基于转录组测序技术挖掘影响布莱凯特黑牛睾丸大小的功能基因

doi:10.7668/hbnxb.20194430

摘要/Abstract

摘要：

旨在通过对布莱凯特黑牛大小睾丸转录组数据进行分析,挖掘影响睾丸生殖功能的关键候选基因,探究睾丸大小对牛生殖功能维持的分子机制。采集12月龄布莱凯特黑牛睾丸,根据质量、长径和短径将其分为2组(大小睾丸组各3头)进行高通量转录组测序;以|log₂(Fold Change)|≥1且P-value≤0.01为阈值筛选差异基因,对差异基因进行GO和KEGG功能富集分析,筛选出影响睾丸生殖功能的关键基因;对其进行CDS区克隆测序,并运用qRT-PCR、Western Blot、免疫组化等方法进行进一步研究。结果显示,差异表达基因共353个,其中114个基因在小睾丸中表达上调,239个基因表达下调。GO功能注释表明,差异基因参与代谢、发育、生殖等过程;KEGG富集分析显示,差异基因主要富集在Wnt信号传导、PI3K-Akt信号通路等与睾丸生殖功能相关的途径。最终筛选出睾丸生殖功能基因CCN4进行进一步研究。qRT-PCR和Western Blot结果显示,CCN4基因和蛋白在大睾丸中表达量显著低于小睾丸。免疫组化结果显示,CCN4蛋白主要在精细胞外的区域(特别是支持细胞)中表达,且大睾丸中的相对表达量显著低于小睾丸,推测其高表达会抑制支持细胞发育,从而影响睾丸生殖功能。综上,研究结果可为深入开展牛睾丸大小对生殖功能影响的分子机制研究提供基础资料。

关键词: 牛, 大小睾丸, 转录组, 差异基因, CCN4

Abstract:

The aim of this experiment was to analyze the transcriptome data of testicle in different size of Shandong black cattle,and discover the key candidate genes affecting the reproductive function of testicles,in order to explore the molecular mechanism of maintaining the reproductive function in cattle testicle.Six bulls were divided into two groups according to different size testicle for transcriptome sequencing.The differential expression multiplier values |log₂(Fold Change)|≥1 and P-value≤0.01 were used as the conditions for selecting the differential expression genes (DEGs),and GO and KEGG analysis were performed to select the candidate gene of reproductive function of testicle.The CDS region of cattle candidate gene was cloned,and qRT-PCR,Western Blot,IHC were used for further confirmation.A total of 353 DEGs were obtained from the sequencing results,including 114 up-regulated genes and 239 down-regulated genes in small testicle.The results of GO functional annotation found that the items related to metabolism,development,reproduction and other processes.The results of KEGG pathway enrichment analysis showed that DEGs were mainly enriched in Wnt signal transduction,PI3K-Akt signaling pathway about reproductive function of testicle,further screening for CCN4 related to testicle reproductive function as candidate gene to validate.The qRT-PCR and Western Blot results showed that the relative expression of CCN4 gene and protein in big testicles was significantly lower than that in small testicle.The IHC results showed that CCN4 protein was mainly positive in the areas outside spermatids (especially sertoli cells) and the relative expression in big testicle was significantly lower than that in small testicle.It is speculated that the CCN4 gene may inhibit sertoli cells to affect cattle testicle function.This study provides a basic data for further study of reproductive function testicle in different size of cattle.

Key words: Cattle, Testicle size, Transcriptome, Differential gene, CCN4

梅艳芳, 刘瑞莉, 韩明轩, 张文琦, 陈振鹏, 王秀源, 苗秀萍, 肖超柱, 张任政, 董雅娟, 柏学进. 基于转录组测序技术挖掘影响布莱凯特黑牛睾丸大小的功能基因[J]. 华北农学报, 2024, 39(1): 191-200. doi: 10.7668/hbnxb.20194430.

