Identification And Characterization Of MicroRNAs Related To Drought Stress In Tea Tree(Camellia Sinensis L.)

Drought is the major natural disaster in the major tea producing area in China. At the same time, tea tree is an important leafy plant. Leaf tea is a plant that grows healthy or not will directly affect the quality of tea. More and more studies show that microRNA (miRNA) plays an important role in the respective plants under drought stress.To identify and characterise the microRNAs related to drought in tea tree tree, we have used the water weighting method to make the Tieguanyin tree under different drought stress. By the high-throughput sequencing, we have microRNA related to drought in Tieguanyin tea tree have been identified. At the same time, the qPCR has been used to analyse have know the information of the miRNA expression in the different drouhgt stress.The main results are as follows:1 The change of the main biochemical composition in the drought stressIn this paper, we have done the normal water (CK), light drought stress(T1), moderate drought stress (T2), severe drought stress (T3) treatment groups by the water weighing method. And we found that the chlorophyll a, chlorophyll b and total chlorophyll content have been decreased when the drouhgt stress increased. The photosynthetic rate of the leaves have been weakened. The drought stress have lead to increased levels of stress membranous damage plants and the MDA content. Because of the electrolyte leakage, the conductivity of the leaves have been enhanced. When Tieguanyin tea tree suffered mild and moderate drought stress, MDA content increased slightly and plant cells slightly enhanced conductivity. When the Tieguanyin tree suffered severe stress, MDA content in leaves and leaf conductance increase was evident.2 Identification and characterization of microRNAs related to drought stress in Tieguanyin treeThe RNA of differernt drouhgt stress for the Solexa sequenceing has been extracted. The results of the Solexa sequenceing are as follows:(1) We have obtained 15777773,13218397,14510362 and 13,452,128 sequences in the normal water, light stress, moderate stress and severe stress the four treated samples. The proportionS of the total sequencing data were 99.86%,99.58%,99.59%, 98.95% respectively. The peak of the sRNA in differrent sample is 24nt, so the data is reliable.(2) Based on the unknown of tea tree genomic data, we have selected the Arabidopsis genome as a reference genome. Simultaneously, we have consulted the Genbank and Rfam3 in order to find much more information of the miRNA. The ratio of the miRNA in the four sample are 5.60%,7.35%,7.66% and 15.78%. There are huge unknown sRNA data than we don’t konwn much about them.(3) For further identification of tea plant miRNAs, we have used Tieguanyin transcriptome data for reference genome. We have found 176 miRNA based on the Tieguanyin transcriptome. There are 7 miRNA belong to the 5 miRNA family have been annotated in miRBase database. The the vast majority of miRNA sequence are unknown or unique miRNA information.(4) In the water treatment process, the vast majority of miRNA is kept in the low abundance status. There are extreme variation in the same miRNA family. The difference of the same gene target miRNA family are greet too.(5) On the basis of expression differences in the high-throughput sequencing, we have find 19 miRNA which may be related to drought in tea. The 19 miRNA includes 12 conserved miRNA and 7 novel miRNA. The 12 conserved miRNAs are miR156/ 159a/165a-3p/166a/166g-3p/167d/2199/398/408b-5p/535a/6170/894. And the 7 novel miRNAs are csi-miR4/7/12/18/24/26/28.Most of the predicted target genes of miRNA are the transcription factors.The transcription factors control the regulating expression of the gene at a specific time and space. Meanwhile, the miRNA which relate to drought in Tieguanyin tea treemay have a regulatory role in protein synthesis and metabolism, plant organ development and morphogenesis and many other life activities.3 The analysis of miRNA qPCR in different drouhgt stress of the Tieguanyin treeWe selected the four RNA as the test materials to do the miRNA qPCR experiment and found csi-miR156, csi-miR398, csi-miR7 showed "ascending" expression in the drouhgt stress, so they play a positive regulatory role. And csi-miR165a-3p, csi-miR159a, csi-miR167d, csi-miR408b-5p and csi-miR6170, csi-miR4, csi-miR12, csi-miR18, csi-miR26, csi-miR28 showed a "first rises then falls" expression trends, csi-miR166a, csi-miR166g-3p, csi-miR435a, csi-miR894, csi-miR2199, csi-miR24 showed negative regulation.4 The cloning and expression analysis of target gene of the miR166 familyIn this study, we have used homology cloning method to obtain THN-14-like, ATHN-15-like gene.The partial cDNA length was 1246bp and 522bp. Specific primers have been designed for qPCR technology to analysis the expression of the ATHN-14-like, ATHN-15-like. Then compared the expression of the miR166a, miR166g-3p, we have found the following conclusion:(1) The expression of miR166a and miR166g-3p show a downward trendency in the four samples, While the HD-ZipIII family ATHN-14-like, ATHN-15-like genes show a upword trendency. When miR166a, miR166g-3p play a negative role in regulating drouhgt stress, ATHN-14-like and ATHN-15-like genes are positively regulated.(2) When the miR166a, miR166g-3p showed negatively relating to drought stress in Tieguanyin tea treein different treatments, the expression of ATHN-14-like and ATHN-15-like gene showed significant increasing trendency. Especially, the ATHN-15-like gene showed the most significant in moderate stress.In the different drouhgt stress, the relationship between ATHN-14-like, ATHN-15-like and miR166, miR166g-3p maybe indicate the HD-ZipIII response to drought in Tieguanyin tea tree.