山羊卵泡發(fā)育相關(guān)基因的篩選及分析

趙園園，李鵬飛，許勤智，安清明，孟金柱

山羊卵泡發(fā)育相關(guān)基因的篩選及分析

趙園園1，李鵬飛2，許勤智1，安清明1，孟金柱1

（1銅仁學(xué)院，貴州銅仁 554300；2山西農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院，山西太谷 030801）

山羊第一卵泡波中的優(yōu)勢(shì)卵泡（dominant follicles, DF）和從屬卵泡（subordinate follicles, SF）是整個(gè)卵泡發(fā)育過(guò)程中最為關(guān)鍵的兩個(gè)階段。隨著卵泡的進(jìn)一步發(fā)育，最終DF可能發(fā)育成為成熟卵泡，直到排卵；SF將走向閉鎖，其中顆粒細(xì)胞的凋亡是導(dǎo)致卵泡發(fā)生閉鎖的關(guān)鍵因素。然而目前對(duì)促進(jìn)卵泡的優(yōu)勢(shì)化或?qū)е缕溟]鎖的分子機(jī)理尚不清楚。【】通過(guò)對(duì)山羊第一卵泡波中DF和SF顆粒細(xì)胞進(jìn)行高通量測(cè)序，旨在篩選影響卵泡發(fā)育的關(guān)鍵基因，為深入探究卵泡發(fā)育的調(diào)控機(jī)制提供理論依據(jù)。選取10只1歲齡健康的貴州白山羊分別注射前列腺素F2α，使其同期發(fā)情，此后每天用B超檢測(cè)并記錄卵泡的生長(zhǎng)情況，發(fā)情3 d后，統(tǒng)一屠宰并采集第一卵泡波中DF (直徑4.5—6 mm)與SF (直徑3 —4.5 mm)，分別分離其中的顆粒細(xì)胞，提取總RNA、構(gòu)建文庫(kù)后通過(guò)Illumina Hiseq 2500平臺(tái)進(jìn)行測(cè)序。利用FastQC對(duì)測(cè)序產(chǎn)出raw reads進(jìn)行質(zhì)量評(píng)估并經(jīng)過(guò)過(guò)濾后，獲得品質(zhì)較高的clean reads；使用Trinity對(duì)得到的clean reads進(jìn)行重新組裝，從而獲得unigenes；使用CLC Genomics Workbench將unigenes與山羊RefSeq數(shù)據(jù)庫(kù)進(jìn)行比對(duì)獲得mRNA；使用DESeq2 軟件對(duì)獲得的mRNA進(jìn)行差異表達(dá)分析；分別采用goseq和kobas軟件對(duì)得到的差異表達(dá)基因進(jìn)行GO分析及KEGG信號(hào)通路分析；最終通過(guò)qRT-PCR對(duì)篩選出的可能影響卵泡發(fā)育的關(guān)鍵基因進(jìn)行驗(yàn)證。分別對(duì)測(cè)序得到的raw reads進(jìn)行過(guò)濾后，在DF顆粒細(xì)胞中獲得43 217 934條clean reads，占raw reads的比例為95.19%；SF顆粒細(xì)胞中獲得40 766 348條clean reads，占raw reads的比例為95.35%。將得到的unigenes與山羊的RefSeq 數(shù)據(jù)庫(kù)進(jìn)行比對(duì)后，共得到33 896條帶有注釋的轉(zhuǎn)錄本，再通過(guò)設(shè)定FPKM＞1, q value＜0.05，共在兩種卵泡顆粒細(xì)胞中獲得13 644個(gè)基因。設(shè)定參數(shù)：FPKM≥1，SF-FPKM/DF-FPKM＞1，＜0.05，獲得695個(gè)差異表達(dá)mRNA，其中233個(gè)在SF顆粒細(xì)胞中表達(dá)顯著上調(diào)，462個(gè)表達(dá)顯著下調(diào)；對(duì)所獲得695個(gè)差異表達(dá)mRNA進(jìn)行GO功能富集分析，共分為三大類(lèi)42組：其中生物學(xué)過(guò)程占47.6%，細(xì)胞組分占47.6%，分子功能占4.8%；KEGG信號(hào)通路分析，發(fā)現(xiàn)20條通路，其中與核糖體通路相關(guān)的基因富集最為顯著。通過(guò)在Genecard中進(jìn)行功能分析后，篩選6個(gè)可能與山羊卵泡發(fā)育密切相關(guān)的基因，其中、、在SF顆粒細(xì)胞中表現(xiàn)為上調(diào)；、、則表現(xiàn)為下調(diào)。qRT-PCR顯示、、、、的表達(dá)趨勢(shì)與高通量測(cè)序結(jié)果一致，且在從屬卵泡顆粒細(xì)胞中的表達(dá)量極顯著地高于優(yōu)勢(shì)卵泡（＜0.01）；、、在優(yōu)勢(shì)卵泡顆粒細(xì)胞中的表達(dá)量極顯著地高于從屬卵泡（＜0.01）。、、和在優(yōu)勢(shì)卵泡和從屬卵泡中表達(dá)量存在極顯著差異，推測(cè)在山羊卵泡發(fā)育過(guò)程中可能促進(jìn)卵泡的優(yōu)勢(shì)化或?qū)е麻]鎖，對(duì)深入探究卵泡發(fā)育的調(diào)控機(jī)制具有重要意義。

