Study On Cloning And Isolation Of The Disease-resistance Gene From Cabbage(Brassica Oleracea.var.capitata)

Cabbage (Brassica oleracea. var. capitata) is a kind of important vegetable in China, there are about several hundred thousand hectare (ht). But the disease of cabbage is serious , which often cause the yield losses . In China the many achievements about the breeding of resistant disease of cabbage have been obtained, such as a lot resistant material and resistant varieties of cabbage have been developed. But the many resistant materials possess disadvantaged characters such as the linkage with some bad characters. The traditional method preventing from disease such as seed sterilization and chemical control not only takes time, but also pollutes the environment.With development of modern bio-technique and study on molecular biology , the plant genetic engineering shows large potentiality , which is taken advantage of plant resistant disease breed. The foreign resistance gene being introduced crops, enhancing the crops resistance to disease becomes reality. The new variety with good economic characters and high-resistance proceed of plant will be developed more. Construction a cDNA. library is the basis of studying on molecular biology, and is the effective mean of cloning specific gene, special the high eukaryotes gene. Isolation and cloning the target gene is very important useful to studying gene structure, expression and the regulating mechanism. Studying the molecular biology of cabbage falls behind that of some crops in China, in order to cloning some useful gene of cabbage , a cDNA library of cabbage was constructed and the part characterization of the library was identified.Many resistant disease genes were isolated successfully from maize , tomato, tobacco, flax, Arabidopsis, rice, sugar beet, wheat et al . These genes show resistance to virus .bacterial , fungi, and nematode. These genes were mainly cloned by means of positional cloning or map-based cloning, transposon tagging; the two methods need more humen and financial resources . But the homologoussequence screening method is economic and rapid , the degenerate primer is designed according to the conservative domain of the resistant disease gene , the resistance genes or the resistance gene fragments are amplified from DNA or cDNA of resistant disease plant material by PCR, they are used as probes to isolate the resistant gene or resistant gene homologous sequence from the constructed cDNA library of resistant gene material . In this experiment , by the homologous sequence screening method , the cDNA library was constructed from resistant cabbage material and was screened, a candidate of resistance gene had been cloned and isolated , is named as TuR2.. It was studied on its sequence, the ami no acid sequence encoded by the TuR2 gene, the characterization and structure prediction of the protein encoded by the TuR2 gene , then this gene had been transformed into mustard (Brassica Ju/jcea) , the following results were obtained: 1 construction of the cabbage cDNA librarycDNA library was constructed by SMART cDNA library construction Kit Manual . The vector is X TriplEx2 , the library host is E. coli XLl-Blue , the part characterizations of the library were analyzed : the titer of amplified cDNA library is 4 x 10 8 pfu/ml, the insert fragment size is more than 1 kb , the recombinant of rate of the library is about 95%.. The cDNA library had been amplified and stored , the indexes of the library are up to standard . The target gene of cabbage can be isolated and cloned from the cDNA library . 2. Isolation of the resistance gene fragment of cabbageThe domain of ami no acid identity in N gene from tobacco , the RPS4 gene from Arabidopsis and the L6 gene from flax were used to design degenerate primers, primer sequence were as follow: 5' primer: 5' -GGIGGIGTIGGIAAIACIAC-3' ; 3' -primer : 5' -A(A/G) IGCTA (A/G) IGGIA(A/G) ICC-3' ; the fragments of 513 bp were amplified by RT-PCR and genomicDNA PCR from resistant material 84075 , then cloned into multi-clone site of pUCm-T, transform into ?coli DH5 a , the recombinants were screened by blue/w...