新西兰白兔CYP11A1基因的克隆、生物信息学分析及其对繁殖相关基因的影响

doi:10.16431/j.cnki.1671-7236.2022.07.006

Abstract

Abstract: 【Objective】 To analyze the bioinformatics function of cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and the regulation of genes related to reproductive performance in New Zealand White rabbit ovarian granulosa cells,in order to lay the foundation for exploring its regulatory function in follicular development.【Method】 According to the sequence of rabbit CYP11A1 gene in GenBank,specific primers were designed to clone CYP11A1 gene and construct pcDNA3.1-CYP11A1 overexpression recombinant vector from ovary tissue of New Zealand White rabbits.Phylogenetic tree was constructed by Mega 5.1 software. The amino acid composition,physicochemical properties,phosphorylation site,conserved domain,secondary structure,tertiary structure,subcellular location,protein cross-linking of CYP11A1 protein were predicted by bioinformatics softwares. Ovarian granulosa cells were further isolated from ovarian tissue of New Zealand White rabbits and identified by immunofluorescence using specific antibody follicle stimulating hormone receptor (FSHR).Three primers (siRNA-1,siRNA-2,siRNA-3) were designed to interfere the expression of CYP11A1 gene,and the one with the highest interference efficiency was screened,and the overexpressed recombinant vector pcDNA3.1-CYP11A1 and siRNA-CYP11A1 were transfected into ovarian granulosa cells.After RNA was extracted from granulosa cells,then Real-time quantitative PCR was used to analyze the effects of CYP11A1 overexpression and knockdown on the mRNA expression levels of reproductive genes such as HSD17B1,BMP15 and FSHR.【Result】 CYP11A1 gene was successfully cloned from New Zealand White rabbits.The full-length sequence of CDS of CYP11A1 gene of New Zealand White rabbits was 1 557 bp,encoding 518 amino acids,among which leucine (10.6%) was the highest,followed by arginine (7.6%),valine (7.4%) and alanine (7.2%).Evolutionary tree analysis showed that the protein sequence of rabbit CYP11A1 was closest to mice,humans and orangutans.Bioinformatics analysis showed that the theoretical isoelectric point of CYP11A1 protein was 7.4,the number of positive and negative charge residues was 50,the aliphatic amino acid index was 82.16,and the instability index was 39.54,among which the number of hydrophilic residues in amino acid sequence was more than that of hydrophobic ones.This protein had 40 potential phosphorylation sites,among which threonine,serine and tyrosine were the most abundant.The secondary structure of CYP11A1 protein consisted α-helix (48.31%),random coil (40.22%),extended chain (7.42%) and β-turn (4.04%),and the tertiary structure was predicted to be curved helix.Subcellular localization predicted that CYP11A1 protein was mainly distributed in mitochondria (52.2%),cytoplasm (17.4%) and nucleus (13.0%).It also had a domain of P450 family and interacted with multiple proteins related to reproductive performance (STAR,CYP21A2,CYP17A1 and FDX1).The expression of FSHR in isolated granulosa cells was positive,which could be used for subsequent experiments.After CYP11A1 gene was overexpressed in granulosa cells,the expression of HSD17B1 and FSHR genes were significantly upregulated (P＜0.05), the expression of BMP15 gene was extremely significantly upregulated (P＜0.01).After CYP11A1 gene was interfered,the expression of BMP15 and FSHR genes were extremely significantly down-regulated (P＜0.01),and the expression of HSD17B1 gene was significantly down-regulated (P＜0.05).【Conclusion】 CYP11A1 gene could regulate the expression of reproductive performance related genes HSD17B1,BMP15 and FSHR in ovarian granulosa cells,suggesting that CYP11A1 played a certain role in the reproductive process of female rabbits.

Key words: New Zealand White rabbits; CYP11A1 gene; bioinformatics; granulosa cells; eukaryotic expression

CLC Number:

BAI Shaocheng, ZHOU Juan, JIN Rongshuai, WANG Fan, LU Tingting, TANG Xianwei, ZHAO Bohao, WU Xinsheng, CHEN Yang. Cloning and Bioinformatics Analysis of CYP11A1 Gene in New Zealand White Rabbits and Its Effects on Reproduction-related Genes[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2484-2496.