山羊；高通量測(cè)序；卵泡發(fā)育；顆粒細(xì)胞

0 引言

【研究意義】哺乳動(dòng)物卵泡發(fā)育是一個(gè)受多種因素調(diào)控的復(fù)雜生物學(xué)過(guò)程，在這個(gè)過(guò)程中有眾多激素或者調(diào)控因子直接或間接參與[1]。通過(guò)基因敲除，在小鼠的卵巢中已經(jīng)發(fā)現(xiàn)有 1 000 多個(gè)基因共同調(diào)控著卵泡的發(fā)育[2]。然而目前對(duì)促進(jìn)山羊卵泡的優(yōu)勢(shì)化或?qū)е缕溟]鎖的分子機(jī)理尚不清楚。通過(guò)對(duì)山羊第一卵泡波中DF和SF顆粒細(xì)胞進(jìn)行高通量測(cè)序，篩選出影響卵泡發(fā)育的關(guān)鍵基因，為深入探究卵泡發(fā)育的調(diào)控機(jī)制具有重要意義?！厩叭搜芯窟M(jìn)展】在哺乳動(dòng)物的一個(gè)發(fā)情周期中，一簇卵泡經(jīng)過(guò)募集、選擇及優(yōu)勢(shì)化過(guò)程發(fā)育成為排卵卵泡[3]。在卵泡的優(yōu)勢(shì)化過(guò)程中，細(xì)胞色素P450側(cè)鏈裂解酶（Pytochrome P450 side chain lyase, P450scc）和細(xì)胞色素P450芳構(gòu)化酶（cytochrome P450 aromatase, P450arom）mRNA能夠促進(jìn)卵泡的發(fā)育，推測(cè)P450arom和P450scc可能促進(jìn)卵泡顆粒細(xì)胞分泌雌二醇（E2），從而參與負(fù)反饋調(diào)節(jié)作用并促進(jìn)卵泡的優(yōu)勢(shì)化[4]。胰島素樣生長(zhǎng)因子（insulin-like growth factor，IGF）會(huì)顯著促進(jìn)顆粒細(xì)胞分泌E2，并加快卵泡選擇與優(yōu)勢(shì)化過(guò)程[5]，然而成纖維細(xì)胞生長(zhǎng)因子（fibroblast growth factor, FGF）與IGF的作用卻相反，它通過(guò)抑制卵泡顆粒細(xì)胞分泌E2和抑制卵泡LH受體的表達(dá)從而抑制卵泡的選擇與優(yōu)勢(shì)化過(guò)程[6]。卵泡一旦確定了優(yōu)勢(shì)化地位后，優(yōu)勢(shì)化卵泡會(huì)通過(guò)分泌E2和INH等調(diào)控因子維持自己的優(yōu)勢(shì)化地位[7]。LI等[8]通過(guò)對(duì)水牛不同大小卵泡的顆粒細(xì)胞進(jìn)行高通量測(cè)序，發(fā)現(xiàn)免疫系統(tǒng)可能在卵泡的成熟和排卵過(guò)程中起了重要作用。TERENINA等[9]通過(guò)對(duì)豬的正常卵泡與閉鎖卵泡顆粒細(xì)胞進(jìn)行轉(zhuǎn)錄組測(cè)序，篩選出了等11個(gè)基因可能在卵泡顆粒細(xì)胞的增殖過(guò)程中起了抑制作用，從而引起卵泡閉鎖。【本研究切入點(diǎn)】山羊第一卵泡波中的DF最終可能發(fā)育成為成熟卵泡，直到排卵；而SF則會(huì)受到各種調(diào)控因子而作用而走向閉鎖，其中顆粒細(xì)胞的凋亡是導(dǎo)致卵泡發(fā)生閉鎖的關(guān)鍵因素[10]。然而目前對(duì)促進(jìn)卵泡的優(yōu)勢(shì)化或?qū)е缕溟]鎖的分子機(jī)理尚不清楚?！緮M解決的關(guān)鍵問(wèn)題】通過(guò)對(duì)山羊第一卵泡波中DF和SF顆粒細(xì)胞進(jìn)行高通量測(cè)序，并通過(guò)qRT-PCR進(jìn)行驗(yàn)證分析，篩選出影響卵泡發(fā)育的關(guān)鍵基因，為深入探究其調(diào)控卵泡發(fā)育機(jī)制提供理論依據(jù)。

