Screening And Application Of Small RNAs Related To Cold Stress In Tea Plant(Camellia Sinensis)

Tea plant(Camellia sinensis)is a kind of shrub or small tree that likes warmth and fears cold.It is often planted in humid tropics,subtropics and temperate zones,and is one of the important economic crops in China.However,low temperature disasters such as "frost" and "late spring cold" have seriously affected the quality and yield of spring tea,so the research on the mechanism of cold resistance has always been a hot issue in the field of tea trees.MiRNA is a kind of non-coding small RNA in eukaryotes,which can play the role of gene regulation through specific degradation of target gene mRNA.In recent years,RNA bio-pesticide based on RNAi principle has developed rapidly.It has the characteristics of green safety,and is one of the most promising biotechnology in the field of pesticides.The purpose of this study is to screen the miRNAs of target genes related to cold stress by small RNA sequencing analysis of tea samples under cold stress,construct the regulatory network of miRNA and target genes,and develop"RNA antifreeze protectant",so as to provide new ideas for the prevention and control of chilling injury in tea plants.The main results are as follows:1.Screening of differentially expressed miRNA under cold stressA small RNA library was constructed from tea samples under cold stress for 0 h,6 h,12 h,24 h and 48 h.A total of 920 mature miRNA sequences were identified after data filtering.Comparison with miRBase database showed that a total of 198 miRNA sequences were similar to the miRNA sequences of known species,and the other 722 miRNA precursors had typical stem-loop structures,which were considered to be new tea miRNA.The expression levels of miRNA before and after cold stress were compared.the results showed that there were 3,12,8 and 8 miRNA differentially expressed at 6 h,12 h,24 h and 48 h,respectively.2.Prediction and functional annotation of target genesA total of 2170 target genes were predicted by the intersection of two target gene prediction software psRobot and TargetFinder,and 1,604,1,763 and 2,133 target genes with biological functions were annotated by GO,KEGG and NR databases,respectively.Functional enrichment analysis of target genes of differentially expressed miRNA showed that these target genes were mainly involved in biological processes such as"transport and catabolism","signal transduction","binding" and "catalytic activity",and there are 5 differential miRNAs associated with brassinosteroid signal transduction.3.Prediction of secondary structure and verification of expression level of miRNAThe precursor sequences of 14 miRNA up-regulated under cold stress were analyzed,and their lengths ranged from 63 nt to 220 nt,in which the shortest miRNA was novel-miRNA311-3p,and the longest miRNA was novel-miRNA-251-5p and novel-miRNA-247-5p.The secondary structure of miRNA is predicted by Mfold online tool,and the results show that these miRNA contain typical stem-loop structure.The expression of miRNA was verified by qRT-PCR,and 14 miRNA were significantly upregulated under different periods of cold stress,which was consistent with the sequencing results.Tissue specificity analysis of four miRNA that may be involved in the regulation of cold stress in tea plants showed that their expression levels were the highest in stems,the second in leaves,and the lowest in flowers.4.Functional verification and application development of cold resistance of miRNAAccording to the miRNA sequence information,four selected miRNA molecules were chemically synthesized,and the effects of root soaking,spraying and injection on the absorption of 3 exogenous miRNA by tea plants were compared.the results showed that after root soaking treatment,the level of miRNA in tea plants was increased by about 8.2 times,which was the best treatment method.After the tea plants were treated with four kinds of miRNA,the expression levels of miRNA and its target genes in tea plants were determined by qRT-PCR.The results showed that the expression level of miRNA was up-regulated by 4.6-8.5 times,and the expression of target genes decreased by 0.26-0.75 times,indicating that exogenous miRNA treatment played a role in regulating the expression of target genes.The results of freezing injury simulation experiment and electrical conductivity test showed that the degree of injury of miRNA treatment group was low under 0℃ freezing injury,the relative electrical conductivity of leaves was 18.3%and 44.1%lower than that of the control group(NM group),and the activity of protective enzyme SOD in leaves was significantly higher than that of NM group,indicating that the four "miRNA protectants" could improve the cold resistance of tea plants.