| Maize dwarf mosaic disease, caused by maize dwarf mosaic virus (MDMV), is widely distributed in the world. In resent years, maize dwarf mosaic disease has become one of major maize diseases in China, and has seriously limited our country various areas corn's yield. MDMV disease in China was mainly caused by Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV), Johnsongrass mosaic virus (JGMV), Sorghum mosaic virus (SrMV). Chemical control of MDMV is ineffective due to the non-persistent mode of virus transmission by aphids. Hence it is very important to genetically control the virus by studying the resistance. So far, genetic improvement and breeding resistant varieties are the most effective ways by transgenic technology. The method of the antisense RNA inhibiting previously used had little resistance effects, but RNA interference (RNAi) could do so, such as high disease-resistant degree, strong specificity and hereditary stability, etc. Now, many genetically modified strains, which have resistance to virus disease of crops, have been breeded.Now, two kinds of methods, gene gun and agrobacterium-mediated transformation, are used in genetic manipulation of maize on a large scale. They have advantages and disadvantages; when they are putted into practice with callus for receptors, there are many restrictions, such as maize genotypes and seasons, and the lengthy cycle in cultivate, transformation, screening and regeneration. Moreover, we should cast light on the low percent of transformation and the bad operation in the transformed exogenous gene. So large-scale transgene technology which doesn't depend on tissue culture awaits breakthrough in transgenosis study.Based on such considerations and the theory of RNAi, we constructed expression vector of RNAi homologous to capsid proteins (CP) genes of MDMV, transformed maize stem tip growing point, and expected to obtain the transgenic corn materials resistance to MDMV.After homology analysis of CP genes, we selected 404 bp DNA fragement in conserved domain as RNAi target, then synthetized it in vivo. A pair of positive and reverse primers were designed and amplificated special fragments, respectively. Then the two DNA fragements were inserted into the middle vector pSK-int so as to form a special vector with hairpin structure. Meanwhile, Ubiquitin promoter and T-nos sequence in pC1300UbiDREB vector were digested by restriction endonucleases, then sub-cloned into pTF101.1 vector, and finally above-mentioned inverted-repeat sequences of middle vector were inserted into the reconstructive pTF101.1. Related experiments by restriction enzymes cut analysis and determination of reverse repeat sequences showed that the structure of pTFAsC404 vector for RNAi was right and could used for genetic transformation of maize.The stem tips of maize inbred line 18-599W were used as receptors and for genetic manipulation in situ by agrobacterium-mediated transformation. We obtained 1140 transformation seedlings in total. By herbicides screening (0.2 g/L),525 transformation seedlings survived, Then they were breeded in field, extracted genomic DNA by CTAB and amplificated special fragments. Out of these amplificated fragments, we sequencd positive PCR product and compared them with purpose gene. Results showed that 12 young plants were positive and the percent of transformation was 1.05%.The results showed that genetic transformation system, which used the stem tip of corn as receptors by agrobacterium-mediated transformation, was feasible and simple. What's more, the method need fewer time than previous method which used callus as receptor. After further identified, these genetically modified (GM) strains can used for resistance breeding. |
PDF Full Text Request