1 材料與方法

本試驗(yàn)于2018年12月至2019年4月在銅仁學(xué)院完成。

1.1 試驗(yàn)動(dòng)物及樣品采集

在貴州省銅仁市沿河土家族自治縣華珍牧業(yè)有限公司，選取10只1歲齡健康的貴州白山羊分別注射前列腺素F2α，使其同期發(fā)情，此后每天用B超檢測(cè)并記錄卵泡的生長(zhǎng)情況，發(fā)情3 d后，統(tǒng)一屠宰并采集第一卵泡波中DF（直徑4.5—6 mm）與SF（直徑3—4.5 mm），迅速置于4℃滅菌杜氏磷酸緩沖液（DPBS）中，迅速運(yùn)輸?shù)姐~仁學(xué)院動(dòng)物學(xué)實(shí)驗(yàn)室。將處于DPBS中的DF和SF分別放到盛有0.9%的生理鹽水的培養(yǎng)皿上，使用眼科剪刀剪開(kāi)并用細(xì)胞刮刀刮取位于卵泡內(nèi)膜上的顆粒細(xì)胞（GCs）后置于-80℃冰箱中保存。

1.2 試驗(yàn)方法

1.2.1 總RNA提取、文庫(kù)構(gòu)建及測(cè)序 將保存在-80℃冰箱中的GCs取出后置于冰盒中解凍，加入1 mL Trizol（購(gòu)自Invitrogen公司，美國(guó)）分別提取兩種卵泡中GCs的總RNA，經(jīng)RNeasy mini kit（購(gòu)自QIAGEN公司，德國(guó)）純化，Agilent Bioanalyzer 2100完整性檢測(cè)，Qubit 2.0 Flurometer測(cè)量濃度后，交由北京諾和致源生物信息科技有限公司進(jìn)行文庫(kù)構(gòu)建并通過(guò)Illumina Hiseq 2500平臺(tái)測(cè)序。

