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绒山羊胚胎期次级毛囊发生发育相关miRNA的筛选及靶基因验证
尚方正,马荣,戎友俊,潘剑锋,王敏,牛舒冉,齐云鹏,李彦伯,李金泉,张燕军
0
(内蒙古农业大学 动物科学学院/农业农村部肉羊遗传育种重点实验室/内蒙古自治区动物遗传育种与繁殖重点实验室/内蒙古自治区山羊遗传育种工程技术研究中心, 呼和浩特 010018)
摘要:
为筛选绒山羊次级毛囊发生发育相关microRNA(miRNA)及靶基因验证,本研究基于课题组前期高通量测序技术得到绒山羊胎儿期(45、55、65、75、95、115和135 d)皮肤组织miRNA表达谱的基础上,鉴定各比对组中差异表达的miRNA并根据毛囊形态发生规律筛选次级毛囊发生发育相关的miRNA,运用RNAhybrid、miRanda和TargetScan软件预测筛选到的miRNA靶基因并与次级毛囊发育相关基因取交集,所得基因进行GO和KEGG富集分析,筛选毛囊发生发育重要通路中的关键基因构建miRNA-mRNA调控网络,进而运用双荧光素酶报告验证关键miRNA-mRNA的靶向关系。结果表明:1)筛选到次级毛囊发生发育相关miRNA 153个,共预测到32 978个靶基因;2)取交集后得到1 352个靶向的次级毛囊发生发育相关的基因,且显著富集在29条KEGG通路中,其中毛囊发生发育重要通路TGF-β同样显著富集;3)构建了chi-miR-26b-5p-SMAD1和 chi-miR-495-3p-SMAD1等232对次级毛囊发生发育可能相关的miRNA-mRNA关系对,双荧光素酶报告系统进一步验证了关键靶向关系。本研究为解析miRNA在绒山羊次级毛囊发育过程中的分子机制奠定了前期基础。
关键词:  绒山羊  次级毛囊  miRNA  靶基因
DOI:10.11841/j.issn.1007-4333.2023.02.08
投稿时间:2022-02-18
基金项目:国家重点研发计划资助(2021YFD1200902);内蒙古自治区科技重大专项项目(2021ZD0012);研究生科研创新项目(B20210175Z)
Screening of miRNAs related to secondary hair follicle development in cashmere goats and validation of their target genes
SHANG Fangzheng,MA Rong,RONG Youjun,PAN Jianfeng,WANG Min,NIU Shuran,QI Yunpeng,LI Yanbo,LI Jinquan,ZHANG Yanjun
(College of Animal Science/Key Laboratory of Mutton Sheep Genetics and Breeding, Ministry of Agriculture/Key Laboratory of Animal Genetics, Breeding and Reproduction/ Engineering Research Center forGoat Genetics and Breeding, Inner Mongolia Agricultural University, Hohhot 010018, China)
Abstract:
In order to screen microRNA(miRNA)related to the morphogenesis and development of secondary hair follicles in cashmere goats and verify their target genes, in this study, based on the previous high-throughput sequencing technology of the research group, the miRNA expression profiles of cashmere goat fetal period(45, 55, 65, 75, 95, 115 and 135 d)skin tissue were obtained. The differentially expressed miRNAs were screened according to the hair follicle morphogenesis rules, and the miRNAs related to the development of secondary hair follicles were identified. RNAhybrid, miRanda and TargetScan software were used to predict the screened miRNA target genes and intersected with the genes related to the development of secondary hair follicles. The intersected genes were enriched and analyzed by go and KEGG to screen the key genes in the important pathway of hair follicle development, construct the miRNA mRNA regulation network, and further verify the targeting relationship of key miRNA mRNA by using the double Luciferase Report. The results showed that: 1)A total of 53 miRNAs related to secondary hair follicle development were screened, and 32 978 target genes were predicted; 2)After the intersection, 1 352 targeted genes related to secondary hair follicle development were obtained, which were significantly enriched in 29 KEGG pathways, including TGF-β, an important pathway of hair follicle development; 3)Similarly, 232 pairs of miRNA mRNA pairs that may be related to the occurrence and development of secondary hair follicles, such as chi-miR-26b-5p-SMAD1 and chi-miR-495-3p-SMAD1, were constructed. The dual-luciferase reporter system further verified the key targeting relationship. In conclusion, this study laid a preliminary foundation for analyzing the molecular mechanism of miRNA in the development of secondary hair follicles of cashmere goats.
Key words:  cashmere goats  secondary hair follicle  miRNA  target gene