牦牛毛色候选基因的筛选及MC1R基因功能验证

发布时间：2018-12-17 05:04

【摘要】：本试验以大通牦牛和天祝白牦牛两种毛色牦牛为研究对象,采用了荧光定量PCR、石蜡切片、建立B16细胞模型等试验方法。利用荧光定量PCR技术测定Agouti、MC1R、MITF和TY尺基因在不同毛色牦牛皮肤组织中相对表达量;通过甲苯胺蓝和HE染色方法观察黑色素颗粒在牦牛不同毛色皮肤组织中分布;用B16细胞作为生物模型,特异性shRNA重组质粒载体导入B16细胞抑制MC1R基因mRNA表达量,然后对转染B16细胞Agouti、MC1R、MITF和TYR基因表达量进行测定和测定B16细胞中黑色素颗粒含量。研究结果如下:(1)实时荧光定量PCR分析得到,Agouti基因表达量大通牦牛与天祝白牦牛无显著差异;MC1R基因表达量大通牦牛是天祝白牦牛的2.4倍,差异极显著(P0.01);MITF基因表达量在天祝白牦牛比大通牦牛高,差异显著;TYR基因表达量大通牦牛比天祝白牦牛高,差异显著。TYR对黑色被毛有一定影响,MITF对白色被毛有一定影响;推测黑色被毛与MC1R的高表达量有重要关系。(2)甲苯胺蓝染色比HE染色较容易观察到黑色素颗粒的分布和皮肤分层结构,但对黑色素细胞进行染色不理想;HE染色对黑色素细胞染色充分,细胞核和细胞质区分明显,能够看到黑色素细胞的细胞核和细胞质,黑色素颗粒在细胞中分布。黑色素细胞出现在表皮与真皮连接处,另外毛囊周围也大量分布。HE染色显示大通牦牛毛囊中与毛发相连毛乳头附近的黑色素含量比毛囊周围其他部位含量高。表皮层黑色素细胞有形成空泡趋势,呈现不规则形态,其一般细胞核比较大。天祝白牦牛表皮的基底层发现少量黑色素颗粒分布,但在毛囊毛球部及周围没有黑色素颗粒分布。在大通牦牛中大量黑色素颗粒分布在表皮基底层和毛囊周围。白毛色是天祝白牦牛毛囊毛球部的黑色素细胞无法生成黑色素颗粒,致使毛纤维中不存在黑色素颗粒呈现白色。(3)本研究用B16细胞作为生物模型,用shRNA对细胞中的目的基因MC1R表达抑制。测定细胞中黑色素颗粒含量,转染shRNA重组质粒后的细胞形态没有发生变化,活性没有受到影响。试验分为三组:对目的基因特异性抑制的shRNA重组质粒试验组、含有无效序列shRNA重组质粒阴性对照组和空白对照组。转染特异性shRNA重组质粒后试验组B16细胞合成的黑色素颗粒含量降低,与空白对照组和阴性对照组差异极显著。从蛋白水平,空白对照和阴性对照分别是试验组的7.5倍和6.83倍,抑制率达到87%。MC1R基因表达量空白对照和阴性对照分别是试验组的11.21倍和10.19倍,试验组表达量低于其它两组差异极显著(P0.01),抑制率91%以上。天祝白牦牛毛囊分布着黑色素细胞及表皮少量黑色素颗粒;MC1R基因表达量对黑色素颗粒的形成起着重要的调控作用。
[Abstract]:In this experiment, Datong yak and Tianzhu white yak were used as research objects. Fluorescence quantitative PCR, paraffin sections were used to establish B16 cell model. Fluorescence quantitative PCR technique was used to detect the relative expression of Agouti,MC1R,MITF and TY ulnar genes in different hairy yak skin tissues, and to observe the distribution of melanin granules in yak skin tissues with different coat color by toluidine blue and HE staining. B16 cells were used as biological model. The specific shRNA recombinant plasmid vector was introduced into B16 cells to inhibit the expression of MC1R gene mRNA. Then the expression of Agouti,MC1R,MITF and TYR genes in B16 cells was measured and the content of melanin granules in B16 cells was measured. The results were as follows: (1) the expression of Agouti gene in Datong yak and Tianzhu white yak was not significantly different from that in Tianzhu white yak by real-time fluorescence quantitative PCR analysis, and the expression of MC1R gene in Datong yak was 2.4 times higher than that in Tianzhu white yak (P0.01). The expression of MITF gene in Tianzhu white yak was higher than that in Datong yak, and the expression of TYR gene in Datong yak was higher than that in Tianzhu white yak, the difference was significant. TYR had a certain effect on black coat, MITF on white coat. (2) toluidine blue staining was easier than HE staining to observe the distribution of melanin particles and skin layering structure, but the staining of melanocytes was not ideal; HE staining showed that the melanocytes were stained well and the nuclei and cytoplasm were distinguished clearly. The melanocytes and cytoplasm of melanocytes could be seen and melanin granules were distributed in the cells. Melanocytes appeared at the junction of epidermis and dermis and were also distributed around hair follicles. HE staining showed that melanin content near hair papilla was higher in Datong yak hair follicles than in other parts around hair follicles. The melanocytes in the epidermis tend to form vacuoles, showing irregular morphology, and their nuclei are relatively large. A small amount of melanin granules were found in the basal layer of the epidermis of Tianzhu white yak, but there were no melanin granules in and around the hair follicle. In Datong yak, a large number of melanin granules were distributed in the basal layer of epidermis and around the hair follicle. White coat color is the melanocyte of hair follicle of Tianzhu white yak can not produce melanin granules, resulting in the absence of melanin granules in hair fibers. (3) B16 cells were used as biological model in this study. The expression of target gene MC1R was inhibited by shRNA. The content of melanin granules in the cells was determined. The morphology of the cells transfected with shRNA recombinant plasmid was not changed and the activity of the cells was not affected. The experiment was divided into three groups: the shRNA recombinant plasmid group which specifically inhibited the target gene, the negative control group containing invalid shRNA recombinant plasmid and the blank control group. After transfection of specific shRNA recombinant plasmid, the content of melanin granules in B16 cells in the experimental group was significantly lower than that in the blank control group and the negative control group. The protein levels of the blank control and negative control were 7.5 times and 6.83 times of that of the experimental group, respectively, and the inhibition rate was 11.21 times and 10.19 times higher than that of the control and negative control, respectively. The expression level in the experimental group was significantly lower than that in the other two groups (P0.01), and the inhibitory rate was over 91%. Melanocytes and a small amount of melanin granules were distributed in the hair follicles of Tianzhu white yak, and the expression of MC1R gene played an important role in the formation of melanin granules.
【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】：S823.85

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