1.2.2 數(shù)據(jù)處理及分析 FastQC（http://www. bioinformatics.babraha m.ac.uk/projects/fastqc/）用于對(duì)測(cè)序產(chǎn)出原始數(shù)據(jù)進(jìn)行質(zhì)量評(píng)估，然后清除原始數(shù)據(jù)（raw reads）中帶接頭的、低質(zhì)量的reads，從而獲得品質(zhì)較高的有效讀段（clean reads）。使用Trinity（http：//trinityrnaseq.sourceforge.net/）對(duì)得到的clean reads進(jìn)行重新組裝，以便得到單一序列（singleton）及重疊群（contigs）此時(shí)得到的序列稱(chēng)為unigenes。使用CLC Genomics Workbench將unigenes與山羊RefSeq數(shù)據(jù)庫(kù)進(jìn)行比對(duì)。基于負(fù)二項(xiàng)分布的DESeq2軟件用于差異表達(dá)mRNA的分析，之后使用goseq軟件對(duì)得到的差異表達(dá)基因進(jìn)行GO分析，使用kobas軟件進(jìn)行KEGG信號(hào)通路分析。

1.2.3 反轉(zhuǎn)錄及引物合成 通過(guò)EasyScript? One-Step gDNA Removal and cDNA Synthesis SuperMix（購(gòu)自北京全式金生物技術(shù)有限公司）將提取到的總RNA反轉(zhuǎn)錄成cDNA，條件為：42℃孵育15 min，85℃加熱5 s失活TransScript RT與gDNA Remover。Primer 5.0設(shè)計(jì)引物（表1），使用作為內(nèi)參基因，引物合成委托生工生物工程（上海）股份有限公司完成。

1.2.4 qRT-PCR分析 qRT-PCR用于驗(yàn)證貴州白山羊DF與SF GCs中差異表達(dá)mRNA的相對(duì)表達(dá)水平。采用3個(gè)樣本重復(fù)，3個(gè)技術(shù)重復(fù)，通過(guò)TransStart? Tip Green qPCR SuperMix（購(gòu)自北京全式金生物技術(shù)有限公司）對(duì)各基因進(jìn)行相對(duì)定量分析，根據(jù)產(chǎn)品使用說(shuō)明書(shū)構(gòu)建20 μL PCR反應(yīng)體系：2×Transstart?Tip Green qPCR super mix 10 μL，上下游引物各0.4 μL，cDNA 4 μL（100 ng），RNA-free H2O 5.2 μL。反應(yīng)程序?yàn)椋?4℃預(yù)變性1 min；94℃ 10 s，60℃ 30 s，72℃ 10 s，45個(gè)循環(huán)。結(jié)果使用2-△△CT法來(lái)計(jì)算各基因的相對(duì)表達(dá)情況。

表1 熒光定量引物基因列表

2 結(jié)果

2.1 RNA-seq數(shù)據(jù)分析

高通量測(cè)序得到的raw reads，經(jīng)過(guò)濾帶接頭的、含N的及低質(zhì)量的reads，最終在DF中獲得43 217 934條clean reads，占95.19%；在SF中獲得40 766 348條clean reads，占95.35%（圖1）。將得到的clean reads比對(duì)到山羊RefSeq數(shù)據(jù)庫(kù)中，共得到33 896條帶有注釋的轉(zhuǎn)錄本。設(shè)定FPKM ＞1, q value＜0.05，在兩種卵泡顆粒細(xì)胞中共獲得13 644個(gè)基因，其中438個(gè)基因高表達(dá)（FPKM≥1），表2中列出了表達(dá)量排名前20的基因。

圖1 DF和SF顆粒細(xì)胞中原始數(shù)據(jù)的分類(lèi)

