CDNA Library Construction Of Triticum Aestivum-Haynaldia Villosa 6VS/6AL Translocation Line, And Molecular Cloning Of Powdery Mildew Resistance Relative Genes In 6VS/6AL Translocation Line

Powdery mildew is one of the severest diseases in wheat production. A Triticum aes(ivum-Haynaldia viiiosa 6VS/6AL translocation line containing powdery mildew resistance gene Pm21 was developed in Cytogenetic Institute of Nanjing Agricultural University,which is effective against all the current biotypes of Erysiphe graminis .The line is also resistant to the new races of Puccinia strijformis (stripe rust) and Puccinia recondita (leaf rust) in China. Cloning of Pm2 I and the related genes for powdery mildew resistance is significance for understand its resistence mechanism and disease resistance breeding. To construct cDNA library is a prerequisite for gene cloning.In this study,two cDNA libraries were constructed using mRNA from leaves of Triticum aestivum- Haynaidia viiiosa 6VS/6AL translocation line, induced and non-induced with Erysiphe graminis infection. The cloning vector was an expression plasmid vector pSPORTI, introduced into E. coil DH I OB.The eDNA library of induced translocation line contains about 300,000 recombinant eDNA clones, with average insert size of 1.2 Kb. the eDNA library of non-induced transloeation line contains about 500,000 recombinant cDNA clones, and the average insert was 1.25 Kb ,ranging basically from 400bp to 2.2 Kb. The library was successfully screened with the probe of wheat thaumatin-like protein gene pWIR cloned by ourselves. mRNA differential-display reverse-transcription polymerase chain reaction (DDRT-PCR) was used to compare mRNA from leaves of the translocation line induced and non-induced with Erysiphe graminis ,total 35 different cDNAs were obtained.Northern blot analysis confirmed that eight cDNAs showed increased signal with mRNA from the induced leaves.Sequenee and database serehes revealed that four of them are homologous to Giycine max protein kinase GmPK6,Dahsca glomerata thiosulfate sulfurtransferase mRNA, Triticum aesfivum mRNA for putative 1n2. 1-like protein and Arabidopsis thaliana BAC clone Fl 5G1 6 DNA in chromosom 3 respectively,They are all new genes cloned in commen wheat.The other three genes represent wheat chloroplast genes rbcl,psal for large subunit of ribulose 1,5- bisphosphate carboxylase/oxygenase , apoprotein I,Rubisco activase gene and rpoA gene respectively. These wheat genes may be involved in powdery mildew resistance interaction. EcoRI, EcoRV and HindIII-cut genomic DNA of Yanginai V and 6VS/6AL translocation line was used for southern hybridization analysis with cloned cDNAs as probes. The result showed that thiosulfate sulfurtransferase gene (NJS22) had polymorphism bands between all three restriction enzyme-cut two genomic DNAs, and the protein kinase gene (NJS4), 1n2-1-like protein gene (NJS35), beclinl like gene (NJS2) all had some extent differences between two genome. Based on cDNA fragments from DDRT-PCR, RACE technique was successfuly used to obtain full-length eDNA sequences of NJS2, NJS4 and NJS22 genes. Then gene specific primers were designed flanking putative open reading frames (OREs), and three full-length eDNA clones of NJS2, NJS4 and NJS22 genes had been obtained by PCR. Protein database search revealed that NJS2 was significantly homologous to a putative protein in Arabidopsls thaliana (accession number: CAB 7101.1). NJS4 was a wheat serine/threonine protein kinase and NJS22 was a wheat thiosulfate sulfurtransferase gene. 4 The cloned plant disease resistance genes of Pt...