Cloning And Functional Characterization Of HOG1 And PBS2 Genes Of The Fungus Alternaria Tenuissima

The high osmolarity and glycerol (HOG) pathway in eukaryotes involves in many processes, suchas expression and regulation of genes induced by drought stress,cell morphology,repression of sexpheromone response and pathogenicity. In this study, we constructed a Alternaria tenuissima cDNAexpression library based on the integrase-excisionase system of bacteriophageλ, screened the library,and identified and characterized the A.tenuissima AtHOG1 and AtPBS2 genes. In addition, weestablished that resistant genes for G418 and hygromycin B could be used as selection markers fordisruption of genes in A. tenuissima. Furthermore, we constructed plasmids for knocking out AtHOG1and AtPBS2 genes using DNA recombination technique, which would provide a basis for further studyon functions of AtHOG1 and AtPBS2 genes. The main results obtained in this study are as fellows:1, The genus Alternaria contains ubiquitous, saprophyte molds, which are difficult for speciesidentification base on morphology. The morphology of the Alternaria sp. strain JH505 isolated in ourlab is similar to that of A. radicina,A. brassicae,A. solani and A. brassicicola. In order to furtherconfirm taxonomic status of Alternaria strain JH505, the rDNA ITS sequence was PCR-amplified fromthe strain. GenBank Database Blast Search and phylogeny tree analysis results indicated that it is astrain ofA. tenuissima.2, A destination vector pRS-DEST42 containing attL1 and attL2 recombination sites wasconstructed. The entry cDNA library of A. tenuissima was transferred into the yeast destination vectorpRS-DEST42 through the LR recombination reaction with the Gateway^? LR Clonase enzyme mixture.As a result, the cDNA expression library has a titer of 2.44×10⁶ (cfu/ml) and a total clones of 2.44×10⁷.The rate of positive recombinants clones was 100% and the size of average insert cDNA was 1.38 kb.3, Twelve transformants of the yeast hog1 mutant could grow on YPD plates containing 1M NaCland contained cDNA clones. DNA sequencing indicated that the 12 cDNA inserts contained the sameopen reading frame encoding the A. tenuissima homologue of ScHOG1 (AtHOG1) with 355 amino acidsin length. AtHoglp shows 96%, 92%, 90% and 88% identities in the amino acid sequence with thehypothetical proteins MG01822.4 (Magnaporthe grisea), UM02357.1 (Ustilago maydis), FG09612.1(Fusarium graminearum), and ScHoglp of S. cerevisiae, respectively. The catalytic domain in ScHoglpis highly conserved in its homologous proteins, and the three catalytic sites as well as the lysine residueinvolved in the ATP-binding in ScHoglp are also present in its homologues. These observations suggestthat Hoglp is highly conserved in these filamentous fungi.4, From 1.6×10⁵ library cDNA transformants we isolated one cDNA clone that could complementthe salt sensitivity of the yeast pbs2 mutant. The cDNA insert was sequenced to have a size of 2,492 bpin length, which encodes a protein of 683 amino acids with 42.6% sequence identity to that of ScPbs2p,which indicates this cDNA sequence encodes the A. tenuissima AtPbs2p. AtPbs2p also shows 55.3%,50.2% and 48.6% sequence identities with its sequence homologues of Aspergillus fumigatus (XP₇52961), Magnaporth grisea (XP₃66048) and F. graminearum (XP₃88867), respectively. Thekinase catalytic domain of ScPbs2p was conserved in its homologues of those filamentous fungi.5, Our study indicates that A. tenuissima could grow on YPD plates containing 1M NaCl. PlasmidcDNA from positive transformants were then individually reintroduced back to the hog1 and pbs2mutant to confirm their sodium-tolerance phenotypes, respectively. As a result, we confirmed that alltransformants contained cDNA clones conferring the sodium-tolerance phenotypes in the yeast hog1and pbs2 mutant. AtHOG1 and AtPBS2 genes complement the functions of ScHOG1 and ScPbs2p insodium tolerance, respectively.6, Drug sensitivity tests indicates that A. tenuissima could not grow on YPD plates containingG418(100μg/ml) and Hygromycin B(100μg/ml) in this study, so we established that resistant genes forG418 and Hygromycin B could be used as selection markers for transformation ofA. tenuissima.7, We constructed recombinant vectors for knocking out AtHOG1 and AtPBS2 genes through DNArecombination, which contains about 500 bp upstream and downstream DNA fragments of AtHOG1 andAtPBS2 genes with the G418 resisitence gene inserted between them. This provides a basis for furtherstudy on functions of these genes in A. tenuissima.