奶牛泌乳早期乳脂肪酸变化及乳腺组织转录调控分析

发布时间：2018-05-01 15:33

本文选题：中国荷斯坦奶牛 + 脂肪酸　；参考：《扬州大学》2017年硕士论文

【摘要】：本课题以中国荷斯坦奶牛为试验对象,测定分析奶牛乳中主要成分和脂肪酸比例的变化规律,采用转录组测序技术和生物信息学方法,研究乳腺组织在泌乳初期(围产期的泌乳阶段)和泌乳盛期这两个关键时期,其分泌细胞从血液中获取脂肪酸(FA import into cells)、脂肪酸从头合成(Denovo synthesis)、外源固醇转运(Xenobiotic and Cholesterol transport)、脂肪酸激活及胞内运送(Acetate and FA activation and intra-cellular transport)、脂肪酸细胞内合成和去饱和(FAsynthesis and desaturation)至脂滴形成和分泌(Lipid droplet formation and secretion)等脂类代谢过程的转录及转录后调控机制,根据转录组测序结果,筛选泌乳早期差异表达的基因。为厘清奶牛乳腺脂类合成、代谢机制,实现乳成分的定向遗传调控,生产优质原料奶奠定理论基础。在扬州大学实验农牧场中分别挑选两组健康的中国荷斯坦奶牛,第一组30头(2-3胎次),用于牛奶中乳成分和脂肪酸的测定,第二组3头(2-3胎次),用于牛奶中乳成分和脂肪酸的测定和乳腺组织的采集。33头奶牛均采用TMR日粮饲养,整个研究期间日粮及饲养管理条件保持相对一致。测定3(初乳)、30、90 d奶牛乳中乳脂率、乳蛋白、乳糖、非脂乳固体和脂肪酸的百分比含量。微创法采集-7(产前)、30、90 d第二组奶牛乳腺组织,利用RNA-seq技术对试验牛乳腺组织进行深度转录组测序。主要结果如下:(1)本实验利用气相色谱法在牛奶中共检测出35种脂肪酸(共41类),对含量高于1%的 18 类显著差异的脂肪酸 C4:0、C6:0、C8:0、C10:0、C12:0、C14:0、C15:0、C16:0、C16:1,cis-9、C18:0、C18:1,cis-9、C18:2,cis-9,12、SFA、MUFA、PUFA、sc-FA、mc-FA和lc-FA着重研究,结果显示:除去C16:1,cis-9的17类脂肪酸的含量在泌乳3 d和30 d存在显著差异(P0.05);除去C4:0、C6:0和sc-FA的15类脂肪酸的含量在泌乳30 d和90 d差异不显著(P0.05);除去C4:0和sc-FA的16类脂肪酸的含量在泌乳3 d和90 d差异显著(P0.05)。(2)对不同时期乳腺组织进行转录组(RNA-seq)测序,新转录本预测分析结果:在不同时期乳腺组织中共预测得到8706个新转录本,其中预测具有编码能力的有3253个。已知基因结构优化结果:在不同时期乳腺组织中共优化了 1664个已知基因结构,大部分为基因起始位置到终止位置序列的延长。差异表达分析结果如下:-7vs30乳腺组织中有2421个差异基因;30vs90乳腺组织中有33个差异基因。(3)通过对差异表达基因进行KEGG富集分析,共筛选到13条与脂肪酸代谢相关通路,包括PPAR信号通路(PPAR signaling pathway)、甘油脂类新陈代谢信号通路(Glycerolipid metabolism signaling pathway)、脂肪酸代谢信号通路(Fatty acid metabolism signaling pathway)和不饱和脂肪酸的生物合成信号通路(Biosynthesis of unsaturated fatty acids signaling pathway)等。(4)对通路中与脂肪酸合成代谢相关的30个基因进行分析,结果显示:CD36、ABCA1、SLC27A6、ACSL1、ACSS1、ACSS2、FASN、AGPAT6、DGAT2、GPAM、GPAT2、LPIN1和SOCS3基因在泌乳启动期间表达量存在显著差异(P0.05);而SOCS3、FABP4、GPAT2、DGAT1和DGAT2基因在泌乳终止期间存在显著差异(P0.05)。把这些基因与奶牛乳中脂肪酸成分之间进行相关性分析,结果显示:除去FADS2、GPAT2、SREBF1和SREBF2外,其他基因均与所测脂肪酸中的一类或几类存在显著或极显著相关。本研究发现,CD36、ABCA1、SLC27A6、ACSL1、ACSS1、ACSS2、FASN、AGPAT6、DGAT2、GPAM、GPAT2、LPIN1和SOCS3基因在泌乳的启动和维持过程中起着重要的作用。PPAR信号通路、甘油脂类新陈代谢信号通路、脂肪酸代谢信号通路和不饱和脂肪酸的生物合成信号通路等多条与脂肪酸代谢相关通路中富集的基因,可作为深入研究乳腺泌乳机制的候选基因群。
[Abstract]:In this study, Chinese Holstein cows were used to test and analyze the changes in the proportion of main ingredients and fatty acids in milk of dairy cows. The two key periods of mammary tissue in the early lactation (perinatal lactation stage) and lactation period were studied by the sequence of transcriptional sequencing and bioinformatics, and the secretory cells were obtained from the blood. Fatty acids (FA import into cells), fatty acids from ab initio synthesis (Denovo synthesis), exogenous steroid transport (Xenobiotic and Cholesterol transport), fatty acid activation and intracellular delivery (Acetate and), fatty acids in fine cell synthesis and desaturation to lipid droplets The transcriptional and post transcriptional regulation mechanism of lipid metabolism processes such as Lipid droplet formation and secretion and other lipid metabolism processes are selected to screen the genes of differential expression in early lactation, to clarify the lipid synthesis and metabolic mechanism of dairy cows, to realize the steady genetic regulation of milk ingredients and to lay a theoretical foundation for the production of high quality raw milk. In the experimental farm of Yangzhou University, two groups of healthy Chinese Holstein cows were selected. The first group of 30 heads (2-3 parity) was used for the determination of milk ingredients and fatty acids in