表2 SF和DF顆粒細(xì)胞中表達(dá)量最高的20個(gè)基因

2.2 差異表達(dá)基因篩選

在差異轉(zhuǎn)錄本分析過(guò)程中，將DF和SF的FPKM （Fragmentsperkilobaseoftranscriptpermillionfragmentsmapped）進(jìn)行標(biāo)準(zhǔn)化，使用DESeq2軟件對(duì)獲得13 644個(gè)mRNA進(jìn)行差異表達(dá)分析，設(shè)定參數(shù)：FPKM≥1，SF-FPKM/DF-FPKM＞1，＜0.05，共獲得695個(gè)差異表達(dá)mRNA，其中233個(gè)在從屬卵泡顆粒細(xì)胞中表達(dá)顯著上調(diào)，462個(gè)則表達(dá)下調(diào)（圖2）。

2.3 GO功能富集

通過(guò)goseq軟件對(duì)得到的695個(gè)差異表達(dá)基因進(jìn)行GO功能富集分析, 共分為三大類(lèi)42組：其中生物學(xué)過(guò)程占47.6%，細(xì)胞組分占47.6%，分子功能占4.8% （圖3）。

圖2 DF和SF顆粒細(xì)胞中差異表達(dá)基因火山圖

1：RNA加工；2：RNA拼接；3：核酸代謝過(guò)程；4：含堿基化合物的代謝過(guò)程；5：核糖核蛋白復(fù)雜生物合成；6：細(xì)胞大分子代謝過(guò)程；7：核糖體生物合成；8：雜環(huán)代謝過(guò)程；9：細(xì)胞芳香族化合物的代謝過(guò)程；10：細(xì)胞氮化合物代謝過(guò)程；11：基因表達(dá)；12：RNA代謝過(guò)程；13：核糖體小亞基生物合成；14：核糖體RNA加工；15：大分子代謝過(guò)程；16：氮化物代謝過(guò)程；17：ncRNA加工；18：ncRNA代謝過(guò)程；19：有機(jī)循環(huán)化合物代謝過(guò)程；20：核糖體RNA代謝過(guò)程；21：下?lián)芎藘?nèi)腔；22：細(xì)胞核部分；23：細(xì)胞核；24：細(xì)胞內(nèi)的細(xì)胞器；25：細(xì)胞器；26：細(xì)胞膜內(nèi)腔；27：細(xì)胞內(nèi)細(xì)胞器內(nèi)腔；28：細(xì)胞器內(nèi)腔；29：細(xì)胞內(nèi)細(xì)胞器的部分；30：細(xì)胞內(nèi)的；31：細(xì)胞內(nèi)的部分；32：細(xì)胞器的部分；33：細(xì)胞內(nèi)膜上細(xì)胞器；34：膜上細(xì)胞器；35：核質(zhì)；36：核糖核蛋白復(fù)合體；37：胞質(zhì)核糖體小亞基；38：胞質(zhì)核糖體；39：核質(zhì)部分；40：大分子復(fù)合體；41：RNA結(jié)合；42：mRNA結(jié)合

2.4 KEGG信號(hào)通路分析

通過(guò)kobas軟件對(duì)差異表達(dá)基因進(jìn)行KEGG信號(hào)通路分析，共發(fā)現(xiàn)20條通路（圖4），其中與核糖體通路相關(guān)的基因最為顯著富集，達(dá)到50個(gè)；另外，還發(fā)現(xiàn)5個(gè)基因參與了卵母細(xì)胞的減數(shù)分裂。

2.5 qRT-PCR驗(yàn)證分析

通過(guò)功能分析，我們篩選出6個(gè)可能與山羊卵泡發(fā)育密切相關(guān)的基因（表3）。其中、、在SF顆粒細(xì)胞中表現(xiàn)為上調(diào)；、、則表現(xiàn)為下調(diào)。QRT-PCR結(jié)果顯示、、、、的表達(dá)趨勢(shì)與高通量測(cè)序結(jié)果一致，且在SF顆粒細(xì)胞中的表達(dá)量極顯著地高于DF（＜0.01）；、、在DF顆粒細(xì)胞中的表達(dá)量極顯著地高于SF（＜0.01），的熒光定量結(jié)果雖然與高通量測(cè)序結(jié)果的表達(dá)趨勢(shì)相反，但不存在顯著差異（＞0.05）（圖5）。

