牦牛SIRT3基因的克隆及其在各组织和睾丸不同发育阶段的表达

doi:10.11843/j.issn.0366-6964.2019.12.007

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (12): 2431-2439.doi: 10.11843/j.issn.0366-6964.2019.12.007

牦牛SIRT3基因的克隆及其在各组织和睾丸不同发育阶段的表达

王斌^1,2, 佟连鑫¹, 秦文昌^1,2, 李键^1,2,3, 殷实^1,2,3,4*

1. 西南民族大学生命科学与技术学院, 成都 610041;
2. 青藏高原动物遗传资源保护与利用教育部重点实验室, 成都 610041;
3. 青藏高原动物遗传资源保护与利用四川省重点实验室, 成都 610041;
4. 西南民族大学现代生物技术国家民委重点实验室, 成都 610041

收稿日期:2019-06-10 出版日期:2019-12-23 发布日期:2019-12-20
通讯作者: 殷实,主要从事动物繁殖学和发育生物学研究,E-mail:raulyinshi@163.com
作者简介:王斌(1993-),男,山西原平人,硕士生,主要从事动物细胞与胚胎工程研究,E-mail:15708451401@163.com
基金资助:
中央高校基本科研业务费专项基金项目（2018NQN33）；四川省科技支撑计划（2017NZ0076）；牦牛遗传资源与保护创新团队（13CXTD01）；青藏高原动物遗传资源保护与利用重点实验室资助

Cloning of Yak SIRT3 Gene and Its Expression Pattern in Different Tissues and Testis at Different Development Stages

WANG Bin^1,2, TONG Lianxin¹, QIN Wenchang^1,2, LI Jian^1,2,3, YIN Shi^1,2,3,4*

1. College of Life Science and Technology, Southwest Minzu University, Chengdu 610041, China;
2. Key Laboratory of Ministry of Education for Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation, Chengdu 610041, China;
3. Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province, Chengdu 610041, China;
4. Key Laboratory of Modern Biotechnology of State Ethnic Affairs Commission, Southwest Minzu University, Chengdu 610041, China

Received:2019-06-10 Online:2019-12-23 Published:2019-12-20

摘要/Abstract

摘要： 旨在克隆牦牛沉默蛋白调节因子3（sirtuin 3，SIRT3）基因，并检测其在牦牛不同组织及不同年龄睾丸中的表达水平，从而为研究SIRT3在牦牛睾丸发育过程中的作用机制提供试验依据。本研究选取胎牛期（5~6个月）、幼年期（1~2岁）、性成熟期（3~5岁）及老年期（7~9岁）健康雄性牦牛共12头（每组各3头），其中来源于同一年龄阶段的3个组织样本视为生物学重复。采集性成熟期牦牛肝、心、肺、脾、脑、肾、肌肉、小肠、睾丸和大肠以及4个时期的睾丸组织。分别提取各组织的总RNA，并利用反转录PCR（reverse transcription PCR，RT-PCR）技术克隆获得牦牛SIRT3基因序列，使用相关生物信息学软件预测其编码蛋白质的结构和功能；通过实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）检测SIRT3 mRNA在牦牛各组织及睾丸4个发育阶段的表达水平。结果表明，SIRT3基因的ORF（open reading frame，开放阅读框）为1 002 bp，编码333个氨基酸，与黄牛和绵羊的同源性分别达到99.9%和96.7%，该基因的mRNA在牦牛的各个组织中广泛表达，在脾、睾丸、肺和肌肉中的表达量较高。随年龄增长SIRT3 mRNA在牦牛睾丸中表达呈现先上升后下降的趋势，其中在性成熟期的表达量最高。本研究为揭示SIRT3在牦牛生长发育，尤其是睾丸发育过程中的调控提供了一定的基础数据。

关键词: 组蛋白去乙酰化, SIRT3, 牦牛, 组织表达谱, 睾丸

Abstract: This research was conducted to clone the sirtuin 3 (SIRT3) gene in yak and to identify its expression pattern in various tissues and testis at different development ages, respectively. This research might provide reliable basis for studying the mechanism of SIRT3 in testis development of yak. A total of 12 healthy male yaks (3 in each group) in fetal (5-6 months), calve (1-2 years old), sexual maturity (3-5 years old) and old (7-9 years old) stages were selected. Three samples from the same age were considered as biological duplications. The samples of liver, heart, spleen, lung, brain, kidney, muscle, small intestine, testicle and large intestine were collected from yaks during sexual maturity stage, and testicles were collected from yaks at 4 different development stages, respectively. Total RNAs in different samples were extracted respectively and the coding sequence of yak SIRT3 gene was cloned by reverse transcription PCR(RT-PCR). The structure and function of the protein encoded by SIRT3 gene were predicted by related bioinformatics softwares. The expressions of SIRT3 mRNA in different tissues and testis at 4 different development stages were determined by quantitative real-time PCR (qRT-PCR). The results showed that the ORF(open reading frame) of SIRT3 was 1 002 bp, which encoded 333 amino acids. The sequence of SIRT3 showed 99.9% and 96.7% homology with that of cattle and sheep, respectively. The mRNA of SIRT3 was widely expressed in various tissues of yak, and its expression was relatively higher in spleen, testis, lung and muscle. The expression of SIRT3 mRNA in the testis of yak increased firstly and then decreased with age, with the highest expression in sexual maturity stage. This study provides basic data for revealing the regulation of SIRT3 in yak growth and development, especially in testicular development.

