Artificial MicroRNA Autoresponder Prevention Technology Of Plant Viruses

Viruses often cause plant abnormal growth and development, even death. In recent years, artificial miRNA technology for control of plant virus disease has made great achievements, often involved foreign artificial miRNA expression driven by a strong promoter in the plant. Those studies always choose a strong constitutive promoter. The transgene under control of a constitutive promoter can be expressed stably and efficiently at different developmental stages. However, high-level expression of foreign genes in the recipient plant often turns to be a burden of plants and leading to abnormal growth and development of receptor plants. Hence, the vector which contain promoter induced by plant viruses and artificial miRNA constructed by endogenesis miRNA is beneficial to enhance the effects of defensing plant viruses. In this experiment, the model plant tobacco was investigated. We try to find the promoter induced by plant viruses and endogenesis miRNA possessed of complete function in tobacco, which perhaps provide a theoretical basis for the prevention and treatment of plant virus diseases.This research found 10 candidate genes induced by virus in tobacco via conculting journal and using BLASTX in order to identifying gene involved in virus resistance response of tobacco. Tobacco under three viruses (Tobacco mosaic virus, Cucumber mosaic virus, Potato virus Y), two abiotic stresses (150 mM NaCl,4°c) and two stress signal materials (1 mM methyl jasmonic acid, MeJA and 1 mM salicylic acid, SA) was investigated in this study. With quantitative analysis of 10 canditate gene expression, the result showed that 06G expression increased remarkable after infection of plant viruses and SA treatment. However, in other condition,06G expression changed non-significant. Furthermore,06G expression at different stages was analyzed after infection of TMV. The result showed that 06G expression could be quickly activated. After 6 h,06G expression reached 500 times compared with the control and almost sustained 20 day. In the conclusion, we speculated that 06G can be involved in virus resistance response and regulated directly by SA in tobacco. Hence, we speculated that candidate genes 06G promoter can be induced by plant viruses.We cloned upstream sequence 1967bp of transcription starting site of 06G via genewalking. Subsequently, we found transcription factor binding site WBOXATNPR1 associated with pathology through the online software PLANTPAN. Different size fragment 06G promoter connecting with a reporter gene GUS was investigated, which called 06GP1215:GUS and 06GP1967:GUS. The results showed that GUS expression levels reached the maximum of 20-fold in transient expression and stable expression of 06GP1215:GUS after treated by 1mM SA. However, the experiment that analyzed the GUS histological activity after staining could’t find the blue part.06GP1967: GUS transient expression showed that the expression level of GUS increased 40-fold, and 200-fold after treated by 1mM SA. Subsequently, the GUS staining showed blue detectable spots. The results demonstrated that the 1215bp length of the 06G promoter showed that hardly promote repoter gene GUS. In contrast, the 1967bp full-length sequences of 06G was identified that has full functionality and enhance the high-level expression of GUS under SA treatment. Quantitative analysis of the previous results identified that SA could promote 06G expression in tobacco. Hence, we speculated that 06GP1967 can be induced specially by plant viruses.Subsequently, we cloned miRNA167 precursor in tobacco. Previous studies demonstrated that overexpression miRNA167 in Arabidopsis thaliana lead to abnormal development of plants, malformation of flower and sterility. In this study, tobacco miRNA167 under the 35S promoter via agrobacterium GV3101 mediated method transformed into Nicotiana tabaccum. Then, this research obtained 10 positive transgenic plants by kanamycin. Quantitative analysis showed that precursor miRNA167 and mature miRNA167 in transgenic plants have a high level. On the contrary, expression level of target gene ARF8 of miRNA167 was significantly inhibited. Morphological observation showed that the transgenic plants have normal form except yellowing leaf and fertility. In the conclusion, we found an inducible promoter 06GP induced specific by virus and conducted a tobacco endogenous miRNA167 functional verification. It is very important for the construction of the autoresponder technology of virus prevention in tobacco.