圖4 DF和SF顆粒細(xì)胞中差異表達(dá)基因KEGG通路富集散點(diǎn)圖

圖5 候選基因在山羊DF和SF顆粒細(xì)胞中的相對(duì)表達(dá)量（**表示P＜0.01）

表3 可能與山羊卵泡發(fā)育相關(guān)的候選基因

3 討論

雌性哺乳動(dòng)物卵巢上的DF持續(xù)生長(zhǎng)、成熟并排出卵子是依賴(lài)于顆粒細(xì)胞中FSH通過(guò)激活cAMP-蛋白激酶A通路，進(jìn)而誘導(dǎo)芳香化酶將膜細(xì)胞分泌的雄激素轉(zhuǎn)化為E2，而SF由于生長(zhǎng)速率和E2分泌降低，從而走向閉鎖[11]。HUSSEIN等[10]研究發(fā)現(xiàn)，顆粒細(xì)胞的凋亡是引起卵泡閉鎖的關(guān)鍵因素；顆粒細(xì)胞受到凋亡蛋白酶激活壞死因子（TNF）、Fas等刺激后，使促凋亡蛋白的構(gòu)象發(fā)生變化，從而由細(xì)胞液轉(zhuǎn)移到線粒體外膜上，并與膜上及膜內(nèi)的抗凋亡蛋白相互作用，最終導(dǎo)致其凋亡[12-13]。LI等[14]通過(guò)對(duì)牛發(fā)生偏差前的最大卵泡（PDF1）和發(fā)生偏差后的最大卵泡（ODF1）顆粒細(xì)胞進(jìn)行轉(zhuǎn)錄組測(cè)序，獲得83個(gè)差異表達(dá)基因，其中、和在卵泡發(fā)育過(guò)程中可能起抑制作用。本研究通過(guò)對(duì)貴州白山羊DF和SF顆粒細(xì)胞進(jìn)行高通量測(cè)序篩選出695個(gè)差異表達(dá)基因，其中233個(gè)在SF顆粒細(xì)胞中表達(dá)顯著上調(diào)，462個(gè)表達(dá)下調(diào)。結(jié)合功能分析，篩選出具有代表性的6個(gè)可能與山羊卵泡發(fā)育密切相關(guān)的基因，qRT-PCR驗(yàn)證結(jié)果表明，、、、和在DF和SF顆粒細(xì)胞中的表達(dá)趨勢(shì)與高通量測(cè)序結(jié)果完全一致，在DF和SF顆粒細(xì)胞中的表達(dá)趨勢(shì)與高通量測(cè)序結(jié)果雖相反，但差異不顯著（＞0.05）。

在哺乳動(dòng)物中，促乳素受體（PRLR）對(duì)維持黃體以及孕酮的分泌起著重要的作用[15]。PRL可能通過(guò)對(duì)子宮內(nèi)膜的直接作用在妊娠中發(fā)揮作用[16]。PRL能夠促進(jìn)排卵、著床及胎盤(pán)發(fā)育的過(guò)程[17], 敲除小鼠卵泡中的，使得卵母細(xì)胞釋放延遲，成熟受損進(jìn)而導(dǎo)致排卵減少[18]。顆粒細(xì)胞中正五聚蛋白3 (PTX3) 基因的表達(dá)也與卵母細(xì)胞及胚胎的發(fā)育能力有關(guān)，是預(yù)測(cè)胚胎發(fā)育能力的可靠指標(biāo)[19-20]。在小鼠排卵前卵泡腔內(nèi)，mRNA表達(dá)主要位于卵丘細(xì)胞中，而在顆粒細(xì)胞中表達(dá)很少[21]，進(jìn)一步證實(shí)了筆者的數(shù)據(jù)準(zhǔn)確性。鈣調(diào)素（Regucalcin，RGN）在牛大卵泡（直徑＞10 mm）中的表達(dá)量是小卵泡（直徑＜5 mm）中的9.8倍，被認(rèn)為是參與了優(yōu)勢(shì)卵泡的形成及提高顆粒細(xì)胞的存活率[22]。這與本研究的結(jié)果產(chǎn)生分歧，可能是物種之間表達(dá)的差異所致。

