The Research Of MicroRNA And Targets Related To Cashmere Cycle In Cashmere Goat

Inner Mongolia Cashmere goats are autonomous local varieties of quality resources, cashmere performance is an important economic traits. There are many factors that influence on the growth of cashmere, melatonin and miRNA are two very important factors. In recent years, the study that melatonin impact on the growth of cashmere found that sustainable implanted melatonin can promote cashmere into anagen in advance, induced two secondary cashmere and changed growth-related gene expression at the molecular level.but for miRN A this important factor in hair growth research had no detailed report. Therefore, this study builds relationships between exogenous melatonin, miRNA and its target genes by microRNA targets Finger Printing, real-time quantitative PCR, RNA-seq and other methods, further elaborates mechanism of melatonin induced the growth of cashmere at the molecular level and provide a theoretical basis for improving the yield and quality of cashmere.The main results are as follows:1the establishment of microRNA Targets Finger Printing (MTFP) methodEstablishing a new method for obtaining experimental miRNA target genes (microRNA Targets Finger Printing, MTFP). On the basis of setting the seed sequence complementary sequence, upstream and downstream anchor sequence which including special linker, through the reverse transcription and special two step PCR, microRNA targets were amplified. The polyacrylamide gel electrophoresis was used to analyze the amplified microRNA targets’ molecular size and their abundance of expression which was used to screen specifically expressed genes in different physiological or experimental conditions;Specific target genes were obtained through DNA extraction and sequencing methods.As examples,the target genes of miR-203,five of them were amplified and sequenced with the size of718bp (JN709494,349bp(JN709495),243bp(JN709496),159bp(JN709497),97bp(JN709498) from goat skin collections. of them, N709494, JN709494, JN709494were homo logy of MBNL1、 ZKSCAN1、SLC7A60S.The novel universal MTFP method could be applied for finding microRNA regulation targets or assessing targets gene expression profile.2Melatonin affect miRNA and its target genesMelatonin significantly alter the expression pattern of miRNA related to the growth of cashmere. With the exception of let-7a,the expression of miRNA occur three transition in a cashmere cycle. In control group, miR-203, miR-205, let-7, miR-96, miR-183in August and September were essentially unchanged at low levels, beginning to rise in October, November reached its highest level throughout a year. after a sharp decline, and in February and June there are two small peak expression; miR-205expression pattern consistent with the rest of the five miRNA, expression also appeared in three peaks in the cashmere cycle,but the highest expression of the annual value appeared in December. In implant group miR-203, miR-205expression levels appeared three distinct peaks, respectively in October, December, February, the highest values of miR-203expression occur in October throughout a year, miR-205is situated in February; Although miR-96, miR-199a have three peaks in October, December, February throughout a year, in addition in October reached its highest level throughout the year, another two peaks fluctuations no obvious; miR-183with inplanting melatonin at a low level throughout the yearMelatonin changed co-expression pattern of miRNAs. The correlation coefficient between miRNAs is the range of0.87-0.99in control group, were greater than0.85. compared with the control group, melatonin significantly weaken similarity between let-7a and miR-96、miR-199a、miR-205, miR-203and miR-96、miR-199a, miR-96and miR-183, miR-183and miR-199a.MTFP not detecte specific bands in inplant and control groupt, but miRNA target genes were qualitatively analysis.3Transcriptome library construction and sequencing of implant group and control group in October skinTwo mixed cDNA library were prepared from October inplanted and control skin sample In Inner Mongolia Cashmere Goats, the transcripts were sequenced using illumina sequencing platform. After cleaning and quality checks, approximately80million100bp clean reads were obtained. Finally,these reads were assembled into60,565contigs with a mean size of656bp,N50of860bp, the longest contigs of19,346bp, and52,331contigs were longer than300bp.393contigs up-regulated(fold_change (MT-10/C-10)>4, Q-value<0.001) and72contigs down-regulated(fold_change (MT-10/C-10)<-4,Q-value<0.001) by RNA-Seq analysis of CLCgenomicWorkbench6.0. of465differentially expression contigs,420contigs with annotation information in the NR database. Digital gene expression analysis from GO and KEGG pathways indicated that16pathways significantly enrichment in up-regulated gene, including Focal adhesion, extracellular matrix- receptor interaction, Regulation of actin cytoskeleton, TGF-β signaling pathway, Wnt signaling pathway and so on. Down-regulated genes not found significant enrichment pathway.4Melatonin mediate miRNA candidate target genesTo further investigate the relationship between melatonin, miRNA and its target genes. In this study, on the basis of October transcriptome data, using miranda3.0software predicted and obtained7miRNA-mediated16,750contigs target sequences. Screened Q value<0.05and|Fold_change|>2by comparing RPKM value between implanted and control groups,912contigs targets were differentially expression. Combining experimental miRNA target data, miR-203and target gene DOST, let-7a and target genes LYPLA1、ND4, miR-183and target gene GLG1, miR-199a and target genes KRTAP5.6, RPL30, MYH9, MYL6play important role in cashmere growth.