Key words: histone deacetylation, SIRT3, yak, tissue expression pattern, testis

中图分类号:

S823.85

王斌, 佟连鑫, 秦文昌, 李键, 殷实. 牦牛SIRT3基因的克隆及其在各组织和睾丸不同发育阶段的表达[J]. 畜牧兽医学报, 2019, 50(12): 2431-2439.

WANG Bin, TONG Lianxin, QIN Wenchang, LI Jian, YIN Shi. Cloning of Yak SIRT3 Gene and Its Expression Pattern in Different Tissues and Testis at Different Development Stages[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(12): 2431-2439.

参考文献 33

[1]	SHEN Y,WEI W,ZHOU D X.Histone acetylation enzymes coordinate metabolism and gene expression[J].Trends Plant Sci,2015,20(10):614-621.
[2]	YIN S,JIANG X H,JIANG H W,et al.Histone acetyltransferase KAT8 is essential for mouse oocyte development by regulating reactive oxygen species levels[J].Development,2017,144(12):2165-2174.
[3]	MILLARD C J,WATSON P J,FAIRALL L,et al.Targeting class I histone deacetylases in a "Complex" environment[J]. Trends Pharmacol Sci,2017,38(4):363-377.
[4]	ALVES-FERNANDES D K,JASIULIONIS M G.The role of SIRT1 on DNA damage response and epigenetic alterations in cancer[J].Int J Mol Sci,2019,20(13):3153.
[5]	ZHOU L,XU D Y,SHA W G,et al.Long non-coding RNA MALAT1 interacts with transcription factor Foxo1 to regulate SIRT1 transcription in high glucose-induced HK-2 cells injury[J].Biochem Biophys Res Commun, 2018, 503(2):849-855.
[6]	MAHMUD Z,GOMES A R,LEE H J,et al.EP300 and SIRT1/6 co-regulate lapatinib sensitivity via modulating FOXO3-acetylation and activity in breast cancer[J].Cancers,2019,11(8):1067
[7]	BOTTA G,DE SANTIS L P,SALADINO R.Current advances in the synthesis and antitumoral activity of SIRT1-2 inhibitors by modulation of p53 and pro-apoptotic proteins[J].Curr Med Chem,2012,19(34):5871-5884.
[8]	KOSCIUK T,WANG M,HONG J Y,et al.Updates on the epigenetic roles of sirtuins[J].Curr Opin Chem Biol, 2019, 51:18-29.
[9]	KATWAL G,BARAL D,FAN X L,et al.SIRT3 a major player in attenuation of hepatic ischemia-reperfusion injury by reducing ROS via its downstream mediators:SOD2,CYP-D,and HIF-1α[J].Oxid Med Cell Longev,2018, 2018:2976957.
[10]	STORDER J,RENARD P,ARNOULD T.Update on the role of Sirtuin 3 in cell differentiation:a major metabolic target that can be pharmacologically controlled[J].Biochem Pharmacol,2019,169:113621.
[11]	CUI X X,LI X,DONG S Y,et al.SIRT3 deacetylated and increased citrate synthase activity in PD model[J].Biochem Biophys Res Commun,2017,484(4):767-773.
[12]	LIU X H,ZHANG L,WANG P,et al.Sirt3-dependent deacetylation of SOD2 plays a protective role against oxidative stress in oocytes from diabetic mice[J].Cell Cycle,2017,16(13):1302-1308.
[13]	YU W,DITTENHAFER-REED K E,DENU J M.SIRT3 protein deacetylates isocitrate dehydrogenase 2(IDH2) and regulates mitochondrial redox status[J].J Biol Chem,2012,287(17):14078-14086.
[14]	PARODI-RULLÁN R M,CHAPA-DUBOCQ X R,JAVADOV S.Acetylation of mitochondrial proteins in the heart:the role of SIRT3[J].Front Physiol,2018,9:1094.
[15]	WEI L,ZHOU Y,QIAO C,et al.Oroxylin a inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization[J].Cell Death Dis,2015,6(4):e1714.
[16]	权凯.牦牛超数排卵技术研究[D].兰州:甘肃农业大学,2004.QUAN K.Study on superovulation of yak[D].Lanzhou:Gansu Agricultural University,2004.(in Chinese)
