The Comparative Study Of Single MiRNA Mediated Virus-resistance To Potato Virus Y And Tobacco Etch Virus

As the largest group of plant viruses, Potyviruses （genus Potyvirus, family Potyviridae）include about200determined and possible species. It can infect many kinds of plants,Solanaceae, Chenopodiaceae and so on, serious damage to the plant growth, and cause hugeeconomic losses. Potato virus Y （PVY） and Tobacco etch virus （TEV） are two typicalmembers of Potyviruses. In recent years, aritificial microRNA provides a new way for plantantivirus strategies. AmiRNA technology has been widely used in plant antiviral research forits advantage, such as nearly immunity; sustaining resistance; no biosafety problem and so on.In our studies, we chose nine target mRNA sequence with high similar and miRNA features,by comparing PVYNand TEV-SD1genome sequences. Through reform miRNA319aprecursor constructed miRNA expression vectors respectively and transformed tobacco. Ourstudy shows that multi-resistance transgenic plants can obtain with a single miRNA, but theresistance and similarity are not completely positive related. Mismatch in different positionshas different dffect for target sequence degradation. This result has provided the basis for thefurther research on amiRNA-mediated virus resistance mechanism, and contributes toantiviral transgenic plants cultivation with the optimal target sequence. The main results andconclusions presented in this thesis are as follows:（1） By comparing PVYNand TEV-SD1genome sequences in similarity, reference thescreening criterion of the amiRNA, we had obtain nine mRNA target sequence located in CI,NIa-VPg, NIb, CP genes respectively. Designed and synthetized nine primers, reformmiRNA319a precursor. The recombinant plant expression vectors were transferred into E.coliDH5α by heat-shock. The recombinant plant expression vectors （pR-amiR-1, pR-amiR-2, pR-amiR-3, pR-amiR-4, pR-amiR-5, pR-amiR-6, pR-amiR-7, pR-amiR-8and pR-amiR-9） wereconstructed successfully.（2） The recombinant plant expression vectors were transferred into Agrobacteriumtumfaciens EHA105, and then transferred into N. benthamiana through Agrobacteriumtumfaciens-mediated transient infection. After three days, Northern blot analysis revealed thatpre-amiRNAs could express amiRNAs successfully in plants.（3） The recombinant plant expression vectors were transferred into Agrobacteriumtumfaciens LBA4404, then transformed tobacco.The transgenic plants had no obvious difference with non-transgenic plants in the growth process. Confirmation with the PCRdetection, we obtained T0generation transgenic plants44,38,36,32,41,32,38,35,32corresponding with pR-amiR-1, pR-amiR-2, pR-amiR-3, pR-amiR-4, pR-amiR-5, pR-amiR-6,pR-amiR-7, pR-amiR-8and pR-amiR-9.（4） The seeds screening in contain kanamycin （100mg/L） medium after T₀selfing. EachT₁transgenic line transplanted some randomly in greenhouse. T₁transgenic plants wereinfection with the PVY^N and/or TEV-SD1isolates, the result reveal that artificial miRNAscan mediate virus resistance against PVY^N and TEV-SD1, but different pR-amiRs havedifferent resistance.（5） The total RNA Northern blot for transgenic plants showed that the trangene havebeen expressed in level of RNA, The RNA accumulation level in resistant plants was lowerthan that of the susceptible transgenic plants. There is a large number of miRNA in resistanceplants, and there is a positive correlation between virus resistance and expression level ofamiRNA, so we knowed that amiRNA can mediated the virus resistance;5′RLM-RACE assayalso has showed that amiRNA could mediated a precise cleavage of their target viral RNAs.（6） The total DNA was extracted from the transgenic plant with susceptible or resistantresponses. Southern blot showed that the transgenic tobacco have different DNA bands, meandifferent copies,1⁴specific hybrid bands respectively, it is further proof that the transgenehas been integrated into the genomes of tobacco. Combined with plant resistance analysis,between copies and virus resistance has no necessarily linked.（7） Analysis impact factors of amiRNA-mediated virus resistance level, amiRNA andtarget sequence has1⁴mismatches can still mediated higher resistance. Less than4mismatches, between resistance and mismatch has no obvious rule that multi-resistance andtarget sequence comparability without fully positive correlation. Study found that mismatchmainly focus on the amiRNA3′end, proof that miRNA exist certain tolerance for mismatchon3′end.（8） Virus resistance assay in the T2generation single copy transgenic plants showed thatthe progeny transgenic plants of resistant plants still exhibited high resistance, it indicated thatamiRNA-mediated virus resistance can be stably inherited in T2generation.