利用iTRAQ蛋白质组学技术筛选与种公鸡繁殖力相关的候选蛋白

发布时间：2018-04-01 07:40

本文选题：北京油鸡种公鸡　切入点：繁殖力　出处：《中国农业科学院》2015年硕士论文

【摘要】：中国地方鸡种肉蛋品质优良,抗病能力强,但繁殖效率较低。进一步调查发现,地方鸡无精症、弱精症比例较高,而且精液量的减少常常伴随着精子浓度降低,提高了地方鸡生产中公母配比上升。增加了动物生产成本。研究发现:鸡精液品质性状为中等遗传力性状,较大程度上受到遗传因素的影响。本研究目的是探究影响繁殖力的遗传因素。蛋白质作为基因表达的产物,直接调控性状。本研究通过筛选低繁殖力公鸡与正常公鸡,比较睾丸发育学和形态学特征,检测低繁殖力公鸡与正常公鸡睾丸组织蛋白表达量的差异,利用同位素标记相对和绝对定量(iTRAQ)技术及生物信息学分析,筛选与繁殖力相关的候选蛋白,并进行蛋白质印迹法验证,为进一步深入研究影响公鸡繁殖力及睾丸发育分子遗传机制提供理论基础和潜在方向。主要研究内容及结果如下:1.低繁殖力公鸡挑选及睾丸特征比较。试验动物为42周龄相同环境下饲养北京油鸡公鸡200只,通过精液品质检测和交配试验挑选出差异显著的低繁殖力与正常种公鸡各25只。52周龄获取睾丸组织,称重,制作组织切片并观测睾丸精细小管形态,综合比较低繁殖力公鸡与正常公鸡睾丸参数即生精上皮长,精细小管直径、面积,约翰逊评分等。结果显示低繁殖力组公鸡睾丸重量、生精上皮长、精细小管直径、面积及约翰逊评分等睾丸参数显著低于正常组。根据试验结果,挑选出3只低繁殖力公鸡及3只正常同胞公鸡,作为iTRAQ试验样本。2.睾丸组织差异表达蛋白筛选、分析及验证。利用iTRAQ技术,对样本睾丸组织总蛋白进行相对及绝对定量分析。对iTRAQ定量结果的分析,进行低繁殖力和正常个体及组间的睾丸组织蛋白表达相互比较。结果发现,低繁殖力组上调倍数较大的蛋白为天冬氨酸氨基转移酶(AspAT)、谷胱甘肽S转移酶(GST)蛋白和组蛋白H2A(Histone H2A),下调倍数较大的蛋白有DNA损伤修复蛋白(DDB2)、T型中心粒蛋白(CENP-T)和G型蛋白酪氨酸磷酸酶(PTPRG)。KEGG Pathway富集分析发现差异表达蛋白显著富集于嘌呤和嘧啶的代谢、精氨酸和脯氨酸代谢等信号通路。利用蛋白免疫印迹法(Western Blotting)验证GST蛋白、AspAT蛋白和Histone H2A蛋白在低繁殖力组和正常组睾丸组织中表达情况,结果发现蛋白表达趋势与iTRAQ技术检测结果一致。综合试验2结果,DDB2、CENP-I、AspAT、GST、S100-A6和Histone H2A为调控精子发生的候选蛋白,其中AspAT、GST、DDB2和Histone H2A为重要候选蛋白。GnRH信号通路、嘌呤和嘧啶的代谢和蛋白质的转运等信号通路可以作为公鸡繁殖力相关的重要候选调控通路。
[Abstract]:Chinese local chicken breeds have good quality and strong resistance to disease, but the breeding efficiency is low. Further investigation found that the proportion of azoospermia and asthenospermia in local chickens is higher, and the decrease of spermatozoa is often accompanied by a decrease in sperm concentration. The ratio of male and female increased in the production of local chickens, and the cost of animal production was increased. The results showed that the quality of chicken semen was a medium heritability trait. The aim of this study was to investigate the genetic factors that affect fecundity. Protein, as a product of gene expression, directly regulates traits. By comparing testicular development and morphological characteristics, the differences of testicular protein expression between low fecundity rooster and normal cock were detected, and relative and absolute quantitative iTRAQ techniques and bioinformatics analysis were used. Candidate proteins associated with fecundity were screened and verified by Western blotting. The main contents and results of this study are as follows: 1. Selection of low fecundity rooster and comparison of testicular characteristics. A total of 200 Beijing fowl roosters were raised in the same environment at 42 weeks of age. Testicular tissue was obtained by semen quality test and mating test in 25 male chickens of low fecundity and 25 normal cocks at the age of .52 weeks. Tissue sections were made and the morphology of testicular fine tubules was observed. The testicular parameters of low fecundity rooster and normal cock were compared, such as the length of spermatogenic epithelium, the diameter of fine tubules, the area and Johnson's score, etc. The results showed that the weight of testis, the length of spermatogenic epithelium and the diameter of fine tubules in the low fecundity group. The testicular parameters, such as area and Johnson's score, were significantly lower than those in the normal group. According to the results of the experiment, three low fecundity roosters and three normal sibling roosters were selected as samples of iTRAQ test. Analysis and verification. Relative and absolute quantitative analysis of total protein in testicular tissue was carried out by using iTRAQ technique. In the analysis of quantitative results of iTRAQ, the low fecundity and the expression of testicular tissue protein in normal individuals and groups were compared. In low fecundity group, aspartate aminotransferase (AST), glutathione S-transferase (glutathione S-transferase) and histone (H2A(Histone H2AN) were the most up-regulated proteins. The down-regulated proteins included DNA damage repair protein (DNA damage repair protein), CENP-T) and G type. The enrichment analysis of protein tyrosine phosphatase (PTPRG). KEGG Pathway showed that the differentially expressed proteins were significantly enriched in the metabolism of purine and pyrimidine. The expression of GST protein AspAT protein and Histone H2A protein in testis of low fecundity group and normal group was detected by Western blotting. The results showed that the trend of protein expression was the same as that of iTRAQ technique. The results of experiment 2 showed that DDB2CENP-IAspATT S100-A6 and Histone H2A were candidate proteins for regulating spermatogenesis, among which AspAT-GST-DDB2 and Histone H2A were important candidate proteins. The signaling pathways of purine and pyrimidine metabolism and protein transport can be used as important candidate regulatory pathways related to fecundity of cock.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831

【引证文献】