Rna Interference-based Transgenic Maize Resistant To Maize Dwarf Mosaic Virus

Maize dwarf mosaic virus (MDMV) is a worldwide pathogenic virus causing serious yield loss in maize. Strategies for the management of the virus diseases normally include control of vector population using insecticides, adjusting seedtime, appropriate cultural practices. However, these methods have their own defects and ineffective because of the non-persistent model of virus transmission by aphids. The deployment of resistant germplasm is an environmentally-sustainable and effective way for controlling virus diseases of maize, but a few germplasms could be used. Furthermore, it is time consuming for the reason that identification and development of resistant inbred lines or hybrids need year-to-year inconsistencies in viral disease pressure.RNA interference (RNAi) triggered by double-stranded RNA (dsRNA) transcribed from transgenic inverted-repeat sequence is a straight forward way for antiviral defense in plants acting as a natural defense mechanism against invasive viruses, and has been proved to be more efficient to defense against viruses than the tranditional breeding for resistant germplasm. In this dissertation, plant expression vectors containing different lengths of inverted-repeat sequences of MDMV CP and P1 genes were constructed and used to transform maize embryonic calli by Agrobacterium-mediated transformation. Regenerated plants were obtained after selection and differentiation. Transgenic plant lines detected by Specific PCR and Southern blotting were conducted by field inoculated evaluation. The maize dwarf mosaic virus gene expression level and the virus protein were indetified by quantitative real-time PCR (qRT-PCR) and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The results about RNA interference-based transgenic maize resistant to maize dwarf mosaic virus were described as follows:1. Hairpin RNA (hpRNA) expression vector containing inverted-repeat sequence was constructed to target to CP and PI gene of MDMVThe MDMV CP and PI gene were confirmed by homologous analysis in GenBank. 100,404 bp conserved CP gene and 150,451 bp P1 gene were selected and synthesized in vitro. Restriction sites were added to the fragments by PCR amplification. The four different length fragments were digested by restriction enzyme then sub-cloned into the vector pSK-int to form the inverted-repeat constructs. The four inverted-repeat constructs were link into plant expression vector pCUbi1390 and pCUbi1300 after appropriate restriction enzyme digestion and the new RNAi vectors were named as pASC100, pASC404, pASP150 and pASP451, respectively.2. Transformation of maize embryonic calli and detection of transgenic regenerated plantsThe immature embryos of maize inbred line 18-599 were used to induce embryonic calli. The embryonic calli as transgenic acceptor system were used for Agrobacterium-mediated transformation. Selection medium with different concentration of hygromycin B were utilized to select the resistant embryonic calli, which were transferred to differentiation and regeneration medium.68,91,46 and 153 regenerated plants were obtained from the resistant calli transformed with pASC100, pASC404, pASP150 and pASP451, respectively.28,33,18 and 20 To plants were certified to be positive by PCR detection, and 21,20,9 and 17 T1 plant lines were confirmed with transgene by Southern blotting, thereafter. Most transgenic plant lines with single transgene copy.Addition of uniconazole S3307 (0.25 mg/L) and ABT root-promoting powder (0.5 mg/L) in regeneration medium showed a significant improvement of hardening in regenerated plantlets, which were stronger and generated a better fibrous root system than the control. Therefore, regenerated and survival plants have been significantly improved.3. Evaluation resistance of the transgenic plant lines4.15,9 and 15 T2 plant lines transformed with pASC100,pASC404,pASP150 and pASP451 together with non-tansformed control "18-599", resistance control "H9-21" and susceptible control line "Mo 17"were grown in a randomized block design.The disease incidence and symptom scale were investigated. The disase index was calculated to identify the resistant grades of the transgenic plant lines. Three out of four lines transformed with pASC100 were identified as intermediate resistance (I), the disease index was significantly lower than non-transformed control "18-599" but significantly higher than the resistant control "H9-21"; Six out of the 15 T2 plant lines transformed with pASC404 were identified as resistance and the resistance scale of four lines were higher than the resistance control "H9-21". However, two lines were susceptible with two copies of transgene in Southern blotting; Resistance scale of all the 15 T2 plant lines transformed with pASP451 were improved compared with non-transformed control "18-599" and six lines were identified as resistance. The resistance scale in four out of the six lines was higher than the resistant control "H9-21". In the eight lines detected as middle resistance, there were two lines with double-copy of transgene. While the only one susceptible T2 line with three copies. The results showed that the resistance of the transgenic lines linked with the length of inverted-repeat sequence and the transgene copies.4. MDMV gene expression level and its protein in transgenic plant linesThe third upper leaf of mechanical inoculated transgenic T2 plant lines transformed with pASC100,pASC404,pASP150 and pASP451 together with control maize inbred lines were used to extract total RNA for reverse transcript reaction. The cDNA was used to detect MDMV expression level by real time quantitative PCR (qRT-PCR). The CP and P1 gene expression level in the resistant transgenic lines and resistant control "H9-21" was significantly lower than the susceptible control "Mo 17" and non-transformed control "18-599". The result of detecting the viral gene expression level by qRT-PCR was in accordance with the field inoculated evaluation.The virus titer of the third upper leaf of mechanical inoculated T2 plant lines transformed with pASP150 together with the control lines was quantified by DAS-ELISA. The virus titers in the resistant transgenic lines and the resistant control "H9-21" were significantly lower than the susceptible control "Mo 17" and non-transformed control "18-599". The result of detecting the viral titers by DAS-ELISA was in accordance with the field inoculated evaluation.