Identification And Expression Analysis Of MiRNAs And Their Target Genes Response To The Protoplast Isolation Of Citrus Reticulata Blanco

MicroRNAs, a kind of endogenous non-coding small RNA molecules, inhibit gene expression or translation through sequence-specific combination with the target mRNAs, which further plays an important role in plant growth and development, metabolism, signal transduction and adversity stress response. The process of protoplasts separation is a stress process, intracellular stress responses will directly affect the performance of protoplasts regeneration. For studying the possible effects of miRNAs in the process of protoplasts separation in Citrus, this study took Ponkan calluse and its protoplasts as the materials, total RNA was extracted to construct the small RNA libraries of two samples; for the identification of miRNA, prediction of target genes and their functions, HiSeq high-throughput sequencing and bioinformatics analysises were used; verificated 8 differential expression miRNAs by poly(A)-Tailed qPCR and took expression analysises of 8 candidated target genes by using RT-PCR. The main results were as follows:1. The screening of methods for extracting total RNA of protoplasts in PonkanThe total RNA of protoplasts were extracted by Trizol method, improved Trizol method, CTAB method and TaKaRa RNA extraction kit. The qualities and yields were detected by agarose gel electrophoresis, ultraviolet spectrophotometer and RT-PCR. The results showed that the improved Trizol method had good integrity, the estimated values of OD260/OD280 and OD260/OD230 were in the normal range and the yield of total RNA was the highest, additionally, the result of RT-PCR validation showed that the improved Trizol method gave the most clear and bright band of the PCR product with the expected size. Therefore, the improved Trizol method can be used for extracting high quality total RNA of citrus protoplasts.2. The dates analysises of small RNA libraries acquired by high-throughput sequencing(1) A total of 17,975,734 and 15,828,591 sRNAs were obtained from the small RNAs libraries of calluses and protoplasts, respectively. The high quality sequences accounted for 99.83% and 99.73% of the original RNA sequences. It meaned that the qualities of our libraries were fully meet the requirements. After the removal of the adaptor, insert, poly(A) and short RNAs of<18nt, obtained 17,899,040 and 15,663,034 clean sRNA sequences, respectively, accounted for 99.74% and 99.22% of the high quality sequences.(2) In the calluses and protoplasts libraries,61 and 60 known miRNAs were identified, and predicted 492 and 477 candidated targets genes, respectively; 265 and 229 novel miRNAs were identified, and predicted 1167 and 1130 candidated targets genes, respectively. In addition,4 miRNAs* were detected in the two libraries.(3) By differential expression analysises, obtained 18 differential expression miRNAs in the known miRNAs, of those,11 were up-regulated and 7 were down-regulated in protoplasts. As for novel miRNAs, there were 64 differentially expressed miRNAs, of those,29 were up-regulated and 35 were down-regulated in protoplasts.(4) By mapping the target genes of miRNAs to the sweet orange genome (http://citrus.hzau.edu.cn/orange/). It found that the predicted target genes related to many transcripts transcription factors and functional proteins.3. Validation of differential expression miRNAs8 differential expressed miRNAs (crt-miR171, csi-miR319, csi-miR172a*, csi-miR535, novel-mir-187, novel-mir-235, novel-mir-172 and novel-mir-98) were selected for validating by using poly(A)-Tailed qPCR. The results showed that 2 were up-regulated and 6 were down-regulated of 8 miRNAs, which were consistent with the results of high-throughput sequencing.4. Validation for target genes of the differentially expressed miRNAs by RT-PCRTo analysis the expression patterns between miRNAs and their target genes, we selected the corresponding target genes of 8 miRNAs for RT-PCR which were validated by fluorescence quantitative PCR. The results showed that the miRNA expression trends and their target genes were opposite in calluses and protoplasts, it ment the miRNAs may have negative regulation on their target genes.This study laid a theoretical foundation for initially revealing the mechanism responsed by Citrus protoplasts separation which may regulated by miRNAs, and provide a theoretical basis for protoplast regeneration regulation.