| The wheat yellow dwarf disease (WYD) is one of the most serious virus disease in wheat. It is caused by barley yellow dwarf virus (BYDV) which was spreaded by aphids. The WYD can reduce the yield of wheat heavily. So prevention and cure for WYD has significance for the high and steady yield of the wheat. Therefor, isolating of rensistance gene against WYD and study on the resisitance mechanisms are widely significant by molecular measure.Two near-isogenic lines, susceptible and resistant to WYDV (wheat yellow dwarf virus) respectively, derived from a F7 line of the cross between the wheat varieties Chuang35050 and Shannong483. And they were employed in this study. We aimed at isolating the gene against BYDV by DDRT-PCR and resistance gene analogs PCR. This study made the following progresses:(1) To strengthen the isolation of resistance gene, seven specific or degenerate primers were designed on the basis of sequences of certain domains conserved among disease resistance genes (R gene). Using these primers instead of the 5′random primers and DDRT-PCR (differential display reverse transcription PCR), the differential display PCR was implemented.The PCR amplification products were separated by 6% denaturing polyacrylamide gels and visualized by silver staining. The results indicated that there were amplification products only amplicated by SP5 and anchor primer pair. We gained and recycled fourteen expressed cDNA fragments.We gained four expressed cDNA fragments Rbdv1-Rbdv4 (registered numbers in GenBank are EU267934-EU267937). The resultes showed close identity to cDNA clone from the seeding, leaves and spike of barley(95%), CDC48 protein from Arabidopsis and rice (81% and 90% respectively) according to BlastN search in GenBank.(2) Regarding Rbdv1 as target sequence, 778bp sequence of 3′end was obtained using RACE (Rapid Amplification of cDNA Ends). We assembled the sequences of the RACE product and Rbdv1 under the software DNAMAN and obtained a 1196bp sequence named A1 (registered number in GenBank is EU267938). The amino acid sequence of A1 included a P-Loop which was the conserved domain of R gene. By BlastN analysis, A1 has show high identity to CDC48 protein from Arabidopsis and rice. CDC48 is one of the AAA-ATPase gene family, and its function is mainly to bind ATP site. Based on alignment of the amino-acid sequences of NBS homologouse (A1) in wheat and the NBS domain of 5 known R genes(N, L6, RPM1, Pib, RPP8) , phylogenetic analysis by DNAstar software showed that A1 had P-Loop domain, but it was not classified together with other 5 R genes.Their relationship was rather disrelated. A1may code one new resistance gene protein.(3)The genomic DNA of two near-isogenic lines for susceptible and resistant to WYDV were amplified by using seven specific or degenerate primers. The results showed that one RGA was obtained. By cloning and sequencing, we obtained a 532bp sequence for RGA. The RGA showed close identity to NBS-LRR resistance gene analogs from wheat (accession number: AF320845) and sorghum (accession number: AF052396) (81% and 90% respectively) according to BlastN search in GenBank. And it also showed close identity to R gene V6 of triticum ventricosum (accession number: AF158635) and Cre3 resisitance analogs protein Cre3-1, Cre3-2 (accession number: AF281284, AF281285) (100%).(4) We amplified the RGA flanking sequence by TAIL-PCR. A 1563bp sequence was obtained and extended RGA by 337bp and 726bp at 5′和3′end by Sequence Assembly of DNAMAN software. By analyzing amino acid of the assemble sequence, its amino acid contained an ORF with 361aa. This ORF contained complete 7 domains of NBS: I, P-Loop; II, kinase-2a; III, kinase-3a; IV, GLPL(A/L)A; V, RCFAYCS; VI, EGF and VII, HDL. Compared with RGA (532bp), TAIL-PCR had successfully amplified the P-Loop and HDL of the upstream and downstream respectively. |
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