Cloning And Homologic Analysi Of Part Sequence Of Rice Blast Resistance Gene

Degenerate oligonucleotide primers were designed on the basis of nucleotide -binding site (NBS) motifs conserved among the disease resistance genes that have been cloned, and two fragments of resistance analogues were obtained by using the genomic DNA from the rice cultivar resistant to blast as template and by means of PCR. By means of thermal asymmetric interlaced PCR (TAIL-PCR), two DNA fragments neighbor to the sequence of Pi-2 (t) gene were also obtained by using the tail end sequence of Pi-2 (t) gene as specific nested primers and a short arbitrary sequence designed on the basis of the conserved amino acid of rice polyubiquitin as degenerate primer. All the obtained DNA fragments were respectively cloned into pGEM-T Easy Vector. Then the recombined vectors were transformed into and amplified in E. coli DH5 a . By screening and confirming with EcoR I digestion, we obtained nine negative clones (designated as CR271. CR272, CR273, CR274, CR275, CR371, CR372, CR373and CR374) contained the resistance gene analogues and two clones (designated as JR2 and JR3) contained fragments neighbor to the sequence of Pi-2 (t) gene. The sequence analysis indicated that eight of the nine analogues contained the conserved motifs of NBS-LRR of disease resistant genes, such as P-loop(Kinase la), Kinase 2, Kinase 3a and trans-membrane domain. The sequence of amino acid of clone CR271 was highly homologic (61% identify and 76% similarity) to blight resistance gene Xal of rice to Xanthomonas oryzae pv. Oryzae. All other fragments showed significantly amino acidhomology to the NBS-LRR type resistance gene that have been cloned. The length of JR2 and JR3 fragments were 1321bp and 896bp respectively, which did not showed significantly homology on the level of amino acid and nucleotide acid to the sequences that have been known.At the same time, using the genomic DNA of blast sensitive cultivar C039 as a template, two fragments were obtained that were the same large DNA sequence as resistance cultivar C101A51. One sequence has conserved motifs of NBS-LRR type resistance genes, the other sequence can' t read through.