MEI Yanfang, LIU Ruili, HAN Mingxuan, ZHANG Wenqi, CHEN Zhenpeng, WANG Xiuyuan, MIAO Xiuping, XIAO Chaozhu, ZHANG Renzheng, DONG Yajuan, BAI Xuejin. Analysis of Reproduction Function Gene of Testicle in Different Size Black Cattle Based on Transcriptome Sequencing[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(1): 191-200. doi: 10.7668/hbnxb.20194430.

http://www.hbnxb.net/CN/Y2024/V39/I1/191

图/表 11

表1 qRT-PCR引物

Tab.1 Primes used for qRT-PCR

基因 Gene	引物序列(5'—3') Primer sequence (5'—3')	产物大小/bp Product length	退火温度/℃ Annealing temperature
CCN4	F: CACACATCAAGGAAGGGAAGAAG R: ACTTGTAGGGGATACAGCACCG	150	60
PFN4	F: ACAGTGCAGCCCTCATCAAAC R: TCGGACATCACTGGGCATTACA	83	61
ITGA4	F: AAGGGGAAACAGGCGATGTGA R: AGGCTGGACTTACAAACCCATGA	99	62
GAPDH	F: GATGCTGGTGCTGAGTATGT R: GCAGAAGGTGCAGAGATGAT	104	60
KCNU1	F: GTTCTGCGCCATCCCTTTCAG R: GCCTGGGCAACTAGAGGTGTT	144	62
RBL2	F: GTGGACCACAGCAGAAACAAGG R: GGCGGACACCTGCTAAGTGATA	114	62
STAR	F: CCCTCAAGGACCAAACTCACCT R: GCGAGAGGACCTGGTTGATGAT	85	62

图1 大小睾丸组织HE染色组织学结构 IC.间质细胞;SS.支持细胞;SP.精原细胞;P.初级精母细胞;SSP.次级精母细胞;RS.圆形精子;S.成熟精子。图10同。

Fig.1 Histological structure of HE staining of big and small testicular tissues IC.Interstitial cells;SS.Supporting cells;SP.Spermatogonia;P.Primary spermatocytes;SSP.Secondary spermatocytes; RS.Round spermatozoa;S.Mature spermatozoa.The same as Fig.10.

图2 差异基因火山图

Fig.2 Differential genes volcano map

图3 差异表达基因GO功能分类

Fig.3 GO functional classification of differently expressed genes

图4 差异基因KEGG富集

Fig.4 KEGG enrichment of differential gene

图5 差异表达基因的qRT-PCR验证

Fig.5 qRT-PCR validation of differentially expressed genes

图6 CCN4基因的PCR电泳 M.Marker 2 000;C.CCN4基因PCR产物。

Fig.6 PCR electrophoretogram of CCN4 M.Marker 2 000;C.PCR product of CCN4 gene.

图7 CCN4基因在各组织中的相对表达量以睾丸为对照组织,不同字母表示不同组织间差异显著(P<0.05)。图8—9同。

Fig.7 Relative expression of CCN4 gene in each tissue Testicle was used as control tissue,and different letters indicate significant differences between different tissues(P<0.05).The same as Fig.8—9.

图8 CCN4蛋白在各组织中的相对表达量 A.CCN4蛋白在各组织中的相对表达量;B.WB验证CCN4和GAPDH在不同组织中的蛋白表达量。

Fig.8 Relative expression of CCN4 protein in each tissue A.Relative expression of CCN4 protein in each tissue;B.WB verification of protein expression of CCN4 and GAPDH in different tissues.

图9 CCN4在大小睾丸中的相对表达量 C.WB验证CCN4和GAPDH在大小睾丸中的蛋白表达量。

Fig.9 Relative expression of CCN4 in big and small testicle C.WB validation of protein expression of CCN4 and GAPDH in big and small testicle.

图10 CCN4在大小睾丸中的免疫组织化学染色(200×) A.小睾丸;B.大睾丸。

Fig.10 Immunohistochemical staining of CCN4 in big and small testicle(200×) A.The small testicle; B.The big testicle.

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