基于全外显子测序的水牛不同时期卵巢转录组研究

Buffalo is a seasonal oestrus animal, and the symptom of oestrus is not obvious, and high percent of follicles atresia before maturation, so most buffalo have low fertility. The present project is designed to investigate the transcriptome of buffalo ovary tissues in different phases with whole exome sequencing technique. A total of eight phases are selected, including three nonage buffalo phases, as fetus ovary, calf ovary and puberty ovary. And five adult phases were selected, including dormant period, recruitment, selection, dominant and red luteum phase ovaries. With bovine genome assembly and refgene as reference sequence, all reads will be mapped to the reference sequences. Novel transcript unit (TU) and alternative splicing (AS) will be analysised in all buffalo ovary expressed genes. A few of functional genes will be confirmed with QRT-PCR, in-situ hybridization and immuno-histochemistry. To further clarify the function of selected genes, the in vitro expressed protein or shRNA of those genes will be added to the culture medium of preantral follicle. Based on the above results the in vito maturation of buffalo oocyte will be optimized. The investigation will help us learn more about the molecular regulation mechanism of folliculogenesis in buffalo, and provide a few of functional genes in improving the reproduction ability of buffalo in the future by genetic modified techniques.

水牛发情季节性强，发情表征不明显，卵泡闭锁率高，繁殖力普遍较低。本项目拟应用全外显子测序技术，对不同时期水牛卵巢转录组进行系统解析。选择未成年水牛卵巢3个时期（胎水牛、犊牛和初情期水牛卵巢），成年水牛卵巢5个时期（卵泡发育静止期、募集期、选择期、优势化期和红体期卵巢），构建8个测序文库进行分别测序分析。以牛的基因组为参考序列进行mapping拼装和注释，分析测序数据中新转录活性区和选择性剪切的类型，挖掘一批新的转录本。分析不同时期差异表达的基因，获知水牛不同时期卵巢差异表达的重要功能基因。筛选注释的重要功能基因进行QRT-PCR、原位杂交和免疫组化验证，并通过添加相应基因的表达蛋白或shRNA，验证其对水牛腔前卵泡和卵母细胞成熟培养的影响，优化水牛体外胚胎生产系统，充分挖掘母水牛生殖潜能。尝试阐明水牛卵泡发生和发育调控的分子机理，为提高水牛繁殖力的遗传改良研究提供丰富的基因素材。

本研究共选择未成年水牛卵巢3个时期，包括未出生胎水牛卵巢、犊牛卵巢和初情期牛卵巢。选择成年水牛卵巢5个时期，包括卵泡发育静止期、募集期、选择期、优势化期和红体期。分别构建了8个RNA-seq文库S18、S5、S1、S7、S2、S16、S6和S11，共产生高质量的reads 140898136条。所有的reads共mapped 到牛的基因为22034个，占牛已知基因的79.65%。共发现新转录本16039个，其中5588个可以在NR库中注释，191个在interproscan中可注释到结构域，另有1394个与已知的非编码基因注释上，其余8866个转录本均无法注释，推测为可能为新基因。比较每个基因在不同水牛卵巢的差异表达情况，其中未出生胎牛卵巢与静止期犊牛卵巢差异表达的基因最多为9337个，其中上调的基因较少1860个，下调的基因特别多高达7912个，相邻文库基因表达差异平均总数为7317个，说明水牛卵泡发育期间基因表达的波动较大。未成年水牛三个文库中共同表达的基因为14206个，S18、S5和S1文库特异性表达的基因分别为916、254和265个。成年水牛四个文库包括成年静止、募集、选择和优势化中共同表达的基因为14394个，S7、S2、S16和S6四个文库特异性表达的基因分别为201、281、438和206个，显示在每个时期特异性表达的基因并不多。本课题成功克隆了水牛卵泡发生相关基因（包含全长CDS）共29个，包括：BMP1, BMP15, GDF9, ALK3, ALK5, ALK6, FOXO3, Follistatin, Rad51, Rad52, Cx37, Wnt4, Pten, Ngf, gf7, Fgf2, Fgf2r, C-kit, Kit, Gdnf, Bmp6, Bmp7, Nobox, Ctgf, Amh, Lif, Foxl2, Pdgfa 和Sohlh2全部递交NCBI数据库。研究发现水牛BMP1基因通过调控水牛颗粒细胞的增殖，抑制颗粒细胞的凋亡，并通过参与调控细胞凋亡信号通路相关因子的表达，以及调控与卵泡选择和优势化相关基因的表达而主要参与了水牛卵泡的选择和优势化的过程。通过对水牛卵巢的转录组研究，水牛卵巢表达相关功能基因的克隆与表达规律验证等，初步探明了水牛卵泡发生过程中基因表达的变化规律。