基于单细胞转录组测序解析FecB突变影响小尾寒羊多羔的分子机制

The litter size is an important trait of sheep breeding. Fecundity Booroola gene (FecB) is the first identified major gene responsible for high ovulation rate and litter size in sheep. However, the exact regulatory mechanism of FecB influencing the ovulation rate in oocyte and granulosa cells is unclear. Therefore, around this question “how does the FecB gene regulate the ovulation rate in oocyte and granulosa cells respectively”, Small Tail Han sheep (STH，Chinese indigenous breed) with three FecB genotypes (++, +B, BB) were used in this project. After the estrous synchronization, preovulatory follicles were derived from STH sheep with different FecB genotypes independently (wild type (++), heterozygous carriers (+B) and homozygous carriers (BB)) by taking minimally invasive surgery, then oocyte and granulosa cells were separated from the same follicle to be performed single-cell RNA-sequencing. The important pathways involved in the FecB mutation will be screened. The differentially expressed genes and transcript isoforms will be analyzed to screen the key genes involved in the FecB pathway. Functional analysis of key genes will be conducted at molecular, cellular and individual levels, and important regulatory pathways will be validated both in vivo and in vitro by overexpression and knock-out strategy. Based on transcriptome data, the heterogeneity and cross talk between oocyte and granulosa cells will be revealed, and the allele-specific expression profile in +B genotype will be elucidated. These results will reveal the molecular mechanism of FecB affecting prolificacy in STH sheep, and offer a theoretical basis for constructing the efficient breeding system in sheep by the introgression of FecB mutation.

产羔数是绵羊育种的重要目标性状。FecB是鉴定出的影响绵羊排卵数和产羔数的第一个主效基因，然而目前FecB基因在卵母细胞和颗粒细胞中影响排卵数的精确调控机制不明晰。因此，围绕FecB基因在卵母细胞和颗粒细胞中如何协同调控绵羊排卵数这一科学问题，本项目以携带FecB三种基因型（++、+B、BB）的地方品种小尾寒羊为试验对象，活体收集并分离同一个卵泡中卵母细胞和颗粒细胞开展单细胞转录组测序，分别筛选卵母细胞和颗粒细胞中受FecB基因突变影响的重要通路，对重要通路中的关键基因从分子、细胞和个体水平进行功能研究，在体外采用过表达和敲除验证相关调控通路。同时，基于转录组数据分析揭示卵母细胞和颗粒细胞的异质性和互作关系，阐释+B型杂合子在排卵数调控中的基因表达偏好性。本项目预期阐明FecB基因影响小尾寒羊排卵数的分子机制，为进一步利用FecB基因构建肉羊高效繁育体系提供理论依据。

FecB是影响绵羊排卵数和产羔数的主效基因，FecB在绵羊卵母细胞和颗粒细胞中互作调控排卵数的机制尚不明晰。因此，本项目以FecB三种基因型（++、+B、BB）小尾寒羊母羊为试验对象，收集同一个卵泡中卵母细胞和颗粒细胞进行单细胞转录组测序，结合发情排卵表型和卵泡液微环境状态，联合下丘脑、垂体、子宫等重要繁殖组织转录组和蛋白组数据构建排卵产羔的内分泌和基因互作通路，并对重要通路中关键基因进行SNPs筛选及表达水平研究。主要结果如下：.FecB突变极显著提高了小尾寒羊的排卵数和产羔数。排卵数适中、产羔数较高和情期更短的FecB杂合型小尾寒羊更值得重视和推广。与卵巢静脉血清相比，FecB突变代谢效应主要体现在卵泡液中，BB型母羊卵泡液具有更高的抗氧化应激能力。.FecB三种基因型卵母细胞和卵丘颗粒细胞的单细胞转录组测序结果表明，FecB突变后，卵泡发育过程中卵丘颗粒细胞中LHCGR基因表达时间提前，其所在TGF-β信号通路下游SMAD4基因表达水平发生改变，卵母细胞中筛选到的差异表达基因富集在减数分裂通路中。FecB突变加速了卵丘颗粒细胞的增殖分化，促进了卵母细胞成熟。.卵泡期和黄体期BB和++型小尾寒羊下丘脑、垂体全转录组测序结果显示，下丘脑中，LINC-676和WNT3-AS顺式作用于DRD2和WNT9B，LINC-219386和IGF2-AS调控DMXL2、IGF2诱导GnRH分泌。垂体中，GRID2、ST14基因参与激素合成，FecB突变后，黄体期LOC105613905、MSTRG.81536和MSTRG.150434分别反式调控TGF-β信号通路TGFβ1、SMAD3、OXT基因表达变化，促进FecB突变母羊产生更多成熟卵泡。.卵泡期和黄体期无FecB突变的不同产羔数小尾寒羊下丘脑、卵巢和子宫全转录组和蛋白组整合分析表明，下丘脑中，GH、IGF1R、HSD11B1和MSTRG.95128调控的FOXG1基因直接或间接调节GnRH分泌促进排卵；卵巢中，STAR、COX7A1和CD81等基因参与卵巢类固醇激素合成和卵泡发育；子宫中，SDS、SDSL和MGST3等基因通过调节氨基酸、能量代谢为胚胎早期发育、胚胎着床提供所需能量。.对高通量组学筛选到排卵产羔相关的11个基因的12个位点进行关联分析，检测7个基因表达，结果表明这些基因及突变与小尾寒羊产羔数显著相关。