Identification And Genetic Transformation Of The Disease-Resistant Gene From Arabidopsis Thaliana Against Bipolaris Oryzae

Rice Brown Spot is one of the more serious and destructive diseases of rice around the world. Rice Brown Spot occured in different degree every year, which accouted for the yield reductions of rice and reduced rice grain quality. Therefore, the control of it already became one of the top-priority tasks of rice production nowadays.Arabidopsis thaliana is a plant of Brassica in Crucifereae. A. thaliana genome is 125 Mb, having five chromosomes, which had been sequenced. Doctor Xia discovered some disease-resistant genes by gene chip technology and screened these mutants of these genes. These mutants and Columbia ecotype were experimental materials. In the test, we found Col-0 ecotype was resistant to Bipolaris oryzae, some mutants were susceptive to it, and some mutants were resistant to it. Several disease-resistant genes from A. thaliana against Bipolaris oryzae had been obtained, based on the different disease-resistant of A. thaliana mutants inoculated by Bipolaris oryzae. It established the foundation for functional study and utilization of the disease-resistant genes from A. thaliana against Bipolaris oryzae.The main results were listed as follows:(1) Col-0 ecotype and the mutants were inoculated by Bipolaris oryzae, we discovered Col-0 ecotype was resistant to the isolation and vaccinal area emerged no spot; the mutants represented resistant. Among them, the vaccinal area of M47 mutant represented evident etiolation one day after inoculation. With the time going, the etiolation area spreaded rapidly and appeared locul necrotic. At last the whole leaf seared and died.(2) No.47 mutant was At4g37260.1 insertional mutagenesis. By inquired about the gene notation on TAIR, we confirmed the gene participated in response to salt stress, ethylene stimulus, gibberellin stimulus, jasmonic acid stimulus, salicylic acid stimulus and so on. The gene belonged to the member of the MYB transcription factor superfamily. Southern blotting showed that No.47 mutant had one copy of T-DNA insertion.(3) By the prokaryotic expression technology, cDNA sequence of At4g37260.1 was subcloned to the expression plasmid pET-28a(+) vector.The recombined plasmid, named pET-28a(+)/At4g37260.1, was transformed to E. coli BL21 and induced with IPTG at 37℃. SDS-PAGE illuminated that about 34.85 kD protein was expressed in E. coli BL21, and the protein was consistent with anticipative protein.(4) The expression vector was constructed by inserting At4g37260.1 gene into pCAMBIA1300. Phenotypic restore lines and overexpression lines were established, transferring At4g37260.1 into A thaliana by Agrobacterium-mediated transformation. It established the foundation of further validating the function of the gene.