RNA Interference-based To Rough Dwarf Diease In The Innovation Of Transgenic Maize

Maize rough dwarf disease is a widespread viral disease in the corn producing areas of China and the world, extremely serious harm to the corn production. The current agricultural practices and chemical control can not effectively control maize rough dwarf disease, breeding resistant varieties is the most fundamental way. However, the rough dwarf disease resistant germplasm resources of current breeding material are limited, only by conventional breeding is difficult to meet the needs of resistant varieties. In this reserch, we introduced the synthesized fragment of maize rough dwarf virus in to RNA interference vector based on the principle of RNA interference and transformed it into immature embryos of HiII maize by Agrobacterium mediated transformation. Transgenic plant lines detected by bar gene PCR,target gene PCR and field inoculated evaluation, expecting to provide the reference for breeding new rough dwarf disease resistant varieties.The main results were summarized as follows:1. RNA interference expression vector containing inverted-repeat sequence was constructed to the gene of maize rough dwarf virus (MRDV). The maize rough dwarf virus and rice black-streaked dwarf virus gene were searched and confirmed by homologous in GenBank database. A size of 558bp gene sequence of maize rough dwarf virus was selected and artificial synthesized in vitro.The fragment was digested by restriction enzyme then sub-cloned into the intermediate vector NPPLT to form the inverted-repeat constructs with intron sequence.The inverted-repeat constructs with intron sequence was link into expression vector PNCXB,which including Cre/loxP recombination system,by appropriate restricuion enzyme digestion and the new RNAi vector was named as PNCXB-848.2. Transformation system of maize immature embryos by Agrobacterium mediat- ed was established. The 1.2-1.8mm immature embryos of HiII maize were used as receptor for Agrobacterium-mediated transformation. OD600 value of 0.5 and 10 minutes infection with Agrobacterium LBA4404 were utilized to transform gene into immature embryos,which were transferred to co-culture medium,resting culture medium,callus induction medium,selection medium containing 3mg/L PPT,selection medium containing 6mg/L PPT and regeneration medium, 112 regenerated plants were obtained from resistant calli. A stable Agrobacterium-mediated transfor- mation system of maize was established.3. System of maize herbicide (PPT) selection and PCR detection was established. In order to determine the appropriate concentration of herbicide (PPT) selection, 30mg/L,100 mg/L,200 mg/L and 300 mg/L , four different herbicide (PPT) concentrations were established. Observed leaf phenotype after five days, 200 mg/L herbicide (PPT) concentration was the lowest concentration of easy to observe. 247 T1 plants were detected by 200 mg/L herbicide (PPT) selection, 92 T1 plants were certified to be positive, the probability was 37.2%. 25 T2 plant lines were detected by 200 mg/L herbicide (PPT) selection, the probability of 11 plant lines were more than 40%,some line even up to 100%.Combined with the detection of bar gene PCR,target gene PCR, a detection system of regenerated plants was established.4. Evaluation resistance of the transgenic plant lines. 13 T2 plant lines together with non-tansformed control W670,susceptible control line Mo17 and resistance control Shen137 were inoculated MRDV in the isolated box by viruliferous planthoppers (Laodelphax striatellus) in the field. The results showed that the probability of susceptible control line Mo17 was 60%, non-tansformed control W670 was 57.1%, resistance control Shen137 showed no obvious symptoms of maize rough dwarf disease disease. Three plant lines were identified as resistance, the probabilities of the three plant lines were 15.8%,0%,0%, respectively, obviously lower than non-tansformed control W 670 and susceptible control line Mo17. Other lines showed varying degrees symptoms of maize rough dwarf disease, A initial identification system of the maize rough dwarf was established.