Construction Of RNA Interference Expression Vector Resistant To Maize Rough Dwarf Disease And Agrobacterium-mediated Genetic Transformation

Maize rough dwarf disease (MRDD) is a worldwide viral disease caused by maize rough dwarf virus (MRDV),Rice black streaked dwarf virus (RBSDV) or Mal de Rio Cuarto virus (MRCV), and persistently transmitted by Laodelphax striatellus Fallen in a circulative manner. In recent years, the disease was epidemic in our part of the main producing areas of corn and caused serious economic losses. 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. Currently, maize rough dwarf disease resistance breeding mainly using genetic resistance of maize germplasm. However, high resistance to rough dwarf virus is a lack of germplasm resources. The work of resistance breeding is a long process. It is difficult to meet the production requirements through conventional breeding.RNA interference (RNAi) is an evolutionarily conserved 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. Therefore, based RNAi principle, this study use part of MRDD outer coat protein gene and the core coat protein gene to construct RNAi expression vector and transform to maize by Agrobacterium-mediated in order to obtain positive transgenic plants. And make it self homozygous for the subsequent identification of disease resistance provide homozygous transgenic lines. The results are as follows:1) The intermediate vectors pSK-303,pSK-452,pSK-291 were constructed with inverted repeats, which could be applied to other RNA interference. Firstly, sequences of MRDV CP genes S10 and RBSDV CP genes S10 and S8 submitted on NCBI were analysised, and 303bp,452bp,291bp fragment of the conserved domain were selected as RNAi target sequence. Two pairs of primer with restriction sites were used to amplify the sense and anti-sense fragments, with double enzyme digestion, the sense and anti-sense fragments were linked into intermediate vectors. In order to produce a intron-hairpin structure, there is a maize intron between sense and anti-sense fragment. 2) According constructed with inverted repeats of intermediate vector pSK-303, pSK-452, pSK-291, became applicable to build three plants expression vector by Agrobacterium-mediated. They are pRNAi-303, pRNAi-452 and pRNAi-291. Using SpeI and XhoI restriction enzyme cut inverted repeat sequence, connected to the plant expression vector pJIM19, construction RNAi expression vector.3) The pRNAi-303 expression vector was transformed into the Agrobacterium strain EHA105 by freeze-thaw method and used to inplanta transform the apical portion of "18-599" mediated by Agrobacterium, and obtained 3 independent positive plants. PCR detection of T1 generation, obtained 23 positive independent transgenic lines. Two other expression vectors were genetic transformation. Maize inbred line "18-599" embryogenic callus was the receptor of genetic transformation. After screening with 1.5,3,5 mg/L Basta, the resistant callus are being differentiated.