[17]	蔡雯祎,熊显荣,陈通,等.KDM2B基因克隆及其在牦牛组织和卵母细胞减数分裂过程中的表达研究[J].畜牧兽医学报, 2018,49(3):534-541.CAI W Y,XIONG X R,CHEN T,et al.Molecular cloning of KDM2B gene and its expression pattern in tissues and the process of oocyte meiosis in yak[J].Acta Veterinaria et Zootechnica Sinica,2018,49(3):534-541.(in Chinese)
[18]	阮崇美.不同发育期白牦牛睾丸蛋白质组学分析及生殖相关候选基因HSP60生物学研究[D].兰州:甘肃农业大学,2017.RUAN C M.Proteomic analysis of white yak testis in different developmental stages and reproductive candidate gene HSP60 biology research[D].Lanzhou:Gansu Agricultural University,2017.(in Chinese)
[19]	SALVATORI I,VALLE C,FERRI A,et al.SIRT3 and mitochondrial metabolism in neurodegenerative diseases[J].Neurochem Int,2017,109:184-192.
[20]	QUAN Y Z,WANG N T,CHEN Q Q,et al.SIRT3 inhibits prostate cancer by destabilizing oncoprotein c-MYC through regulation of the PI3K/Akt pathway[J].Oncotarget,2015,6(28):26494-26507.
[21]	WEI T,HUANG G J,GAO J,et al.Sirtuin 3 deficiency accelerates hypertensive cardiac remodeling by impairing angiogenesis[J].J Am Heart Assoc,2017,6(8):e006114.
[22]	PAULIN R,DROMPARIS P,SUTENDRA G,et al.Sirtuin 3 deficiency is associated with inhibited mitochondrial function and pulmonary arterial hypertension in rodents and humans[J].Cell Metab,2014,20(5):827-839.
[23]	PENG Y H,YANG C,SHI X L,et al.Sirt3 suppresses calcium oxalate-induced renal tubular epithelial cell injury via modification of FoxO3a-mediated autophagy[J].Cell Death Dis,2019,10(2):34.
[24]	CHEN C J,FU Y C,YU W,et al.SIRT3 protects cardiomyocytes from oxidative stress-mediated cell death by activating NF-κB[J].Biochem Biophys Res Commun,2013,430(2):798-803.
[25]	方妙玉.琼玉膏经MAPK/ERK1/2信号通路延缓大鼠脾脏衰老机制研究[D].广州:广州中医药大学,2016.FANG M Y.Study on Qiongyugao aging mechanisms via MAPK/ERK1/2 signal pathway on the spleen of aging rats[D].Guangzhou:Guangzhou University of Chinese Medicine,2016.(in Chinese)
[26]	CORONA J C,DUCHEN M R.PPARγ and PGC-1α as therapeutic targets in Parkinson's[J].Neurochem Res,2015, 40(2):308-316.
[27]	EDGETT B A,HUGHES M C,MATUSIAK J B L,et al.SIRT3 gene expression but not SIRT3 subcellular localization is altered in response to fasting and exercise in human skeletal muscle[J].Exp Physiol,2016,101(8):1101-1113.
[28]	GUERRIERO G,TROCCHIA S,ABDEL-GAWAD F K,et al.Roles of reactive oxygen species in the spermatogenesis regulation[J].Front Endocrinol (Lausanne),2014,5:56.
[29]	DI-LUOFFO M,BROUSSEAU C,BERGERON F,et al.The transcription factor MEF2 is a novel regulator of gsta gene class in mouse MA-10 leydig cells[J].Endocrinology,2015,156(12):4695-4706.
[30]	BANIHANI S A.Effect of coenzyme Q10 supplementation on testosterone[J].Biomolecules,2018,8(4):172.
[31]	MALMIR M,SOLEIMANI MEHRANJANI M,NADERI NOREINI S,et al.Protective antioxidant effects of N-acetylcysteine against impairment of spermatogenesis caused by paranonylphenol[J]. Andrologia,2018, 50(10):e13114.
[32]	KWON Y,KIM J,LEE C Y,et al.Expression of SIRT1 and SIRT3 varies according to age in mice[J].Anat Cell Biol, 2015,48(1):54-61.
[33]	TATONE C,DI EMIDIO G,VITTI M,et al.Sirtuin functions in female fertility:possible role in oxidative stress and aging[J].Oxid Med Cell Longev,2015,2015:659687.

