Environmental RNAi Applied To Effectively Preventing Tobacco Virus

Since the late 1990s, virus disease of tobacco smoking in China occurred year after year.It not only caused great loss of tobacco production, but also a serious decline in the quality of tobacco. To cultivate disease-resistant varieties, cultivation and selection of control techniques, such as antiviral agents and trace elements in tobacco prevention and control HIV disease is not convergence. Application of genetic engineering means to cultivate disease-resistant transgenic varieties of the existence of security problems. This study explored the application of environmental RNAi tobacco prevention and control of new means of virus disease. Were infected with the virus from tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), potato Y virus (PVY) in tobacco leaves of three clones of the virus coat protein gene (⊿ cp). TMV⊿ cp cloned gene sequence with the Genebank in TMV⊿ cp gene sequences (EF122500) of more than 99% similarity.Three types of virus mRNA by RT-PCR amplification⊿ cp gene, and PFG19-T simple vector connected.⊿ cp gene and the sac I with kpn I digestion, received⊿ cp gene fragments linked with the L4440 vector, has been named as the recombinant plasmid L4440-TMV-⊿ cp, L4440-CMV-⊿ cp, L4440-PVY-⊿ cp, restructuring plasmid was transformed into E. coli RNAaseⅢ-deficient (dy330), the use of IPTG-induced expression of dsRNA. Was able to express the dsRNA transformants. Laboratory to determine the best culture conditions is 37℃, 220rpm/min, after 16h culture, the expansion of training 4h, IPTG induction of 4h, the dsRNA extract preparation was achieved in the OD260.When seedling leaves grow to 4-5, it will be extracted from TMV dsRNA agent sprayed on the seedling infection of TMV. Through the DAS-ELISA detection of the virus content of tobacco leaves. The results showed that positive control is the content of 1.474, and then spraying poison and dsRNA at the same time extract the content is 1.04, and then one day after the spraying of toxic extract dsRNA content of 1.109, and then three days after spraying poison dsRNA extract content is 1.199, then drug seven days after the spraying of dsRNA extract content is 1.275. DsRNA sprayed on the seedling seedling infected with the virus have different degrees of inhibition, the virus content of tobacco leaves infected with the virus than the positive control must be low, the virus content of the positive control than the highest 30 percent lower. This experiment does not exist and transgenic plants with the potential dangers, but also did not like the chemical substances polluting the environment, the basic preparation is zero. Is a more secure and efficient control of tobacco virus disease agents.The final outcome of this experiment did not achieve the best suppression of RNA silencing efficiency of -95%, which may be expressed by the experiment did not double-stranded RNA stem-loop structure. The existence of stem-loop structure and the stability of the double-stranded RNA.To be in the follow-up experiment required the use of PCR amplification of the sense chain and antisense chain and intron to connect to the plasmid and thus able to build a hairpin RNA expression of the recombinant plasmid to enhance the inhibition of RNA silencing efficiency.