【作者】 张小伟；

【导师】 黄锡霞； 田可川；

【作者基本信息】 新疆农业大学 ， 畜牧学， 2021， 硕士

【摘要】 毛囊的生长发育和周期性的变化是受多种因子调控的复杂分子调控过程,lnc RNA和mi RNA是一类新发现的非编码RNA,它们不被翻译成蛋白质,而是调节各种细胞和生物过程,mi RNA、lnc RNA和m RNA之间相互作用构成复杂的调控关系。越来越多的研究报道了mi RNA和lnc RNA在毛囊发育及周期性生长中的作用,但是对毛囊生长发育的调控网络研究还较少。因此本试验通过挖掘苏博美利奴羊皮肤组织样本转录组数据,分析lnc RNA、mi RNA与m RNA间存在的调控关系。构建lnc RNA-mi RNA-m RNA分子调控网络,进一步对调控网络靶基因进行功能分析,筛选寻找毛囊发育相关nc RNA调控网络中的关键基因并进行验证。(1)通过构建不同时期苏博美利奴羊毛囊发育相关lnc RNA-mi RNA-m RNA调控网络,并对lnc RNA-mi RNA-m RNA调控网络中的靶基因进行功能分析,筛选调控网络中的关键基因,发现G2vs.G1组中靶基因TGFB2和NOTCH1参与毛囊形态发生(GO:0031069)的生物学过程。(2)荧光定量结果表明:mi R-133、mi R-433-3p与NOTCH1表达趋势相反,TCONS＿00008348与mi R-154b-3p、mi R-485-3p、mi R-539-3p表达趋势相反。mi R-154b-3p、mi R-485-3p、mi R-539-3p与靶基因MAPKAPK3、DUSP1、PTPRR的表达趋势相反。TCONS＿00008348与MAPKAPK3、DUSP1、PTPRR表达趋势一致。因此可初步推测TCONS＿00008348和mi R-154b-3p、mi R-485-3p、mi R-539-3p及靶基因MAPKAPK3、DUSP1、PTPRR之间具有靶向关系。(3)双荧光素酶报告系统系显示:NOTCH1-WT+mi R-133 mimics与NOTCH1-WT+NC mimics组相比萤火虫荧光素酶活性值显著下降,NOTCH1-WT+mi R-433-3p mimics与NOTCH1-WT+NC mimics组相比萤火虫荧光素酶活性值也显著下降,说明oar-mi R-133和oar-mi R-433-3p可以靶向结合NOTCH1基因的3’UTR。(4)对NOTCH1基因核苷酸和氨基酸序列进行生物信息学分析,发现绵羊NOTCH1基因位于第3号染色体上,其CDS区长度为9487bp,编码2541个氨基酸。绵羊NOTCH1蛋白的p I为4.95,为中性蛋白。不稳定系数为52.17,所以该蛋白质不稳定。绵羊NOTCH1蛋白含有信号肽,其在21和22之间的切割位点概率为0.52。跨膜螺旋数为1,说明NOTCH1蛋白为跨膜蛋白。含有8个O-糖基化位点,9个N-糖基化位点及37个磷酸化位点。其二级结构由70.64%的无规则卷曲,12.71%的α螺旋,16.65%的延伸链组成。综上所述,本研究通过构建苏博美利奴羊毛囊发育相关的nc RNA分子调控网络,将mi RNA、lnc RNA与毛囊的生长发育紧密联系起来,从转录组学层面对毛囊生长发育进行探讨,通过找寻与羊毛生长发育调控的相关基因,对苏博美利奴羊毛囊发育的分子作用机制进行系统性挖掘。更多还原

【Abstract】 The growth and development and periodic changes of hair follicles were complex molecular regulation processes that were regulated by a variety of factors.Lnc RNA and mi RNA are newly discovered non-coding RNA.They are not translated into proteins but regulate various cellular and biological processes.The interaction between mi RNA,lnc RNA and m RNA constitutes a complex regulatory relationship.More and more studies had reported the role of mi RNA and lnc RNA in the development and periodic growth of hair follicles,but there were few studies on the regulatory network of hair follicle growth and development.Therefore,this experiment used bioinformatics methods to analyze the regulatory relationship between lnc RNA,mi RNA,and m RNA in the development of Subo merino wool sac by mining the transcriptome data of Subo merino sheep skin tissue samples.Construct the lnc RNA-mi RNA-m RNA molecular regulatory network,further analyzed the function of the target genes of the regulatory network,screened and find the key genes in the nc RNA regulatory network related to hair follicle development and verify it.(1)Construct the lnc RNA-mi RNA-m RNA regulatory network related to the development of Submerino wool follicles at different stages,and conduct functional analysis of the target genes in the lnc RNA-mi RNA-m RNA regulatory network,screen the key genes in the regulatory network,found that the target genes TGFB2 and NOTCH1 are involved in the biological process of hair follicle morphogenesis(GO:0031069).(2)Fluorescence quantitative results showed that the expression trends of mi R-133,mi R-433-3p,and NOTCH1 were opposite.The expression trend of TCONS＿00008348 is opposite to that of mi R-154b-3p,mi R-485-3p,and mi R-539-3p.The expression trends of mi R-154b-3p,mi R-485-3p,and mi R-539-3p were opposite to the target genes MAPKAPK3,DUSP1,and PTPRR.TCONS＿00008348was consistent with the expression trend of MAPKAPK3,DUSP1,and PTPRR.Therefore,it could be concluded that TCONS＿00008348 had a targeting relationship with mi R-154b-3p,mi R-485-3p,mi R-539-3p,and the target genes MAPKAPK3,DUSP1,PTPRR.(3)The dual luciferase reporter system showed that the firefly luciferase activity value of NOTCH1-WT+mi R-133 mimics was significantly lower than that of NOTCH1-WT+NC mimics group.Compared with NOTCH1-WT+mi R-433-3p mimics and NOTCH1-WT+NC mimics group,the value of firefly luciferase activity also decreased significantly.This indicates that oar-mi R-133 and oar-mi R-433-3p can target the 3’UTR of the NOTCH1.(4)Bioinformatics analysis of the nucleotide and amino acid sequences of the NOTCH1 gene revealed that the sheep NOTCH1 gene is located on chromosome 3,and its CDS region is 9487 bp,which encodes 2541 amino acids.p I=4.95,which is a neutral protein.The instability coefficient is 52.17,and the protein is unstable.The sheep NOTCH1 protein contains a signal peptide,and its cleavage site probability between 21 and 22 is 0.5269.The number of transmembrane helix is 1,which is a transmembrane protein.It contains 8 O-glycosylation sites,9 N-glycosylation sites and 37 phosphorylation sites.Its secondary structure is composed of 70.64% random coils,12.71% alpha helices,and 16.65% extended chains.In summary,this study established a non-coding RNA molecular regulatory network related to the development of Subo merino wool follicles,closely linked mi RNA and lnc RNA with the growth and development of hair follicles,and discussed the growth and development of hair follicles from the transcriptomics level.By searching for genes related to the regulation of wool growth and development,the molecular mechanism of subo merino wool sac development was systematically explored.更多还原