Dickkopf WNT信號(hào)通路抑制劑3（dickkopf WNT signaling pathway inhibitor 3，DKK3）是一種旁分泌蛋白，它可以通過(guò)Wnt信號(hào)通路參與胚胎發(fā)育[23]。表觀遺傳沉默，會(huì)破壞正常Wnt/β-catenin 信號(hào)傳導(dǎo)和細(xì)胞凋亡調(diào)控[24]。醛脫氫酶1家族成員A2 （Aldehyde Dehydrogenase 1 Family Member A2，ALDH1A2）啟動(dòng)子中存在雌激素反應(yīng)元件位點(diǎn)[25]，在切除大鼠卵巢的子宮內(nèi)，E2可以促進(jìn)表達(dá)，但卻抑制表達(dá)[26]。是調(diào)節(jié)性腺中腎RA合成的主要酶，通過(guò)釋放全反維甲酸（RA）的酶來(lái)啟動(dòng)細(xì)胞的減數(shù)分裂。構(gòu)建和雙敲除小鼠與全反維甲酸反應(yīng)元件（RARE）報(bào)告小鼠雜交表明，在缺乏這兩種酶產(chǎn)生RA情況下，雌性小鼠可發(fā)生減數(shù)分裂，雄性小鼠不可發(fā)生減數(shù)分裂[27-30]。目前對(duì)全反維甲酸受體應(yīng)答器1（retinoic acid receptor responder 1，RARRES1）的報(bào)道較少，作為RA釋放的下游基因，可能在細(xì)胞的減數(shù)分裂過(guò)程中起重要作用。

4 結(jié)論

本研究在貴州白山羊DF與SF顆粒細(xì)胞中共獲得695個(gè)差異表達(dá)mRNA，其中233個(gè)在SF顆粒細(xì)胞中表達(dá)顯著上調(diào)，462個(gè)則表達(dá)下調(diào)。通過(guò)功能分析，篩選出6個(gè)可能與山羊卵泡發(fā)育密切相關(guān)的基因，qRT-PCR結(jié)果發(fā)現(xiàn)，、、和在DF和SF顆粒細(xì)胞中表達(dá)量存在極顯著差異，推測(cè)在山羊卵泡發(fā)育過(guò)程中可能促進(jìn)卵泡的優(yōu)勢(shì)化或?qū)е麻]鎖，對(duì)深入探究調(diào)控卵泡發(fā)育機(jī)制具有重要意義。

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Screening and Analysis of Follicular Development Related Genes in Goat

ZHAO YuanYuan1, LI PengFei2, XU QinZhi1, AN QingMing1, MENG JinZhu1

(1Tongren University, Tongren 554300, Guizhou;2College of Life Science, Shanxi Agricultural University, Taigu 030801, Shanxi)

