Studies On Maize Dwarf Mosaic Virus Gene Genetic Transformation

Maize(Zea mays L.) is one of the most important crops, staple food , forgage crop and industry materials. However, huge yield loss and deteriotation of quality of cultivated plants were caused due to various diseases. The crop breeding of disease resistance is limited because of lacking the resistance resource and the hereditary isolation of different plant species. Plant gene transformation can solve the proplem to improve genetic melioration and enhance resistance to various diseases and stress of maize by gene engineering technology. The current advancement of biotechnoloy has ushered agricultural reasearch into a new ear of transgenic organisms. Great attention has been paied to maize gene transformation at home and abroad. The maize inbred lines with insect resistance and herbicide resistance have been released in commercial production. However, there is a few reports about transgenic maize with MDMV sesistance . Today, Maize Dwarf Mosaic virus(MDMV) disease remains one of the major factors limiting crop prodution world wide. MDMV disease in China was mainly caused by Sugarcane mosaic virus(SCMV), Sorghum mosaic virus (SrMV) and Pennisetum Mosaic Virus(PenMV). There are many strategies to obtain resistant-virus plant. Many researchs showed that RNA interference (RNAi ) was an effective way to obtain virus resistant plants with high level resistance or immunity to virus and long duraion.In order to select and establish the regeneration and transformation systems of maize elite inbred lines, to investigate effects of RNAi in Maize Dwarf Mosaic virus-resistance gene engineering, immtature embryos of 22 maize inbred lines were cultured to compare the efficiency of induced embyrogenic callus and regenerated plants. The effect factors (genotype, hormone content of medium, immature embyo length, scutellum direction, subculture time ,ect.) influcing the induction and regeneration of embryogenic callus were systematically studied. The relationship between inheritance and maize immature embyo culturing capacity was analyzed using 14 SSR primers with rich polymorphism. The genotypes with the high efficiency of embryogenic callus and plant regeneration were screened.We inserted PenMV HC-Pro and SCMV CI gene-specific sequences into the binary vector pCAMBIA3301 in the sense and antisese orientations (named p3301HCIR and p3301CIIR) on the basis of RNAi. Then the p3301HCIR and p3301CIIR were transformated into maize by Agrobacterium tumefaciens-mediated. The fators (A. tumefaciens cell density, infectrion duration, cocultivation duration, cocultivation temperature, treatment of negative pressure, sonication treatment, ect. ) influencing the efficiency of transformation mediated by A. tumefaciens were researched. The main results were summarized as follow:1. The two expression vectors (p3301HCIR and p3301CIIR ) were constructed on the basis of RNAi.The conserved sense sequence of HC-Pro gene was confirmed by homologous analysis between PenMV-B and PenMV-C. The conserved sequence of CI gene was confirmed by the comparison of SCMV–BJ, SCMV-ZJ, SCMV-SX, SCMV-SD gene sequence. The fragments of HC gene and CI gene to construct hairpin dsRNA were cloned from cDNA of PenMV HC-Pro and SCMV CI by the method of PCR on the basis of the conserved domain of HC-Pro and CI gene, respectively. Then we inserted PenMV HC and SCMV CI gene-specific sequence into the binary vector pCAMBIA3301 in sense and antisese orientiation(named p3301HCIR and p3301CIIR). The two vectors were transformed to A. tumefaciens by liquid nitrogen freeze-thaw method.2. The more effective acceptor system of immature embyo culturing was established of maize inbred line.The immature embryos of 22 maize inbred lines were cultured to compare the efficiency of induced embyrogenic callus and regenerated plants. The factors influencing maize immature embryo culturing were optimized. The results showed that the genotype, subculture time, hormone concentration in medium, AgNO3 concentration, ect. were important influencing factors for the induction and regeneration of embryogenic callus. 