The Application Of NtWRKY40,amiRNA And AsODN Technology In Plant Resistance To Virus

Plant virus diseases cause severe damage to the host plant through the inhibition of the expression of genes related to plant growth and development,and is very difficult to control.Plants have acquired defensive system in response to this,which involves in many aspects,e.g.WRKY transcription factors and micro RNAs.NtWRKY40 was found to rapidly response to Tobacco Mosaic virus infection,thus the role(s)of NtWRKY40 were detailed studied and artificial micro RNA(amiRNA)based anti-sense oligonucleotide(asODN)technology was applied here for plant virus disease defense in this study.amiRNAs were widely used and thought to be a good technology in controlling plant virus diseases in recent years.amiRNA technology was developed based on miRNA can bind and degrade its target genes,according to this,replacing the mature miRNA sequence using gene fragment of specific genes was named amiRNA,thus the produced mature sequence was changed and can bind and degrade the specific gene.asODN technique,which uses short fragments of a specific gene,was developed based on miRNA and small RNA(siRNA),its effects are closely related to the fragment used,which also true for amiRNA.Thus asODN predicating program was employed here to select high efficiency fragment for TMV and PVY virus diseases defense,and amiRNA was then employed to get high resistance plants towards TMV and PVY virus diseases.NtWRKY40 was inserted into pBI121 binary vector,and seven transgenic tobacco plants,PCR and quantitative PCR verified,were obtained using Agrobacterium tumefaciens-mediated transformation,named as W1 toW7.Using nature AtmiR167 e as a skeleton,overlapping PCR and restriction enzyme cut,35S::amiR167-PVY was constructed based on PVY coat protein and then 10 amiR167-PVY transgenic tobacco plants were obtained,3 of them were chose for further studies,named as PVY-1,PVY-2 and PVY-3.Results showed that the expression of NtWRKY40 was significantly increased in wide type tobacco plants after virus infection for 9 days.However,the expressions of virus gene were even higher in transgenic plants than in wide type after virus infection,indicating that NtWRKY40 is a negative regulatory factor and increased the sensitivity of tobacco plants to virus.The transcript level of miRNA167-PVY precursor and mature sequences were significantly higher in transgenic plants compared to wild type control.After PVY virus infection,the expression levels of amiRNA167-PVY mature sequence and PVY-CPshowed a negative correlation,the incidence of transgenic plants was lower than wild type,and the symptom developed slower than wild type.Thus indicating amiRNA167-PVY successfully inhibited virus gene expressions,and has defensive effects to PVY infection.The three chosen asODN fragments showed different effects on TMV defense and asODN-3 was the best.It was the first time to show that asODN can potentially defense virus disease in this study.Above results showed WRKY40 has a negative effect on virus disease defense and the technology combined asODN with amiRNA is effective in developing virus resistance plants.