Identification Of Cashmere-related LncRNA XLOC₀05914 In Liaoning Cashmere Goats And Its Effect On Target Gene Expression

Liaoning cashmere goats are a valuable resource species that bans gene outflows in China.The domestic cashmere production of Liaoning cashmere goats ranks first in China and has great economic benefits.According to the current research on the velvet traits of Liaoning cashmere goats,the research content is mainly concentrated on the macroscopic and shallow areas,especially to study the molecular regulation mechanism of LncRNA and target genes has not been reported at home and abroad.Therefore,the research of this subject has important theoretical basis and practical guiding significance for accelerating the cultivation of Liaoning cashmere goats with high cashmere yield,fine cashmere fineness,and superior cashmere quality.In the previous research on Liaoning cashmere goats,our laboratory screened a differentially expressed signaling pathway—NF-?B signaling pathway through RNA-Seq sequencing technology,GO and KEGG and other bioinformatics analysis techniques.In this pathway,the differentially expressed LncRNA(XLOC?005914)corresponding to the differentially expressed target genes related to villous growth(IL8,NFKB2,TNF?,TNFAIP3,and NFKBIA)was identified.Treatment of cashmere goat skin fibroblasts with melatonin,the final determination of the concentration of 0.2g / L and the treatment time of 72 h is the most conducive to the growth of villi.Based on this,we conducted a qualitative experiment on the obtained LncRNA MTC to verify that it does not have a protein coding function,and is indeed a non-coding RNA;then we detected the expression of target gene of LncRNA MTC under the action of melatonin drugs by overexpression lentiviral technology,it's reveals the regulatory role of LncRNA associated with the growth of Liaoning cashmere goat hair on target genes from a completely new perspective.The main research results of this experiment are as follows: enter the LncRNA MTC sequence into the NCBI website to query its sequence related information,and after performing the sequence Blast(https://blast.ncbi.nlm.nih.gov/Blast.cgi),a highly coincident sequence was found,And the overlapping sequence is suspected to be a variable reading frame(Ovis aries musimon ribose-phosphate pyrophosphokinase 1-like,66 k D)that can encode a protein.Therefore,we first constructed a prokaryotic expression vector of the target sequence(p ET28a–LncRNA MTC)and transfected it into BL21 cells,and no expression of fusion protein was found.After that,we constructed a eukaryotic expression vector(p EGFP-C1-LncRNA MTC),transfectedit into 293 T cells,and no expression of fusion protein was found.It proves that LncRNA MTC does not have protein coding ability,and is a non-coding RNA.The expression levels of LncRNA MTC target genes were detected in skin cells overexpressing LncRNA MTC.The q PCR results showed that the expressions of IL8,NFKB2,and TNF? were up-regulated;the expressions of TNFAIP3(A20)and NFKBIA(IKB?)were down-regulated.In summary,our research confirmed that the LncRNA MTC identified by the previous research team did not have protein coding function,which not only clarifies the accuracy of the previous experimental project,but also lays a solid foundation for continuing the research on the role of non-coding RNA in the growth and development of cashmere goat hair;and LncRNA MTC can regulate the expression of its target genes,which also provides new methods and ideas for the in-depth study of the regulation mechanism of the cashmere traits of Liaoning cashmere goats.