Identification And Premilinary Study Of HIS3 And LEU2 Genes Of The Fungus Alternaria Tenuissima

Alternaria tenuissima is a plant pathogenic fungi,it has enormous harm in agriculture,but also used in biological control,environmental protection,and so on.It is important to study the regulatory mechanism of functional gene in Alternaria tenuissima. In Eukaryotic microorganisms,LEU2 and HIS3 genes is an important marker of nutrition. In this study,we screened the Alternaria tenuissima cDNA expression library in auxotroph yeast,and identified and characterized the A.tenuissima AtLEU2 and AtHIS3 genes.A recombinant vectors for knocking out AtLEU2 and AtHIS3 genes was constructed,and inserted the G418 resisitence gene as selection markers.This provide a basis for further study on functions of AtLEU2 and AtHIS3 genes.The main results obtained in this study are as fellows:1.Alternaria tenuissima cDNA expression library was tansformed into yeast strain with LEU2 deletion mutant,and there were 3 transformants could grow on SD-LEU medium.The result of DNA sequencing indicated that the 3 cDNA inserts contained the same open reading frame encoding the A.tenuissima homologue of ScLEU2(AtLEU2) with 363 amino acids.Atleu2p shows 94%,74%,71%and 69%identities in the amino acid sequence with CAB72262(P.nodorum),EDN21270(B.fuckeliana), XP₃86851(Gzeae) respectively,and also has homology with beta-isopropylmalate dehydrogenases of XP₃67617(M.grisea)2.Alternaria tenuissima cDNA expression library was tansformed into yeast strain with HIS3 deletion mutant,2 transformants were obtained from SD-HIS medium.The cDNA insert have a size of 717 bp in length,which encodes a protein of 238 amino acids. It was homologue of Sc tHIS3,the protein encided by AtHIS3 was named as AtHis3p. AtHis3p also shows 100%,90%,68%,65%,63%,64%and 66%identities with BAF69039(A.alternata),EAT83561(P.nodorum),EDN23196(B.fuckeliana), XP₃86269(G.zeae)),XP₃67617(M.grisea),P34041(T.harzianum)) and XP₉61386(N.crassa) respectively.3.We constructed recombinant vectors for knocking out AtLEU2 and AtHIS3 genes through DNA recombination,which contains about 500 bp upstream and downstream DNA fragments of AtLEU2 and AtHIS3 genes with the G418 resisitence gene inserted between them.This provides a basis for further study on functions of these genes in A. tenuissima.4.Drug sensitivity tests indicates that A.tenuissima could not grow on YPD plates containing G418(100μg/ml).The genetic transformation system was established base on the test,which used G418 resisitence gene as selection marker.