【作者】 程杰；

【导师】 陈宏；

【作者基本信息】 西北农林科技大学 ， 农业推广硕士（专业学位）， 2019， 硕士

【摘要】 肌肉发育是一个高度协调的生物学过程,目前对肌肉发育的遗传基础已经有了较清晰的认识。其中涉及PAX基因(paired-homeobox transcription factor)家族,肌生成调节因子(myogenic regulatory factors,MRFs)家族,肌细胞增强因子(myocyte enhancer factor 2,MEF-2)家族和各种复杂的信号通路。虽然骨骼肌形成的遗传基础很清楚,但是由于肌生成涉及的调控网络极其复杂,这一过程中涉及的一大部分调控因子仍然未知。因此,发掘新调控因子和进一步解析骨骼肌发育的分子机制仍然是迫切需要的。近些年来,随着组学研究的不断地深入,大量研究表明FGFR1基因对于肌肉发育有着非常重要的作用,本研究在其它家畜上有少量的报道,但是在牛上FGFR1基因对于肌肉发育的调控作用鲜有报道。因此,本研究利用载体构建,细胞培养,双荧光素酶报告系统,亚硫酸氢盐测序PCR,CCK-8,EdU,qPCR,RNA干扰,蛋白免疫印迹(Western Blot)和流式细胞术等技术对FGFR1基因调控成肌细胞增殖分化的分子机制开展了深入的研究。主要研究结果如下:1.FGFR1基因启动子活性区域筛选为了确定控制FGFR1基因转录的核心启动子,设计启动子不同区域的引物,将不同长度的截短体与线性的pGL3-Basic相连,将构好的载体转染C2C12和293T两个细胞系,测定双荧光,筛选出FGFR1基因的启动子活性核心区域位于FGFR1基因上游202 bp至509 bp和794 bp至1295 bp之间。利用在线软件JASPAR2018对活性区域794bp至1295 bp内结合的转录因子进行预测发现,在活性区域内具有多个与细胞发育相关的转录因子结合位点,如Sp1、EGR1和KLF5等转录因子。2.秦川牛FGFR1基因核心启动子区甲基化研究为了了解秦川牛不同生长时期肌肉组织中FGFR1基因核心启动子区是否存在差异甲基化及其对mRNA表达量的影响。运用亚硫酸氢盐测序PCR技术检测秦川牛胎牛、犊牛和成年牛三个生长时期肌肉组织中FGFR1基因启动子区甲基化状态,同时采用qPCR技术检测秦川牛三个不同生长时期肌肉组织中FGFR1基因mRNA的表达水平。秦川牛胎牛期(0.595%)肌肉组织中FGFR1基因启动子区甲基化水平显著低于犊牛时期(2.14%)(P<0.05);秦川牛胎牛时期肌肉组织中FGFR1基因mRNA表达水平显著高于犊牛时期(P<0.05)。因此,在秦川牛肌肉组织中FGFR1基因启动子区存在差异甲基化,并且启动子区的甲基化程度越高越能抑制FGFR1基因表达。3.FGFR1调控成肌细胞增殖分化的研究为了研究FGFR1基因对牛成肌细胞增殖分化的影响,利用EdU及CCK-8试剂盒法对细胞的增殖进行检测,发现与NC对照组相比,干扰FGFR1基因后细胞数目显著减少。利用流式细胞术对细胞周期进行检测,发现与NC对照组相比,siFGFR1基因G1期的细胞数显著增加,S期的细胞数显著减少。并且干扰FGFR1后,对增殖标志基因的mRNA和蛋白水平进行检测,与NC对照组相比,发现CylincD1,CDK2的表达量和蛋白量显著降低。对分化标志基因的mRNA和蛋白水平进行检测,MyoD和MyHC均有升高的趋势,但不显著,MyoG有下降的趋势,所以不足以证明它对分化的调控作用。以上结果表明,FGFR1对牛成肌细胞增殖有显著的促进作用。本文详细探究了FGFR1基因的转录调控及其对牛成肌细胞增殖分化的影响,为后续肉牛肌肉发育相关基因的研究提供了新的思路和研究基础,对于揭示中国地方黄牛肌肉发育的调控网络,为中国专门化肉牛品种的培育提供理论支持。更多还原