牦牛SIRT3基因的克隆及其在各组织和睾丸不同发育阶段的表达

Cloning of Yak SIRT3 Gene and Its Expression Pattern in Different Tissues and Testis at Different Development Stages

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 33

相关文章 15

编辑推荐

Metrics

本文评价

[1]	肖艺梅, 王胜男, 许悦雯, 何晓琳, 尹福泉. 热应激对雄性哺乳动物生殖机能影响的研究[J]. 畜牧兽医学报, 2024, 55(1): 11-21.
[2]	刘益丽, 唐娇, 闵奇, 杨露, 王泽宁, 胡莲, 赵迪, 江明锋. 基于转录组数据挖掘牦牛皱胃发育代谢的关键候选基因[J]. 畜牧兽医学报, 2024, 55(1): 153-168.
[3]	姚颖, 周应聪, 杜培岩, 李一娟, 钱文洁, 李柳杨, 余志鹏, 崔燕, 余四九, 樊江峰. 基于TMT技术的牦牛妊娠期血清蛋白质组学分析[J]. 畜牧兽医学报, 2024, 55(1): 192-206.
[4]	张颖, 袁莉刚, 陈国娟, 张芳, 杨大鹏. ET-1/eNOS表达差异在牦牛隐睾发生中的作用分析[J]. 畜牧兽医学报, 2024, 55(1): 207-217.
[5]	李在文, 李响, 李小伟, 李飙, 江明锋. 基于GBS简化基因组测序数据重建麦洼牦牛保种群系谱[J]. 畜牧兽医学报, 2023, 54(9): 3710-3721.
[6]	王静瑜, 潘阳阳, 徐庚全, 张瑞, 张文兰, 王筱珊, 乌仁套迪, 照日格图, 崔燕, 余四九. 牦牛Fas相关因子1多克隆抗体制备及初步应用[J]. 畜牧兽医学报, 2023, 54(8): 3369-3382.
[7]	胡婷, 张永红, 侯晓林, 姚华, 崔德凤, 潘早早, 张凌宇, 张家希, 吴琼. 基于转录组学研究双酚A对猪睾丸支持细胞炎症和氨基酸代谢通路的影响[J]. 畜牧兽医学报, 2023, 54(7): 2858-2871.
[8]	卿恩华, 唐彬铖, 牛添, 王珺琦, 陈朝颜, 胡继伟, 何桦, 李亮, 王继文, 胡深强. 不同旱养方式对鹅睾丸及外生殖器组织形态学的影响[J]. 畜牧兽医学报, 2023, 54(7): 2872-2885.
[9]	张鹏, 王明秀, 敬科民, 彭巍, 田园, 李雨谦, 付长其, 舒适, 钟金城, 蔡欣. FGFs/FGFRs及其介导信号通路基因的异常表达影响犏牛未分化精原细胞增殖活性[J]. 畜牧兽医学报, 2023, 54(7): 2886-2897.
[10]	焦正兴, 潘阳阳, 王萌, 王靖雷, 马文斌, 高翔, 张晖, 崔燕, 余四九, 王立斌. 牦牛LC3B蛋白多克隆抗体制备及在生殖器官表达检测中的应用[J]. 畜牧兽医学报, 2023, 54(6): 2436-2447.
[11]	孙俊峰, 潘建秋, 张焯深, 江丹莉, 沈栩, 陈蓉, 许丹宁, 田允波, 黄运茂. OPN5-TSH-DIO2/DIO3和VIP-PRL在短光照抑制公山麻鸭睾丸活性中的表达规律[J]. 畜牧兽医学报, 2023, 54(5): 2001-2012.
[12]	常益铭, 汤承, 岳华. 牦牛源牛呼吸道合胞体病毒的分子流行病学调查[J]. 畜牧兽医学报, 2023, 54(5): 2030-2041.
[13]	王涵, 蒙利洁, 刘文娇, 徐永健, 龚婷. 香猪睾丸间质细胞TAS1R3基因干扰后对自噬相关因子的影响[J]. 畜牧兽医学报, 2023, 54(4): 1525-1534.
[14]	孟朝轶, 王运路, 徐业芬, 牛家强, 索朗斯珠, 郭敏, 席广银. 牦牛lncRNAENSBGRT00000000387.1慢病毒载体构建及其对卵泡GCs凋亡的影响[J]. 畜牧兽医学报, 2023, 54(3): 1058-1070.
[15]	薛鸿雁, 杨孟雨, 杨欢, 董丽君, 蔡霞清, 赵泽民, 王鲜忠. ALOX15B-JNK在热应激诱导支持细胞氧化应激和凋亡中的作用[J]. 畜牧兽医学报, 2023, 54(12): 5056-5065.