References

[1] O'HARA L,YORK J P,ZHANG P M,et al.Targeting of GFP-Cre to the mouse CYP11A1 locus both drives cre recombinase expression in steroidogenic cells and permits generation of CYP11A1 knock out mice[J].PLoS One,2014,9(1):e84541.
[2] REDDY K R,DEEPIKA M L N,SUPRIYA K,et al.CYP11A1 microsatellite (tttta)(n) polymorphism in PCOS women from South India[J].Journal of Assisted Reproduction & Gennetics,2014,31(7):857-863.
[3] XU Q,SONG Y D,CHEN Y,et al.Molecular cloning and expression patterns of the cholesterol side chain cleavage enzyme (CYP11A1) gene during the reproductive cycle in goose (Anas cygnoides)[J].Journal of Animal Science and Biotechnology,2016,7(2):141-148.
[4] 张艳萍.略阳鸡孕酮合成相关基因表达的研究[D].杨凌:西北农林科技大学,2020. ZHANG Y P.Expression of progesterone synthesis related genes in Lueyang chicken[D].Yangling:Northwest A & F University,2020.(in Chinese).
[5] LAN Y Y,ZHANG S P,GONG F,et al.The mitochondrial DNA copy number of cumulus granulosa cells may be related to the maturity of oocyte cytoplasm[J].Human Reproduction,2020,35(5):1120-1129.
[6] JOHNSON A L,WOODS D C.Dynamics of avian ovarian follicle development:Cellular mechanisms of granulosa cell differentiation[J].General and Comparative Endocrinology,2009,163(1-2):12-17.
[7] 吴琦.猪HSD17B1基因启动子活性分析及其表达调控的相关研究[D].广州:华南农业大学,2017. WU Q.Expression and regulation mechanism of porcine genes HSD17B1[D].Guangzhou:South China Agricultural University,2017.(in Chinese)
[8] SU Y Q,SUGIURA K,WIGGLESWORTH K,et al.Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes:BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells[J].Development,2008,135:111-121.
[9] 杨佳栋,刘月琴,张英杰.山羊FSHR基因编码蛋白的结构及功能分析[J].河南农业科学,2017,46(7):120-124. YANG J D,LIU Y Q,ZHANG Y J.Structure and function analysis of goat FHSR gene-coding protein[J].Journal of Henan Agricultural Sciences,2017,46(7):120-124.(in Chinese)
[10] LIU H,XU X,HAN T,et al.A novel homozygous mutation in the FSHR gene is causative for primary ovarian insufficiency[J].Fertility & Sterility,2017,108(6):1050-1055.
[11] DUVAUD S,GABELLA C,LISACEK F,et al.Expasy,the Swiss Bioinformatics Resource Portal,as designed by its users[J].Nucleic Acids Research,2021,49(W1):W216-W227.
[12] DELEAGE G.ALIGNSEC:Viewing protein secondary structure predictions within large multiple sequence alignments[J].Bioinformatics,2017,33(24):3991-3992.
[13] 张炜,杭柏林,董萌萌,等.牛源防御素类抗菌肽的生物信息学分析[J].现代畜牧兽医,2018,7:1-9. ZHANG W,HANG B L,DONG M M,et al.Bioinformatics analysis of antimicrobial peptide in bovine defensins[J]. Modern Journal of Animal Husbandry and Veterinary Medicine,2018,7:1-9.(in Chinese)
[14] MARCHLER-BAUER A,DERBYSHIRE M K,GONZALES N R,et al.CDD:NCBI's conserved domain database[J].Nucleic Acids Research,2015,43(D1):D222-D226.
[15] WATERHOUSE A,BERTONI M,BIENERT S,et al.SWISS-MODEL:Homology modelling of protein structures and complexes[J].Nucleic Acids Research,2018,46(W1):W296-W303.
