so we prior chose comp23886 for further research.By the NCBI BLAST we found that aspC is similar to the gene in Fusarium fujikuroi coding aspartate aminotransferase with the homology of 99%.Reference Orherrin named gene of E.coli encoding aspartate aminotransferase as aspC,we also named comp23886 as aspC in this study.The sequences of aspC were analyzed to see the same between Foc1 and Foc4,the full DNA length of aspC were both 1314 bp in Foc1 and Foc4 and their coding regions were 1218 bp with the homology of 98.7%,all of them have 3 exons and 2 introns.The flanking sequences of Foc1-aspC,Foc4-aspC were obtained by TAIL-PCR,by which we constructed the knockout vector,then we used ATMT transformation,successfully obtained the Foc1-aspC and Foc4-aspC gene deletion mutant Foc1-△aspC,Foc4-△aspC.The shape of the Foc1-△aspC and Foc4-△aspC on PDA medium plate was similar to the wild,while the growth velocity and sporulation quantity is lower than the wild type.In this study,the pathogenicity of knockout mutants was verified by root dip inoculation method,the wild-type Foc and Foc1-△aspC,Foc4-△aspC knockout mutant were inoculated four-leaf stage banana.Although the banana fusarium wilt occurred after inoculatting,while the disease after Foc4-△aspC inoculated banana occurred later than the wild type,and the growth velocity and sporulation quantity of the gene deletion mutant is lower than the wild type,indicated aspC also has an effect on the virulence of Foc4.