Functional Analysis Of MiR395 Related To Eggplant Verticillium Vilt Defense

Verticillium wilt caused by Verticillium dahliae is a notorious wilt disease and often leads to the reduction of eggplant products or even the death.At present,there are still no proven cures for this disease since the mechanisms of Verticillium wilt remain poorly understood.MicroRNAs(miRNAs),a class of small(about 22 nt in length),can regulate gene expression negatively at the post-transcriptional levels.One of them miR395 has been proved to respond to sulfur-deficient stress and sulfur is well known to have relationship with plant disease resistance.Therefore,this study focuses on the research about the possible role of miR395 in Verticillium wilt,and our work will lay the foundation for further analysis and provide a hypothesis for the possible molecular mechanism of V dahliae-caused defense responses in plants.The main results are as follows:To identify the expression change of miRNAs in eggplant infected by Verticillium dahliae under different time courses,we deducted the qRT-PCR assay and found the miR395 showed a gradually reduced trend with the time of infection goes,which predicted that miR395 may be involved in defense to V dahliae.Meanwhile,online software psRNA Target was used to get the potential targets genes of miR395,and the expression of these genes showed a negative correlation with the changes of miR395.For now,we hypothesized that miR395 could play important roles in eggplant against Verticillium wilt.The library of eggplant ESTs is still limited,so we have difficulty in finding the sequence of pre-miR395.However,the mature sequence of miR395 is very conservative,so we cloned pre-miR395 gene from Arabidopsis thaliana with PCR approach and transferred into the eggplant Solanum melongena L.cv.Suqi by Agrobacterium tumefaciena-mediated approach to explore the function of miR395 in eggplants.The result showed that there were four transgenic plants identified by PCR and qRT-PCR.The antibacterial test of the transgenic eggplants proved that overexpression of miR395 resulted in enhanced sensitivity to V.dahliae infection in eggplant.The disease index and disease percentage was higher in transgenic lines than in wild-type control.The predicted miR395 target genes are related to sulfate assimilation,because the terminal of sulfur transport mechanism is glutathione,we examined the content of glutathione in transgenic plants,and the results showed that the content of glutathione was increased obviously in the early infection of V dahliae in plants,and then decreased,which is consistent with the content of GSH in wheat-powdery mildew.We also detected some physiological parameters in vivo:SOD and POD.The results show that in overexpressed eggplants the index of SOD/PODwas lower than that of the wild type under normal condition,but after the V.dahliae infection,The activity of various enzymes in the wild type was increased rapidly,and its amplitude was higher than that of the transgenic plants.The results suggested that infection might activate active oxygen seavenging system in wild eggplant,then produeing certain defensive substances(such as lignin)quiekly,and then caused toaminimum harm.