【Abstract】 Muscle development is a highly coordinated biological process,and at present there is a in-depth understanding of the genetic basis of muscle development.This process is involved by the PAX gene(Paired-homeobox transcription factor)family,the muscle-generating regulatory factor(myogenic regulatory Factors,MRFs)family,and the muscular cell enhancement factor(the Myocyte Enhancer factor 2,MEF-2)family and a variety of complex signaling pathways.Although the genetic hierarchy of skeletal muscle formation has been intensely studied,a large proportion of the regulatory factors involved in this process are still unknown due to the complexity of the regulatory networks involved in muscle generation.Therefore,the discovery of new regulatory factors and the further understanding of the molecular mechanisms during skeletal muscle development are still urgently needed.In recent years,with the continuous deepening of the genomics study,the very important role of FGFR1 in muscle development is beginning to be realized.To date,there is a small amount of reports on domestic animals,but its regulatory role in cattle muscle development has not been reported yet.Therefore,this paper used vector construction,cell culture,dualfluorescein enzyme reporting system,bisulfite sequencing PCR,CCK-8,EdU,quantitative PCR(qPCR),RNA interference and Western Blot and other techniques to carry out an in-depth study on the molecular mechanism of FGFR1 gene in the proliferation and differentiation of myoblast cells.The main findings are as follows:1.Screening of the active promoter region of FGFR1 gene in muscle.We designed primers in different regions of the 2000 bp genomic region upstream the TSS,and cloned into vector of pGL3-Basic,and then transfected the C2C12 and 293 T cell lines.The active promoter region of the FGFR1 gene was identified as 202 bp to 509 bp and 794 bp to 1295 bp in the upstream of the FGFR1 gene TSS.Using the online software JASPAR2018 to predict transcription factors in active area 794 bp to 1295 bp,it was found that there were multiple transcription factor binding sites associated with cell differentiation in the active region,such as Sp1,EGR1 and KLF5 transcription factors.2.Study on the methylation in the promoter region of the FGFR1 gene in Qinchuan cattle.In order to understand whether there was a negative correlation between methylation in the core promoter region of FGFR1 gene and the expression of mRNA in muscle tissue in different growth periods of Qinchuan cattle.The methylation status of FGFR1 gene promoter region in muscle tissue of three growth periods(fetal,calf and adult)was detected by bisulfite sequencing PCR.Besides,the corresponding expression level of FGFR1 mRNA in these muscle tissues was detected by qPCR.The methylation level of fetal bovine(0.595%)in the promoter region of FGFR1 gene was significantly lower than that of calf bovine(2.14%)(P < 0.05),while the mRNA expression level of FGFR1 gene in muscle tissue of fetal bovine was significantly higher than that in calf bovine(P < 0.05).Therefore,there is a different methylation level in the promoter region of FGFR1 gene in the muscle tissue of Qinchuan cattle,and the higher the methylation degree,the lower the FGFR1 gene expression.3.Study on proliferation and differentiation effects of FGFR1 in myocytes.In order to study the effect of FGFR1 gene on proliferation and differentiation of cattle myoblasts,the cell proliferation was detected by EdU and CCK-8 methods,and it was found that the cell number of si FGFR1 decreased significantly compared with that of NC control group.The cell cycle was detected by flow cytometry,and it was found that the number of cells in the G1 phase and the S phase of siFGFR1 gene increased and decreased significantly respectively compared with that of NC control group.And after siFGFR1,the mRNA and protein levels of the proliferative marker gene were detected,and the mRNA and protein abundance of CylincD1,CDK2 were found to be significantly lower than that of NC control group.The mRNA and protein levels of the differentiation marker gene were tested.MyoD and MyHC had an elevated trend and MyoG had a falling trend,which was not significant,so these data were not enough to prove that its differentiation functions.The above results show that FGFR1 has a significant effect on the proliferation of bovine myoblasts.In this paper,the regulation mechanism of FGFR1 gene on bovine myoblasts was probed,which will provide a new idea and basis for the study of genes related to muscle development of cattle,and provide theoretical support for the cultivation of local cattle in China.更多还原