Functional Analysis Of Csi-miR858-3p＿L-1 And Its Target Gene CsMYBPAR After E. Sorghumii Infected Tea Leaf Tissue

Tea（Camellia sinensis（L.）O.Kuntze）is an important cash crop.The tea leaf spot caused by Epicoccum sorghinum was firstly isolated by our research group in Dushan County,Guizhou Province.The pathogen could infect the tender leaves,fresh shoots and mature leaves of tea.However,there are lacking of effective control measures against E.sorghinum at present.Therefore,it is of great significance to excavate and identificate the disease-resistance genes in tea trees for helping us breeding new varieties resistant to E.sorghinum and green prevention and control in tea plantation.In this research,we studied the pathogenic mechanism of E.sorghinum in tea by interaction between mi RNAs and target genes.The main research results are as follows:1.We performed comprehensive sequencing of mi RNA and transcriptome of a resistant tea during E.sorghinum inoculation.It was been screened that the different expressed csi-mi R858-3p＿L-1 and their target gene prediction Cs MYBPAR by database.2.Transient transformation technology was used to transform p BI121-35S::csi-mi R858-3p＿L-1 and p BI121-35S::Cs MYBPAR or p BI121-35S::Cs MYBPAR gene overexpression vector into leaves of was used to transform by agrobacterium-mediated method.According to the result of GUS histochemical staining,it was found that csi-mi R858-3p＿L-1 is negatively regulated target gene Cs MYBPAR.3.Transient transformation technology was used to transform Cs MYBPAR gene was heterologous overexpression vector instantaneously in leaves of tobacco plants by agrobacterium-mediated method.The q RT-PCR results showed that the expression of Cs MYBPAR gene was higher than the control group in tobacco plants.The cotyledon of transiently expressing Cs MYBPAR inoculated by B.cinerea with smaller lesion area,and NBT and DAB staining sepia lighter color,lower H₂O₂and O₂^-.It demonstrated that transiently overexpressed tobacco plants were more resistant to the disease.Transgenic tobacco plants with higher expression than wild-type plants were obtained by genetic transformation of the constructed overexpression vector into Nicotiana benthamiana.It will lay a foundation for further identification of biological function of target gene Cs MYBPAR.4.By Virus-induced gene silencing（VIGS）technology to reduce the expression of the homologous gene Nb Myb123-like of the target gene Cs MYBPAR in tobacco,the expression level of this gene was silenced by more than 60%.It was found that the phenotypes of the control and the silent plants were significantly different at 15 days,Compared with the control TRV2 plants,the silenced gene Nb Myb123-like showed obvious wilting and necrosis.After B.cinerea was inoculated leaves,the stain area of was relatively larger.DAB and NBT staining has a darker color,accumulation of H₂O₂ and O₂^-were greater,indicating that plants effectively silencing Cs MYBPAR homologous gene were more susceptible to disease.The involvement of Cs MYBPAR homologous gene was further verified in disease resistance response.