Study On Isolation Of Resistance Gene Homologous Sequences In Cabbage (Brassica Oleracea Var Capitata L)

The sequences of cloned plant disease resistance genes show that majority , whether conferring resistance to viral, bacterial, fungal pathogens or nematode, contain similar sequences and structure motifs . According to the similarities among resistance genes to design the primers, large quantity of resistance gene homologous sequences(RGHSs) have been obtained by polymerase chain reaction from many plant species . It is possible to use these amplification products as probes for the isolation of genes which conferring resistance to different types pathogens from the cDNA or genomic DNA library.In this paper, resistance gene homologous sequences were amplified by PCR from first cDNA strand and genomic DNA in cabbage 84075 which conferring highly resistance to TuMV (Turnip Mosaic Virus). The product of PCR were cloned and sequenced. Using the products of RT-PCR as probe, the genome orgnization of these RGHSs on cabbage was studied, and cDNA library was screened .The main results are as follows: 1.Isolation of resistance gene homologous sequences in cabbageRegion of amino acid identity in the N gene from tobacco, the RPS2 gene from Arabidopsis and the L6 gene from flax were used to design degenerate primers. Primer sequences were as follow:5' primer,5'-GGIGGIGTIGGIAAIACIAC-3'; 3' primer,5'-A(A/G)IGCTA(A/G)IGGIA(A/G)ICC-3'. Two fragments of 513bp were amplified by RT-PCR and genomic DNA PCR, then cloned into multi-clone site of pUCm-T. The cloned vectors were named as pBorl (RT-PCR products) and pBor2(genomic DNA PCR products), respectively. 2.Seqnencing and analysis of Borl and Bor2The results of DNA sequencing show that the identity between Borl and Bor2 are 71.3% at nucleic acid level and 61.4% at amino acid level, respectively. Homologous analysis using BLAST software showed that this two RGHSs were homologous not only to the resistance genes RPS2 and N but also to the genes 12 and L6, all of which are member of the same structural class of plant resistance genes containing NBS-LRR. 3.Restriction fragment length polymorphismsUsing Borl as a probe, genomic Southern blots on cabbage 84075 were detected . The result exhibited that except for two strong bands, many weak polymorphic bands were observed .So we think Borl fragment belong to a multigene family of cabbage . Moreover, they are so homologous between Borl and Bor2 that they should be describe as member of the same multigene family.4.Screening of the cDNA library of cabbageBorl fragment was used as probe for screening of the cDNA library of cabbage. Through two successive screenings, several positive cDNA clones were achieved. One of them was sequenced. The full length of this cDNA cloned is 828bp. It contained an open reading frame of 504bp encoding a sequence of 168 amino acids. Homologous analysis show that it is highly homologous at the amino acid level to several plant analogs of TCTP(Translationally controlled tumor protein). The overall amino acid sequence of this clone shares 90.5%, 77.4%, 77.4% 77.4%, 76.2% and 73.2% identity with TCTP analogs from Arabidopsis, pea, potato, melon, rice and tobacco, respectively. Amino acid homology score of 39.3%, 36.9% and 37.5% were obtained for the TCTP analog of cabbage and proteins from human ,chicken and yeast, respectively.