Construction Of MRDV RNAi Expression Vector And In Planta Transformation To Maize Apical Portion

Maize rough dwarf disease (MRDD) is a worldwide viral disease caused by maize rough dwarf virus (MRDV), and persistently transmitted by Laodelphax striatellus Fallen in a circulative manner. In recent years, the disease was epidemic in our main maize cultivating areas and caused serious economic losses due to poor resistance of maize varieties and farming system changes. Therefore, how to control MRDD is an issue of great pratical significance. Screening and cultivating resistant resource is the most economic and effective way to control MRDD. However, because the maize resistance mechanism to viral disease is unclear, as well as the genetic effect is complex, it is difficult to meet the production requirements through conventional breeding.RNA interference (RNAi) is an evolutionarily conserved defence mechanism targeted against invasive or mobile RNA elements. It was demonstrated that silencing can be effectively triggered when invert repeat (IR) fragments deriving from pathogen was introduced in host plants, and endowed transgenic plant resistance to the virus and similar virus. In this study, we explore the use of RNAi to confer maize resistance to MRDV.Firstly, sequences of MRDV CP genes submitted on NCBI were analysised, and 303 bp fragment of the conserved domain was selected as RNAi target sequence. According to the principle of overlapping PCR method, we designed five pairs of ends complementary primers and linked them into the full-length target sequence. After that, another two pairs of primer with restriction sites were used to amplify the sense (R1) and anti-sense fragments (R2), with double enzyme digestion, R1 and R2 were linked into pSKintron to construct the intermediate vector pMRNAi, in order to produce a intron-hairpin structure, there is a maize intron between sense and anti-sense fragment. After that, the fragment contaning "sense fragment-intron-anti-sense fragment" was cut and connected to the pJIM19 vector with SpeI and XhoI digestion. This new vector is the RNAi expression vector and named pERNAi.The pERNAi expression vector was transformed into the Agrobacterium strain C58 by freeze-thaw method and used to inplanta transform the apical portion of "chang 7-2" mediated by Agrobacterium, after screening with 0.2g/L glufosinate (PPT), the resistant plants were transplanted to field. Detected with specific PCR primer of selection marker gene bar and the target gene,3 positive plants were obtained and positive rate was 0.096%. The sequence results indicated that the target gene has been transformed into maize genome. In order to reduce the dependence on tissue culture and genotype limitations in the transgenic process, we have studied inplanta transformation with apical portion as receptor, and optimized the transformation system. It was found that Agrobacterium suspension concentration OD6000.7,11 min infection time and 0.02% surfactant SilwerL-77 were the optimal conditions to obtain resistant plants.