Resistance Genes Screening In Uncinular Necator Induced CDNA Library Of Chinese Wild Grape

With the material of cDNA library been prepared from Uncinula necator resistant grape, Chinese wild Vitis pseudoreticulata clone Baihe-35-1 leaves,the homology sequence of disease resistant gene has been used to screen the resistant genes in the cycled cDNA library.The novel findings are as follows:1. On the basis ofλTriplEx2 can be transformed into plasmid pTriplEx2, the original cDNA library were amplified and then transformed into cycled plasmid library. The library cycling method was optimized as follows: 200μLcompetence E.BM25.8 should mixed with amplified library of less than 104 pfu resulted well isolated and adequate colonies for further screening, more or less cDNA volme caused none or insufficient cycled library colonies.2. The degenerated primers were designed according to the conservative domain GVGKTT in P-Loop of disease resistant genes (N,L6,RPS2), colony PCR was employed to screen the circled cDNA library, positive colonies were identified by plasmid PCR assay, positive plasmids were sequenced and 36 resistance gene analogs were obtained.3. Homology analysis by searching Genbank with NCBI-Blast, and the phylogenetic tree was constructed with ClustalW in the software of DNAStar. Blast result showed that most of them were unknown sequence. 36 resistance gene analogs were devided into two group, one was resistance-related gene, some were homology to registered Chinese wild grape sequence, some were homology to Vitis vinifera water deficit response gene and some were highly homologous to grape Pierce's disease resistance gene; the other was relevant to the berry development,which included 07-No.239,No.80,No.235,No.160,No.100,No.76,No.391,No.190,No.241,No.155,No.178,No.238. Disease related gene can be subdevided into two class:known functional gene and unknown functional gene .