卵泡期梅山猪中等卵泡选择时段延长相关lncRNA的鉴定及其调控作用研究

Ovulation rate is the first restrictive factor of litter size. The difference in ovulation rate between breeds with high and low ovulation rate originate from the differences during mid-late follicular phase in dynamics of follicular growth. In sows with high ovulation rate, the follicle selection phase is prolonged and make more preovulatory follicles emerge. The dynamic process involving selection and maturation of follicles is regulated and controled by a highly synchronized and exquisitely timed cascade of gene expression. Since lncRNAs have important and extentive function of regulation and control, and play a crucial role in mammalian reproduction. The difference in ovulate rate betwwen Meishan and Duroc sows is influenced by the differential lncRNAs expression in their follicles to some extend. In this study high-throughput sequencing will be constructed to detect tissue-specific lncRNAs and to screen differentially expressed lncRNAs in follicles M1 (with a diameter of 3.0mm-4.9mm), follicles M2 (with a diameter of diameter 5.0mm-6.9mm) between Meishan and Duroc sows; target genes and interaction network will be constructed, spatio-temporal expression will be characterized, and the SNP will also be analyzed. This study will be helpful to comprehensively understand the development of ovarian follicle, and will obtain a better and further understanding on the genetic basis and molecular mechanism of high prolificacy in pigs.

排卵数是猪窝产仔数的第一个决定因素。高排卵数与低排卵数品种母猪在卵泡期中后段表现出卵泡发育动力学差异，前者中等卵泡选择时段延长并产生更多排卵前卵泡。卵泡选择和成熟的动态变化受高度同步、精确的基因表达调控。鉴于lncRNA具有广泛而重要的表达调控功能和对动物生殖的重要作用，高产猪种在排卵数上的优势可能受卵泡中lncRNA表达差异的调控。本项目拟对卵泡期不同时段猪卵巢组织RNA池进行高通量测序，鉴定特异表达lncRNA；对卵泡期第4天的梅山、杜洛克猪中等M1卵泡（直径3.0-4.9mm）、M2卵泡（直径5.0-6.9mm）分别高通量测序，筛选品种间差异表达lncRNA；分析lncRNA-mRNA互作调控网络；对重要lncRNA在细胞中研究其对靶基因及表型的调控，并进行时空表达模式、SNP遗传效应分析。本项目有助于全面理解猪卵泡发育调控机制和深入解析影响猪排卵数和繁殖力的分子调控网络。

高排卵数与低排卵数品种母猪在卵泡期中后段表现出卵泡发育动力学的差异。卵泡选择和成熟的动态变化受高度同步、精确的基因表达调控。lncRNA对动物生殖有广泛而重要的表达调控作用，高产猪种在排卵数上的优势可能受卵泡中lncRNA表达的调控。.本研究对卵泡期第0天、2天、4天的杜洛克猪卵巢组织和第4天梅山猪卵巢组织RNA分别进行高通量测序，检测到在猪卵巢组织表达的lncRNA 2076个（新发现714个）和mRNA 25491个，鉴定出在不同阶段杜洛克猪卵巢间差异表达的lncRNA 140个和mRNA 1554个，鉴定出在卵泡期第4天梅山与杜洛克猪卵巢间差异表达的lncRNA 508个和mRNA 1779个。对卵泡期第4天梅山与杜洛克猪中等M1卵泡（直径3.0mm-4.9mm）和中等M2卵泡（直径5.0mm-6.9mm）RNA进行了高通量测序，检测到在猪中等卵泡中表达的lncRNA 共3554个（新发现1557个）。在梅山与杜洛克猪中等M1卵泡间得到差异表达的lncRNA 136个和mRNA 748个，在梅山与杜洛克猪中等M2卵泡间得到差异表达的lncRNA 161个和mRNA 877个。通过测序鉴定到在梅山和杜洛克M2卵泡间差异表达的miRNA 56个，数据挖掘获得了11个差异表达的circRNA。.对差异表达lncRNA的靶基因和mRNA、miRNA、circRNA进行网络调控分析，发现差异表达转录本在繁殖相关的Wnt、TGF-β、PI3k、FoxO、类固醇激素生物合成等信号通路中富集。.克隆获得了lncRNA-ENSSSCT00000018610 全长序列1731bp，构建了过表达载体和干扰载体，在PK-15细胞和猪卵巢颗粒细胞中发现其通过靶向调控位置相关靶基因TNIPI、GPX3和表达相关靶基因COL11A2、CYP2J2、DHRS4、SCARB1的表达来抑制猪卵巢颗粒细胞的增殖，进而影响猪卵泡发育。.在猪LTBP1、PGAM2、ENO3基因上发现了5个错义突变位点，其中3个多态位点对猪母猪繁殖性状有显著影响，可作为候选分子标记。.本研究系统分析了在卵泡期中后段猪卵巢和中等卵泡品种间以lncRNA为主的转录组差异，为深入理解猪卵泡发育调控机理打下基础，为继续研究猪繁殖性状相关lncRNA、影响猪排卵数相关性状的分子机理与调控网络提供了新的知识和视野。