Studies On Regulation Technique Of RNA Interference Against SCMV And MRDV In Maize

Maize is an important agricultural crop with diverse applications, including its primary uses in livestock feed, human consumption, and ethanol production (as well as other biofuels). Maize production in China is second in the world, and has now reached nearly one half that of the United States. However viral diseases, particularly maize dwarf mosaic disease caused by Sugarcane mosaic virus and maize rough dwarf disease are among the most damaging diseases affecting maize production and quality in China. It is urgency to cultivate and popularize disease-resistant cultivar and search for new methods. RNA interference (RNAi), as an important RNA-based reverse-genetic system, has been used for gene function and genetic melioration studies in plants. RNAi has displayed great advantage in antiviral of breeding which has been proved to be a technology distinctiveness, high efficiency, economy, and inhibiting gene expression, and has become the focus of scientist researches. In order to select and establish the prokaryotic expression system and the in planta transformation system of the cp gene from virus, and to investigate effects of RNAi in maize antivirus genetic engineering and to find a new molecular breeding method in maize, we explored a basic principle of inverted-repeat expression vector design, and report an efficient method to produce dsRNA using a bacterial expression system. Two marker-free siRNA complex expression vectors of pDTBU SM from SCMV cp and MRDV cp were constructed, and transformed into Agrobactirium tumefaciens. Our work illustrates some basic principles for protecting plants against viral diseases in vitro and in vivo, as well as the study of gene silencing mechanisms in plant viral infections. The integration of target gene was analyzed, and we investigated the effect of RNAi and amylase content. The main results are summarized as follow:1. Two fragments of the SCMV cp gene were amplified by RT-PCR, and cloned into the inverted-repeat cloning vector pUCCRNAi, and subsequently joined to generate LMCP. The two recombinant plasmids were transformed individually into E.coli HT115, and dsRNA was induced by IPTG. The results showed that the expression products were the dsRNA by treating with RNase A or DNase I to remove single-stranded RNA or DNA, respectively. Meanwhile, an IPTG concentration of0.4～0.6mmol/L and induction time of4h was the most optimal expression condition.2. The crude extracts of E.coli HT115containing large amounts of dsRNA were sprayed to plants and the experiment confirmed a preventative efficacy. Our findings demonstrated that spraying crude dsRNA-containing extracts inhibited SCMV infection, and the dsRNA derived from an upstream region (cp1) was more effective than was dsRNA derived from a downstream region(cp2) of the SCMV cp gene. The results showed that plants inoculated with the virus and LMCP preparation, diluted1/5or less, and treated at or before5days prior to viral inoculation, displayed decreased disease symptoms. Besides, dsRNA-containing extracts with penetrating agent of suitable selection concentration could Inhibit virus infection effectively.3. Based on the coat protein gene sequence of SCMV and MRDV in GenBank and MaizeSeq database, six pairs of specific primers were designed and six specific fragments were amplified by RT-PCR. To prepare siRNA that corresponded to part of the SCMV cp or MRDV cp gene. In the first cloning step, an inverted repeat sequence of pUCCRNAi+2F was constructed. Next, two inverted repeat sequences were inserted into pBluscript SK in series to generate pBluscript+SM. Meanwhile, Ubiquitin promoter and Nos termination were cloned into pDTB to generate pDTBU. In the third step, pDTBU and pBluscript+SM plasmids were digested and joined to generate pDTBU SM. The study presented here provides a valuable tool for plant viral control using RNAi and the PTGS approach.4. RNA interference expression vector of SCMV cp and MRDV cp gene were transformed into maize inbred lines8112by Agrobacterium tumefaciens and the results of PCR-Southern and Southern blot proved integration of the targeted segment into the8112genome successfully, and four transgenic plants were obtained finally.5. Total RNA was extracted from different maize leaves4LP (4-leaf-period) or different organs of florescenceand, and cDNA segment of cp was obtained by real-time quantitative PCR, which resulted in a reduction of endogenous cp mRNA. The results showed that the expression of the cp gene fragment in transgenic plants was different. And the highest expression was in male flowers and leaves, while the lowest was in the roots.6. Transgenic plants of4-leaf-stage in T2generation were inoculated with maize dwarf mosaic virus, and total RNA was extracted from the leaves at different times after inoculation, and the expression of endogenous cp gene was detected by fluorescence quantitative PCR when induced by exogenous virus. The results showed that the cp gene fragment in transgenic plants could be transcribed correctly which leaded to mRNA content in alternate, and the hpRNA structure transcripted by the cp gene could interference SCMV infection subsequently.7. Transgenic plants of4-leaf-period were inoculated with maize dwarf mosaic virus, and ELISA results were detected at14dpi、21dpi and30dpi (days post inoculation). The results showed that almost transgenic plants could inhibit SCMV infection, while more than87%non-transgenic plants displayed typical disease symptoms.In summary, maize inbred line8112was inoculated with crude extracts of bacterially expressed dsRNAs to protect maize plants against SCMV infection and viral infection was detected. RNAi technique system of dsRNA in vitro was filtered and constructed. Marker-free siRNA complex expression vector against SCMV and MRDV was constructed and in planta transformation to maize inbred line8112by mediated Agrobacterium tumefaciens. Transgenic plants were obtained and RNAi effect was in evidence. And the resistance of the transgenic plants was improved. Based on those above results, our study provides a reference on utilizing of the RNAi technique to protect maize against SCMV infection and constructing a new breeding method of high resistance maize.