Studies On Molecular Mechanism For Resistance To Maize Dwarf Mosaic Virus

MDMV (Maize dwarf mosaic virus) is one of the most important virus causing serious yield losses for susceptible maize cultivars in the world. Owing to the non-persistant transmission, control of aphid vector by chemical means is not effective and, therefore, cultivation of resistant maize varieties is the most efficient method of virus control. MDMV stain A, B and KS1 distribute widely in the United States and Europe, but, only MDMV-B (MDMV strain B) was universally found in China, nevertheless, it has been becoming economically important for maize losses in many areas in China. To prevent the yield losses caused by the pathogen, and to screen and cultivate the resistant materials, it is necessary that carrying out the research on molecular mechanism for resistance to MDMV-B. In this paper, the resistance to MDMV for maize was studied with two means, one was locating resistant QTLs (quantitative trait loci) by SSR (simple sequence repeat) marker, the other was transforming MDMV CP gene (coat protein gene of MDMV-B) into maize genome via particle bombardment. To date, the results were as follows:1. A F₉ RIL (recombinant inbred line) population including 239 RILs were developed by a cross of Mol7 (highly susceptible to MDMV) X Huangzaosi (highly resistant to MDMV).2. A total of 370 microsatellite (SSR) markers mapped on the 10 maize chromosomes were chosen to screen the parental lines Mol7 and Huangzaosi for polymorphism . As a result, 124 polymorphic SSR markers were identified and employed in assay of the RIL population. At last, 103 SSR markers were placed on a framework with Mapmaker version 3. Ob software with a total map length of 1455. 4 cM, the RIL map has an average SSR marker density 14. 1 cM. the marker order was quite consistent and in agreement with published IBM map. So, the map can be used to locate QTLs.3 . 239 segregant RILs and parents were identified by artificial rub-inoculating MDMV-B in the three-leaf or four-leaf stage, the inoculated leaf is the youngest one for every plant. After two week, each plant was inoculated once more. DI (Disease index) was evaluated for genetic analysis after tasselling. The results showed DI ranged from 0 to 100% among the population, CV (coefficient variance) was 62. 19%, furthermore, two kurtosises displayed in the distributing map of DI, this suggested that major genes were maybe involved in the resistance to the virus.4. We used the method of CIM (Composite Interval Mapping) of Windows QTL Cartographer version 2. 0 software, to scan QTLs with intervals of 2 cM between markers and putative QTLs with a window size of 10 cM, and to analyze the geneticeffect of putative QTLs. The results were that 2 QTLs (named Qmdmvla and Qmdmvlb)linkaged with Umcl753 and Ylssr were detected, the LOD scores were 58.41, 45.12, respectively; the additive effects were -37.19%, -37.14% respectively; and the determination coefficients were 75.43%, 75.23%, respectively.5. The MDMV CP gene was cloned by RT-PCR method. Sequencing showed that this gene is 920 bp , with open-reading-frame encoding 219 amino acids (GeneBank accession number: AF540989) . By comparing its sequence with related genes in GenBank, we concluded that the cloned cDNA fragment derived from MDMV strain B.6. We cloned MDMV CP gene into the expression vector pMCB01-3 harboring Bar expression cassette. For MDMV CP gene , the promoter is 35S (cauliflower mosaic virus 35S promoter), and the terminator is Nos (nopaline synthase terminator). The expression plasmid can be used to transform plant via particle bombardment.7. The MDMV CP gene was introduced into the embryonic calli derived from immature embryos of elite inbred 18⁵99hong and 18-599bai via particle bombardment. Bombarded calli were selected on selection medium containing 5 10 mg/L ( PPT ) Bialaphos. From resistant calli, 79 plantlets were regenerated. At last, 18 of 79 were grown and harvested.8. PCR (Polymerase chain reaction) was used to demonstrate the introduced gene for To regenerated plants, the results showed the expected about 920 bp fragment in 15 plants, including 12 18-599hong and 3 18-599bai, but the fragment was not present in the nontransformed 18⁵99hong. Furthermore, we chose 7 of the 15 plants for Southern hybridyzation analysis, the results was that all the chosen plants were positive with one to four copies of CP gene. So, MDMV CP gene has been integrated into the genome of the resistant regenerated plants.9. PCR-positive progeny plants were artificially inoculated with MDMV-B, and the average chlorosis of the functional leaves of each plant was investigated. The typical symptoms were observed from the leaves of the control inbreds. While, the presence of the MDMV CP gene provided resistance to inoculation with MDMV-B. For the T2 and T3 progenies origined from Tl transgenic plants expressing highly resistant to MDMV, they were still highly resistant to MDMV, the results showed that the resistance could be heritable.