牦牛TMEM18基因多态性及其功能研究

发布时间：2018-05-21 04:42

本文选题：牦牛 + TMEM18　；参考：《甘肃农业大学》2017年硕士论文

【摘要】：我国是世界牦牛拥有量最多的国家,但牦牛的生长周期长,繁殖性能远远低于其他牛种,这限制了牦牛畜牧业经济的进一步发展。跨膜蛋白18(TMEM18)是一个末端为低聚嘧啶的基因,该基因与动物生长发育和繁殖性能密切相关。鉴于此,本研究对有关牦牛TMEM18基因多态性及其功能的进行研究。通过荧光定量PCR和蛋白免疫印迹法分别检测了TMEM18在牦牛不同组织中mRNA和蛋白的表达情况,并利用免疫组织化学染色技术对牦牛不同组织中TMEM18蛋白定位;以192头牦牛血样DNA构建混合池,扩增TMEM18基因内含子1和外显子5的序列。运用高分辨率熔解曲线分析技术(high resolution melting curve,HRM)统计基因型分型以及运用SPSS 21.0分析基因多态位点与生产性能关联;在质粒pIRES2-EGFP的多克隆位点插入TMEM18基因构建真核表达载体pIRES2-EGFP-TMEM18,用脂质体技术转染牦牛胎儿成纤维细胞。利用蛋白免疫印迹法、荧光定量PCR技术检测牦牛胎儿成纤维细胞转染前后TMEM18基因的表达量。研究结果如下:(1)TMEM18蛋白在牦牛各组织器官中均有表达,其中睾丸中mRNA和蛋白表达量极显著高于其他组织(P0.01);在牦牛心肌细胞、肝细胞、脾脏红髓、脾小梁、肺终末细支气管、肺腺体、肺平滑肌、肾脏皮质肾小管、骨骼肌细胞及卵巢颗粒细胞中呈TMEM18阳性免疫反应;而在睾丸中精原细胞、初级精母细胞和次级精母细胞中TMEM18阳性反应明显高于其他组织(P0.01)。(2)牦牛TMEM18基因内含子1处存在2个多态位点,分别是:861(A/G)和1267(C/T),外显子5处存在1个多态位点为4447(C/T)。关联分析表明,牦牛TMEM18基因861(A/G)位点的不同基因型与牦牛的体高、体重、胴体重和屠宰率差异性显著(P0.05);1267(C/T)位点的不同基因型与牦牛的体高、体重和屠宰率差异性显著(P0.05),而牦牛TMEM18基因4447(C/T)位点的不同基因型与牦牛的体斜长、管围、胸围、体重、胴体重、净肉重、净肉率和屠宰率均存在显著性差异(P0.05)。(3)成功构建出pIRES2-EGFP-TMEM18真核表达载体,验证发现转染成纤维细胞TMEM18蛋白和mRNA的表达量显著提高(P0.01)。本研究初步筛选出TMEM18与牦牛生产性状相关的多态位点并成功构建的真核表达载体pIRES2-EGFP-TMEM18,为今后牦牛遗传改良和克隆技术以及基因的功能研究提供基础。
[Abstract]:China is the country with the largest number of yaks in the world, but the growth cycle of yaks is long, the reproductive performance is far lower than other cattle breeds, which limits the further development of yak animal husbandry economy. Transmembrane protein 18 (TMEM18) is a terminal oligopyrimidine gene, which is closely related to animal growth, development and reproductive performance. In view of this, we studied the polymorphism and function of yak TMEM18 gene. The expression of TMEM18 in different yak tissues was detected by fluorescence quantitative PCR and Western blotting, and the localization of TMEM18 protein in different yak tissues was detected by immunohistochemical staining. The mixed pool was constructed with DNA from 192 yak blood samples and the sequences of intron 1 and exon 5 of TMEM18 gene were amplified. High-resolution fusion curve analysis technique was used to analyze genotype typing of high resolution melting and SPSS 21.0 was used to analyze the relationship between gene polymorphism and production performance. The eukaryotic expression vector pIRES2-EGFP-TMEM18 was constructed by inserting TMEM18 gene into the polyclonal site of plasmid pIRES2-EGFP and transfected into yak fetal fibroblasts by liposome technique. The expression of TMEM18 gene in yak fetal fibroblasts before and after transfection was detected by Western blot and fluorescence quantitative PCR. The results showed that TMEM18 protein was expressed in all tissues and organs of yak, and the expression of mRNA and protein in testis was significantly higher than that in other tissues (P0.01A), and in yak cardiomyocytes, hepatocytes, red pulp of spleen, trabeculae of spleen and bronchioles of lung. TMEM18 positive immunoreactivity was found in lung glands, pulmonary smooth muscle, renal cortex, renal tubules, skeletal muscle cells and ovarian granulosa cells, while spermatogonium cells were found in testis. The positive reaction of TMEM18 in primary spermatocytes and secondary spermatocytes was significantly higher than that in other tissues (P0.01. 01. 2) there were two polymorphic loci in intron 1 of TMEM18 gene of yak, which were: 1: 861 A / G) and 1267 C / T, and 1 polymorphic locus was 4447 C / T at exon 5. The association analysis showed that the different genotypes of the 861 TMEM18 gene and the body height, body weight, carcass weight and slaughter rate of yak were significantly different between the different genotypes of P0.05 and 1267 C / T loci and the body height of yaks. The difference between body weight and slaughter rate was significant (P0.05A), while different genotypes of TMEM18 gene 4447C / T were associated with yak body length, tube circumference, chest circumference, body weight, carcass weight and net meat weight. The eukaryotic expression vector of pIRES2-EGFP-TMEM18 was successfully constructed. The results showed that the expression of TMEM18 protein and mRNA in fibroblasts was increased significantly. The eukaryotic expression vector pIRES2-EGFP-TMEM18 was successfully constructed by screening the polymorphic loci of TMEM18 associated with yak production traits, which provided the basis for the genetic improvement and cloning of Yak and the functional study of the gene.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;S823.85

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