Identification And Expression Of Cold-Responsive Micrornas And Their Targets In Tea Plant (Camellia Sinensis)

Tea plant(Camellia sinensis (L.) O. Kuntze) is one of the most important commercial beverage crops in China. Cold stress is one of the common abiotic factors affecting tea plants, which adversely affects the growth, development, and spatial distribution. Plant microRNAs (miRNAs) are approximately 19-24 nucleotides noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs, that highly conserved, timing and tissue specificity, have critical functions in growth, development, differentiation, apoptosis, metabolism, and abiotic stress responses with specific target mRNAs. Some tea plant miRNAs and their target genes have been identified using computability by other researchs. However, there is no systematic examination of miRNA performed on tea plant In this work, two tea plant cultivars,’Yingshuang’(YS, a cold-tolerant tea plant cultivar) and’Baiye 1’(BY, a cold-sensitive tea plant cultivar) were used as experimental materials. The miRNAs and their target genes expression in tea plant were systematically and comprehensively studied by high throughput, bioinformatics, miRNA microarray, qRT-PCR, and degradome sequencing. The main results are summaried as follows:1. Leaves of two tea plant cultivars, YS and BY at 4℃ for 0,1,4,8,12,24, and 48 h were collected as materials. Depth sequencing was conducted through HiSeq technology in the mix library. A total of 3,145,122 unique reads representing 9,700,042 redundant sequences were obtained from a short RNA library generated from small RNA extracted from tea leaves. The sequencing results were statistically analyzed, then the sequencing fragments were screened by Rfam (10.0), Genbank database, and repeat sequence, all the fragments were classified. Finally, miRNAs were found in the small RNA library including known and unknown miRNAs. To align the sequencing data with known plant miRNAs, a total of 112 conserved C. sinensis miRNAs were characterized. Based on sequence similarity, hairpin structure prediction, base distribution, and MFEI,215 potential candidate miRNAs were found, among which 111 candidates with star sequences were chosen as novel miRNAs.2. Total of 172 differential expression miRNAs were identified using miRNA microarray screenings from each cultivars. There were 34 miRNAs up-regulated and 43 miRNAs down-regulate in’Yingshuang’,50 miRNAs up-regulated and 45 miRNAs down-regulate in ’Baiye 1’. A certain degree of overlap of cold-stress response pathway of miR168, miR529, and miR2936 observed by comparing miRNAs expression patterns between different cultivars. By contrast, the expression of miR164, miR408, miR1511, miR5368, miR172, miR482, miR529, and miR1160 were significant differences in the two cultivars. To confirm the microarray results, the abundance of several miRNAs was further analyzed by qRT-PCR. The results of qRT-PCR and abundance profiles of the microarray shared similar trends.3. miRNA target genes from cold-treated mRNA library (+C library) and cold-free mRNA library (-C library) were discovered using degradome sequencing method. A total of 5,376,257 unique reads representing 6,736,820 redundant sequences were obtained from +C library and 7,439,589 unique reads representing 9,224,714 redundant sequences were obtained from -C library. Total of 514 target genes sliced under the induction of 13 known miRNAs conservative families and 249 target genes frome 40 novel miRNAs were identified by the degradome sequencing. These target genes involved in the regulation of processes including plant growth and development, transcriptional regulation, stress responses, hormone signaling, plant metabolic pathways can be categorized into three types: cell composition, molecular function and biological process. Comparison of the mRNA species, quantity and expression characteristics in the two libraries, the tea plant miRNA target genes expression regulatory networks was constructed under cold stress.In summary, this study identified a large number of known and new miRNAs in tea plant at the first time; Comprehensive genome-wide miRNA profiling under cold stress were also exhibited. Those results indicated that miRNA play important roles in the regulation of tea plant resisting to abiotic stress. Further researches on abiotic resistance of tea plant could be carried out based on our hypothesis and genomic database.