[16] NAKAI K,IMAI K J E O B,BIOLOGY C.Prediction of protein localization[J].Encyclopedia of Bioinformatics and Computational Biology,2019,2:53-59.
[17] SZKLARCZYK D,FRANCESCHINI A,KUHN M,et al.The STRING database in 2011:Functional interaction networks of proteins,globally integrated and scored[J].Nucleic Acids Research,2011,39:D561-D568.
[18] 肖河生.胆固醇侧链裂解酶CYP11A1在尼罗罗非鱼中的表达及功能研究[D].重庆:西南大学,2020. XIAO H S.Expression and function of cholesterol side chain lyase CYP11A1 in Nile tilapia[D].Chongqing:Southwest University,2020.(in Chinese)
[19] 郭玲玲,张晓磊,张进顺,等.刚地弓形虫ROP18基因的克隆及生物信息学分析[J].中国病原生物学杂志,2015,10(5):6. GUO L L,ZHANG X L,ZHANG J S,et al.Cloning and bioinformatics analysis of ROP18 gene of Toxoplasma gondii[J].Journal of Pathogen Biology,2015,10(5):6.(in Chinese)
[20] JIANG J,BRIEDE J J,JENNEN D G J,et al.Response to cytochrome P450-derived versus mitochondrial oxidant stress in acetaminophen hepatotoxicity[J].Toxicology Letters,2015,235(3):218-219.
[21] IWADE R,MARUO K,OKADA G,et al.Elevated expression of P450c17(CYP17) during testicular formation in the frog[J].General and Comparative Endocrinology,2008,155(1):79-87.
[22] 刘淑敏.DEHP对大鼠孕酮合成及StAR、P450scc基因表达影响的研究[D].武汉:武汉科技大学,2014. LIU S M.The research on the effect of DEHP on the progesterone synthesis and the StAR,P450scc mRNA expression of ovaries in rats[D].Wuhan:Wuhan University of Science and Technology,2014.(in Chinese)
[23] JIANG C,DIAO F,SANG Y J,et al.GGPP-mediated protein geranylgeranylation in oocyte is essential for the establishment of oocyte-granulosa cell communication and primary-secondary follicle transition in mouse ovary[J]. PLoS Genetics,2017,13(1):e1006535.
[24] KUMAR G G,PAUL S F D,MOLIA C,et al.The association between CYP17A1,CYP19A1,and HSD17B1 gene polymorphisms of estrogen synthesis pathway and ovarian cancer predisposition[J].Meta Gene,2022,31:100985.
[25] 张云鹏,白希壮.BMP家族的研究近况[J].解剖科学进展,2008,14(3):3. ZHANG Y P,BAI X Z.What's new in the research of bone morphogenic proteins family[J].Progress of Anatomical Sciences,2008,14(3):3.(in Chinese)
[26] 忽平.卵巢组织中BMP15、GPR30的表达与PCOS卵泡发育障碍的关系[J].实验与检验医学,2021,39(4):834-837. HU P.Relationship between the expression of BMP15 and GPR30 in ovarian tissue and PCOS follicular development disorder[J].Experimental and Laboratory Medicine,2021,39(4):834-837.(in Chinese)
[27] KASHINO C,HASEGAWA T,NAKANO Y,et al.Involvement of BMP-15 in glucocorticoid actions on ovarian steroidogenesis by rat granulosa cells[J].Biochemical and Biophysical Research Communications,2021,559:56-61.
[28] 冯雪.兔卵巢数字基因表达谱的构建及繁殖相关基因的筛选[D].郑州:河南农业大学,2014. FENG X.Construction of digital gene expression profile of rabbit ovary and screening of reproduction related genes[D].Zhengzhou:Henan Agricultural University,2014.(in Chinese)