3mg/L 2,4-D, 0.2 mg/L IAA and 8~12 mg/L AgNO3 added in induction medium were favorale to induction embryogenic callus; 1mg/L KT and 0.5mg/L IBA added in regeneration medium were favorable to plant regenerating and rooting. The induced callus was subcultured for 5~7 times(each time for 20 days) with more high efficiency of embyogenic callus and regenerated plants. 5 genotypes, E28, Lv9, Qi319, Huangzao4, 502, were screened with the efficiency of about 83.5% embryogenic callus and 80% regenerated plants.3. Molecular system to select the immature embryo culturing capacity of maize inbred was established.The genetic relationships of 22 inbreds were analyzed using 14 SSR primers (mmc0022, bnlg1671, phi308707, phi96100, umc1165,bnlg1018, phi090, phi37418, umc1122, mmc0071, umc1359, phi080, bnlg1538, phi015) with rich polymorphism which were further chosen from 30 ploymorphism SSR primers. Results showed the consistencies were average 88% with the clustered results on the single character(embryogenic callus efficiency and regenerated plant efficiency)and both characters. The inbreds with more close genetic relationship was more similar to immature embryo culturing capacity. Therefore, the maize immature embryo culturing capacity could be correctly estimated by polymorphic loci from the14 SSR primers. The clustering method by the SSR provided a basis for forecasting and selecting the immature embryo cultuting capacity of maize inbred line.4. The system optimized relatively of genetic transformation of maize immature embyo mediated by A. tumefacienens was preliminarily established.Embryogenic callus of maize inbred lines E28, Lv9, Qi319, ect. were used as aceptor materials. The expression vector p3301HCIR was researched for maize gene transformation mediated by A. tumefacienen. The factors influcing the efficiency of transformation mediated by A. tumefaciens were systemically studied. Results showed the trains of A. tumefacienen, A. tumefaciens cell density, infection duration, coculturation duration, treatment of negative pressure, sonication treatment, AS concentration and screening method were important influencing factors for transformation of maize meidiated by A. tumefacienen. The concentration of A. tumefacienen strain LBA4404 at OD600=0.5~0.7, 100μg/L acetosyringon(AS) in both infection medium and cocultivation medium, the duration of infection for 20~25min, the duration of cocultivation for 3 days, and screening for 3 cycles with PPT 5mg/L-10mg/L-5mg/L were optimal for transformation. With teatment of negative pressure for 10min during infection of embryogenic callus, the ratio of resistant callus was highest (about 20.2%). The frequency of resisant callus was affected by A. tumefacienen concentration, infection duration and cocultivation duration together. But the A. tumefacienen concentration and infection duration were more important factors. 18 transgenic plants ( E28 5, Qi319 11, Lv9 2 ) had been obtained via the detection of PCR. In this study , the frequency of resistant callus and transgenic plants obtained from Qi319 were more than that of from E28 and Lv9. So Qi319 was better as acceptor for maize gene transformation .5. The detection and genetic analysis of transgenic maize plants.8 independent transgenic plants were obtained from 9 plants after Southern bloting hybridization screening. The result showed the PenMV HC-Pro gene has integrated into maize genome including 6 single copy and 2 double copies plants. The frequency of single copy was 66.7%. Finally, 4 transgenic plants( E28 2 plants, Qi319 2 plants) seeded naturally. The segregations of PenMV HC-Pro in T1 progeny of transgenic maize were different. Q1,Q2 and E2 transgenic maize lines showed Mendelian segregations. The T1 plants showed different level resistance to PenMV after inoculation. Trangenic maize lines provided good germplasm resource of resistant-virus for maize genetic improvement.6. SCMV CI gene was transformed into maize genome by A. tumefacienen.The expression vector p3301CIIR was transformed into maize inbred E28 by A. tumefacienen by the established genetic transformation system. The results of PCR and PCR-Southern blotting primarily proved intergration of the targeted segment into the E28 genome. The frequency of transformation was 1.25% and 5 independent transgenic maize plants were obtained. Therefore, the established genetic transformation system laid a foundation for maize genetic improvement by gene engineering technique in future.