milk, second groups 3 heads (2-3 parity). The milk ingredients and fatty acids in milk and the mammary gland tissues were collected by TMR diet, and the whole study was carried out. The dietary and feeding management conditions were consistent during the period. The percentage of milk fat, milk protein, lactose, non fat milk solid and fatty acid in milk of 30,90 D dairy cows was measured in 3 (colostrum), milk protein, lactose, non fat milk solid and fatty acid. -7 (prenatal), 30,90 d second group of dairy cow mammary tissues were collected by minimally invasive method, and the deep transcriptional group of bovine breast tissue was sequenced by RNA-seq technology. The results are as follows: (1) in this experiment, 35 fatty acids (41 kinds of fatty acids) were detected by gas chromatography in the milk, and the fatty acids C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C15:0, C16:0, C16:1, cis-9, and C18:0 were studied. There was a significant difference in the content of 17 kinds of fatty acids in C16:1 and cis-9 in lactation 3 D and 30 d (P0.05); except C4:0, the 15 fatty acids of C6:0 and sc-FA were not significant in lactating 30 d and 90 d (P0.05), and the content of 16 types of fatty acids removed from C4:0 and 16 was significant in lactating 3 and 90. RNA-seq sequencing and new transcriptional prediction analysis: 8706 new transcripts were predicted in different stages of breast tissue, of which 3253 were predicted to have the ability to encode. The optimization results of the known gene structure: 1664 known gene structures were optimized in different mammary tissues at different stages, most of which were from the beginning of the gene to the end. Extension of position sequence. The results of differential expression analysis are as follows: there are 2421 differentially differentially genes in -7vs30 breast tissue; there are 33 differential genes in 30vs90 breast tissue. (3) through KEGG enrichment analysis of differentially expressed genes, 13 pathways related to fatty acid metabolism, including PPAR signaling pathway (PPAR signaling pathway), and triglycerides, were screened. Glycerolipid metabolism signaling pathway, fatty acid metabolism signaling pathway (Fatty acid metabolism signaling pathway) and biosynthetic signaling pathway of unsaturated fatty acids (Biosynthesis of unsaturated). (4) 3 of the pathways associated with fatty acid synthesis and metabolism in the pathway 0 genes were analyzed, and the results showed that CD36, ABCA1, SLC27A6, ACSL1, ACSS1, ACSS2, FASN, AGPAT6, DGAT2, GPAM, GPAT2, there were significant differences in the expression of LPIN1 and genes during lactation initiation. The correlation analysis between fatty acids showed that the other genes were significantly or highly correlated with a class or a few classes in the fatty acids that were removed from FADS2, GPAT2, SREBF1 and SREBF2. This study found that CD36, ABCA1, SLC27A6, ACSL1, ACSS1, ACSS2, FASN, AGPAT6, AGPAT6, The maintenance process plays an important role in the.PPAR signaling pathway, the metabolic signaling pathway of triglycerides, the fatty acid metabolism signaling pathway and the biosynthetic pathway of unsaturated fatty acids, which are enriched in the fatty acid metabolism pathway, which can be used as a candidate gene group for the deep study of mammary gland lactation mechanism.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823;TS252.2

【参考文献】