Screening Of Specific Long Non-coding RNAs Regulating Periodic Growth Of Cashmere

Long non-coding RNAs(lncRNAs) are a series of RNA molecules that do not encode proteins and have transcripts longer than 200 nt in length. They can regulate gene expressions at various levels including the epigenetic inheritance, the transcription progress and the post-transcriptional, etc. Considering the only special occurrence of many lncRNAs in the development of cashmere goats and the cyclical growth of cashmere, this study conducted denovo sequencing via RNA-seq at anagen and telogen stages, respectively. After analysing the results of the sequencing, 10 lncRNAs were screened and performed by qRT-PCR to verify. Finally, 5 verified lncRNAs were constructed to the overexpression vectors, which would provide some basic information for studing the function of the specific lncRANs and regulating mechanisms of cyclical growth of cashmere in the respect of lncRNAs.The results were as follows:1. The RNA-seq reads at anagen and telogen stage were 51,531,778 and 61,750,544, and the transcripts were 37,574 and 37,619, separately. 37,649 genes expressed in these two stages, among which 977 genes expressed differently(P<0.05), 559 genes up-regulated and 418 genes down-regulated. 6127 lncRNAs were transcribed, among which 54 lncRNAs were differently transcribed, 32 up-regulated and 22 down-regulated. Go-analysis indicated that the transcripts of samples obtained from goat skin tissues were related with the growth and development of skin in the function and cellular component, including HoxA、HoxB、HoxC and HoxD family genes, BMPs family and KAPs、FGFs genes and their receptor genes,etc. KEGG analysis results indicated that there were some lncRNAs concerning with sensing signal mechanism. Furthermore, there existed significant difference between the quantity of mRNA and lncRNAs from differencial expression genes, demonstrating that there might be some certain relevance of many non-coding genes with encoded proteins.2. The qRT-PCR results of 10 differently-transcribed lncRNAs showed similar trend with the RNA-seq data. However, lncRNA9686 didn’t show significant difference between the two stages.3. Polymerase chain reaction(PCR) combined with restriction enzyme digestion were used to construct 5 over expression vectors for lncRNA9686, lncRNA14672, lncRNA19457, lncRNA15479 and lncRNA13025. Plasmid pB-CMV-DsRed-CMV-GFP.BS.with repeats was used as backbone. After confirming these vectors via double restriction enzyme digestion and sequencing, the overexpression vectors were successfully constructed, which could be used in further study.