Cloning and Bioinformatics Analysis of CYP11A1 Gene in New Zealand White Rabbits and Its Effects on Reproduction-related Genes

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	WANG Saiqiao, ZHAO Lu, ZHAI Zhenhan, ZHANG Binglei, JIA Wanhang, WANG Yuqin. Tissue Expression and Bioinformatics Analysis of miR-449a/b Precursor Sequences in Sheep [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2175-2184.
[2]	WEI Mingbang, BIANBA Qiongda, XIAO Qingqing, DUAN Mengqi, CHAMBA Yangzom, SHANG Peng. Cloning,Bioinformatics and Expression Analysis of CDKN1B Gene in Tibetan Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2196-2206.
[3]	XU Yuanyuan, ZHENG Haiying, YANG Chunyan, DENG Tingxian, HUANG Chenqian, FENG Chao, LU Xingrong, DUAN Anqin, MO Xia, MA Xiaoya, SHANG Jianghua. Effects of Inhibition of BMP/Smad Signaling Pathway on the Growth and Steroid Hormone Synthesis of Buffalo Ovarian Granulosa Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2354-2362.
[4]	XIAO Peng, SHANG Jianghua, YANG Chunyan, LI Mengqi, DUAN Anqin, MA Xiaoya, FENG Chao, HUANG Chenqian, ZHANG Bo, ZHOU Jinchen, WEI Kelong, ZHENG Wei, ZHENG Haiying. Effects of Alpha-linolenic Acid on Buffalo Ovarian Granulosa Cells Cultured in vitro [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2380-2387.
[5]	LI Yongbin, YANG Huilin, SONG Zihao, ZHANG Yu, YANG Jin, MENG Jia, CUI Xiaozhen, LYU Xiaoling, ZHENG Mingxue, BAI Rui. Cloning and Bioinformatics Analysis of MIF Gene in Eimeria tenella [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2450-2459.
[6]	ZHANG Dianqi, MA Xinhao, YANG Zhimei, MEI Chugang, ZAN Linsen. Bioinformatics Analysis of PAI1 Gene and Its Effect on the Proliferation and Differentiation of Intramuscular Adipocytes in Qinchuan Beef Cattle [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1729-1741.
[7]	PIAN Huifang, DU Xubin, LI Yan, ZHANG Yuchen, LIU Fei, YU Debing. Sequencing Analysis of the Full-length Transcriptome of Granulosa Cells of Small Yellow Follicle in Hy-Line Variety Brown [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1754-1763.
[8]	WANG Haorui, YU Baojun, YANG Jiayi, WEI Rong, FU Xi, WANG Chuanchuan, ZHANG Di, LI Desheng, CAI Zhengyun, GU Yaling, ZHANG Juan. Bioinformatics Analysis and Tissue Expression of DERA Gene in Jingyuan Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1764-1773.
[9]	CHEN Meiting, ZHENG Congsen, LIANG Zexian, LIN Qiaoer, CHANG Chuanzhe, ZHOU Jun, FENG Jun, LI Linlin, QIN Limei. Cloning and Bioinformatics Analysis of env Gene of Avian Leukosis Virus Subgroup J Strain [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1785-1795.
[10]	PAN Pengcheng, LEI Zongquan, LI Xian, HU Xiangyun, QIN Qiantao, QIN Zhaoxian, GUAN Zhihui, CHEN Baojian, XIE Bingkun. Bioinformatics Analysis,Eukaryotic Expression Vector Construction and Tissue Expression of MYL2 Gene in Luchuan Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1796-1806.
[11]	LIU Jialong, ZHANG Yiwen, ZHANG Yueran, SUN Qijuan, CHEN Jian, HE Lei, JIA Yanyan, DING Ke, YU Zuhua. Cloning,Prokaryotic Expression and Bioinformatics Analysis of ELAVL1 Gene in Chicken [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1807-1817.
[12]	ZHUO Na, ZHU Zhongwu, LI Jing, HU Shixiang, WANG Jianchang, LIN Hua, CHEN Chaolin, HAN Diangang, AI Jun, CHEN Peifu. Bioinformatics Analysis and Eukaryotic Expression of ORF2 Protein of Swine Hepatitis E Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1959-1970.
[13]	TAO Qinghua, HUANG Caiyun, CHEN Chao, LIU Wei, SUN Ningning, LI Ang. Cloning and Tissue Expression Difference Analysis of LIF Gene in White Muscovy Duck [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1296-1306.
[14]	FAN Bingfeng, LI Wen, LIU Lixiang, ZHAO Xiangyuan, SHAO Jing, LIN Lefeng, SUN Huimin, ZHANG Xulin, XU Baozeng. Cloning and Bioinformatics Analysis of AANAT Gene in Mink [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1307-1318.
[15]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.