| Bacterial blight caused by Xanthomonas oryzae pv. oryzae is a devastating disease in rice worldwide. The resistance gene Xa4 has been widely used in breeding programs and played an important role in protecting rice from this disease. Using 642 highly susceptible individuals and a random sample of 255 individuals from an F2 population developed from a cross between IRBB4 and IR24, the Xa4 gene was genetically mapped to a region less than 1 cM. A contig map was constructed for the Xa4 region consisting of six non-redundant bacterial artificial chromosome (BAC) clones and spanning more than 500 kb in length. Analysis of recombination events in the Xa4 region located the gene locus to one BAC, 3H8. Assay of the recombinants using the subclones of 3H8 in combination with sequence analysis further narrowed the Xa4 locus down to a 47 kb fragment.Sequence analysis of MH63/3H8 showed the 47 kb fragment contained an LRR-Kinase gene that is homologous with the Xa21 gene and some other genes that don't express. This LRR-Kinase (RKa) may be the allele of the Xa4 resistance gene. The analysis of the mutant found in FI population of IR24/IRBB4 also showed Rka gene in IRBB4 is the Xa4 resistance gene. To obtain the RKa in IRBB4, the total DNA was digested with Spe I and separated with electrophoresis, and the 12 kb region that contain RKa gene was recovered and insert into the pCLD04541. We screened the little library with PCR by the primers designed with the MH63/RKa sequence and obtained a fragment that contain RKa gene. Sequence analysis showed this gene is identical to TQ/RKa in TQ/26N11 screened from TQ/BAC library with MH63/RKa probe. The two fragments were insert into pCAMBIA 1301 and the complement experiment is still in progress. Meanwhile, the MH63/RKa and MH63/RKb were also transformed into the MDJ 8, which is susceptible to the X. 0. oryzae races 1 (PXO61). The transgenic plants were evaluated and found some transgenic plants exhibited a strong resistance to PX061, and this resistance cosegarated with the MH63/RKb.A few reports indicated the Xa3 gene in the IRBB3 was the allele of the Xa4. Xa22 in ZCL cosegregated with the R1506 near the Xa4 locus. A 19 kb fragment containing the RKa and RKb was also isolated from the IRBB3 and ZCL respectively with the same strategy cloning RKa from IRBB4 and sequenced. The result showed IRBB3, ZCL and MH63 share the identical sequence in the coding region. These imply the Xa3, Xa22 and MH63/RKb gene are the same gene. That the resistance of this gene in MH63 shows weaker resistance than that in IRBB3 and ZCL may be due to some repression factor in the MH63.The IR24/RKa was also sequenced. Comparing the sequence with MH63/RKa and IRBB4/RKa, we found an insert of amino acids at COOH-terminal, which resulted from duplication of a short nucleotide sequence. Except this difference, four different amino acids from IRBB4 and eighteen amino acids from MH63 wereidentified, but only Arg in the LRR21 is specific to IR24.The expression of RKa, RKb and RKc was examined with RT-PCR in IRBB4, IR24, MH63, IRBB3 and ZCL, and RKa and RKb express in the leaves at the booting stage in all varieties and RKc don't. The expression of RKa and RKb are not induced by pathogens. We also examined the expression of RKa and RKb in different tissure and found they expessed in the root and leaves at tillering stage and leaves and shelth at seeding and didn't in the panicles and stem at the booting stage.Comparing the MH63/haplotype with TQ/haplotype, the notable difference of the organization of gene family was found, whereas the order and transcription direction of RKa, RKb and RKc is same between two haplotypes. There were some remains of receptor-like kinases (RLKs) between RKa and RKb, RKb and RKc, behind RKc in the TQ/haplotype, but there were not in the MH63/haplotype. The homologous regions between two haplotypes were mainly found in the coding region. Using TQ sequence and MH63 sequence as probe to search the rice genome database with blastn, we obtained some 93-11 sequences. T...
|
PDF Full Text Request