Identification Of MiRNAs Genes Related To Drought Stress Response In Camellia Sinensis

MicroRNAs(miRNAs)are a type of non-coding RNA with a length of about 19-24 bp,which widespread in eukaryotic organisms.In plants,miRNAs mainly negatively regulate the expression level of target genes through complementary pairing with target gene sequences,thereby participating in the regulation of plant growth and development,signal transduction,hormone secretion,and response to external stresses and other biological processes.Tea plants is an important economic crop,on which basic research of growth,development regulation and mechanism of response to adversity will provide a theoretical basis for how to improve the quality of tea and select high-quality tea varieties.In order to screen the miRNAs that regulate the formation of quality-related components and the response to stress in tea plant,the research group selected exogenous sucrose to treat tea seedlings in the early stage,and sequenced the miRNAs of the post-treated tea seedlings.In this paper,we first screened seveal miRNAs that may be involved in the quality-related component formation and stress response from 36 known miRNAs in tea plants,and used gene expression analysis and model plant genetic transformation systems to verify their functions.The main conclusions are as follows:1.The transcriptome data showed that compared with the untreated control sample,the gene expression of 36 known miRNAs showed significant changes.Among them,miR858 a and miR156 are predicted to be closely related to phenol metabolism.Compared with the control group,the gene expression levels of miR156 and miR858 a in tea seedlings were significantly reduced after treatment.2.We cloned 9 tea plant CsmiRNAs genes including miR156 and miR858 a,and analyzed their sequence and stem-loop structure.The target genes of 9 tea plant CsmiRNAs were predicted and screened.The amplification efficiency of fluorescent quantitative primers for the target genes of CsmiRNAs was verified.3.We analyzed the characteristics of tissue specificity and induced expression of 9CsmiRNAs and their target genes by fluorescence quantitative technology.The results of the study showed that the 9 CsmiRNAs and their target genes responded to sucrose treatment,salt treatment and PEG4000 treatment with different degrees,indicating that these CsmiRNAs may have a certain relationship with plant resistance.4.The Arabidopsis transformation experiment of 5 CsmiRNAs was implemented,obtained transgenic plants;the transgenic plants were treated with mannitol,and the results revealed that the lateral roots of some CsmiRNAs overexpression plants increased significantly,which further proved that these CsmiRNAs are related to plant resistances.5.The function of CsmiR156＿2 gene was verified by tobacco genetic transformation system.The experimental results suggested that CsmiR156＿2 is indeed involved in the regulation of plant anthocyanin synthesis.6.The regeneration system of petunia was optimized,and the most suitable callus,budding and rooting induction medium for petunia were obtained;at the same time,a genetic transformation system of petunia was established,and the most suitable ones were selected suitable screening pressure.Using the petunia genetic transformation system,we overexpressed CsmiR156＿2 and obtained transgenic plants,the results showed that CsmiR156＿2 has an effect on the anthocyanin synthesis of petunia.