【】 Dominant follicles (DF) and subordinate follicles (SF) were the most important two stages in the follicle development in the first follicle wave of goat. With further development of follicles, DF may eventually develop into mature follicles until ovulation, and SF will move towards atresia, while apoptosis of granulosa cells is the key factor leading to follicular atresia. However, the molecular mechanisms of promoting follicle dominance or causing its atresia are still unclear. 【】 This study was aimed to screen the key genes affecting follicular development and provide a theoretical basis for further exploring the regulation mechanism of follicular development by high-throughput sequencing of DF and SF granulosa cells in the first follicular wave of goat.【】Ten healthy Guizhou white goats were selected (1 year old), and the prostaglandin F2αwere injected respectively for estrus synchronization. B-type ultrasonography was used to detect the follicle growth situation, and then all of the goats were slaughtered when estrus had appeared for three days. DF (4.5-6 mm in diameter) and SF (3-4.5 mm in diameter) were obtained, granulosa cells were separated in the first follicle wave, respectively. Total RNA were extracted, and libraries were constructed and sequenced by Illumina Hiseq 2500 platform. FastQC was used to evaluate the quality of raw reads sequenced and filter them to obtain clean reads with high quality. Trinity was used to assemble clean reads from scratch to obtain unigenes. mRNA was obtained by comparing unigenes with goat RefSeq database using CLC Genomics Workbench. DESeq2 software was used to analyze the differential expression of the obtained mRNA. The goseq and kobas software were used for GO analysis and KEGG signal pathway analysis. Finally, qRT-PCR was used to verify the selected key genes that might affect the follicle development. 【】After the raw reads which obtained by sequencing were filtered, 43 217 934 clean reads were obtained from DF granulosa cells, accounting for 95.19% of raw reads. 40 766 348 clean reads were obtained from SF granulosa cells, accounting for 95.35% of raw reads. When the unigenes were compared with the RefSeq database of goat, a total of 33 896 annotated transcripts were obtained. Setting FPKM＞1 and q value＜0.05, a total of 13 644 genes were obtained in two types of follicle granulosa cells. By setting parameters: FPKM≥1, SF-FPKM/DF-FPKM＞1,＜0.05, 695 differentially expressed mRNAs were obtained, of which 233 were significantly up-regulated and 462 were down-regulated in SF granulosa cells. GO functional enrichment analysis was performed on 695 differentially expressed mRNAs and concentrated in 42 groups of three major categories: biological processes accounted for 47.6%, cellular components 47.6%, and molecular functions 4.8%. KEGG signaling pathway analysis revealed 20 pathways, among which ribosome pathway related genes were most significantly enriched. Six genes that might be closely related to follicle development in goats were screened out, among which,andwere up-regulated in subordinate follicles granulosa cells.,andwere down-regulated. qRT-PCR showed that the expression trend of,,,andwas consistent with high-throughput sequencing results, and the expression level ofin SF granulosa cells was significantly higher than that of dominant follicles (＜0.01). The expression levels of,and＜0.01).【】,,andhad extremely significant differences in the expression levels of dominant follicles and subordinate follicles. It was speculated that those genes might promote the predominance of follicles or lead to atresia during the follicle development of goat, which was of great significance to further explore the regulation mechanism of follicular development.

goat; high-throughput sequencing; follicle development; granulosa cells

10.3864/j.issn.0578-1752.2020.17.016

2019-06-24；

2020-07-10

貴州省教育廳青年科技人才成長(zhǎng)項(xiàng)目（黔教合KY字[2018]348）、貴州省普通高等學(xué)?？萍及渭馊瞬胖С钟?jì)劃（黔教合KY字[2017]089）、銅仁學(xué)院博士啟動(dòng)基金項(xiàng)目（trxyDH1601）、銅仁學(xué)院生態(tài)畜牧創(chuàng)新團(tuán)隊(duì)項(xiàng)目（CXTD[2020-19]）、貴州省農(nóng)業(yè)科技示范園區(qū)項(xiàng)目（黔科合農(nóng)園字[2014]5007號(hào)）、沿河土家族自治縣科技合作計(jì)劃項(xiàng)目“良種山羊與沿河白山羊雜交組合試驗(yàn)與示范推廣”

趙園園，E-mail：84840293@163.com。通信作者孟金柱，E-mail：mjz122021@126.com

（責(zé)任編輯 林鑒非）