牦牛RBM3的基因克隆及其在不同繁殖时期卵巢、输卵管、子宫中的表达定位

doi:10.11843/j.issn.0366-6964.2022.07.017

摘要/Abstract

摘要： 旨在研究RBM3在牦牛(Bos grunniens)生殖相关调控中的作用。本研究以卵泡期、黄体期和妊娠期4~6岁健康雌性牦牛各3头为采集对象，采集其卵巢、子宫和输卵管共9组试验样品，每组设置3个生物学重复。利用基因克隆技术克隆牦牛RBM3基因并进行生物信息学分析；利用实时荧光定量PCR (quantitative real-time PCR,qRT-PCR)和蛋白免疫印迹(Western blot,WB)分别在基因层面和蛋白层面检测RBM3在牦牛子宫、卵巢和输卵管中的相对表达量；利用免疫组织化学(immunocytochemistry,IHC)方法检测RBM3蛋白在试验样品中的分布定位。牦牛RBM3基因(GenBank No.:MF142258.1)被成功克隆并预测出二、三级结构，发现其与野牦牛亲缘性最近，基因编码区第51位、98位核苷酸不同于常见哺乳动物，生物信息学分析预测其编码的蛋白质为稳定的非跨膜蛋白。RBM3蛋白在本试验9组样品的表达量有如下差异：在卵巢中，黄体期显著高于卵泡期和妊娠期(P＜0.05)；输卵管中，卵泡期显著低于黄体期和妊娠期(P＜0.05)；子宫上，卵泡期显著高于黄体期和妊娠期(P＜0.05)。免疫组织化学结果显示，RBM3在卵巢中的主要表达位置是卵泡膜、颗粒层和黄体细胞；在输卵管的表达位置是黏膜上皮；子宫上的表达主要以子宫内膜和子宫腺为主。本研究结果提示，RBM3可能参与牦牛个体发情周期和妊娠过程的调控以及妊娠识别等过程，为RBM3这一冷应激调节因子参与牦牛对高原环境的适应性生理机制方面的研究提供参考。

关键词: 牦牛, 生殖调控, RNA结合基序蛋白3, 冷应激

Abstract: The study aimed to investigate the role of RBM3 in the reproductive-related regulation in yak (Bos grunniens). Three healthy female yaks aged 4-6 years old in different reproductive periods (follicular, luteal and pregnancy phases) were used in this study. Nine groups of experimental samples were collected from ovaries, uterus and fallopian tubes, and 3 biological replicates were set in each group. The RBM3 gene of yak was cloned by gene cloning technology and analyzed by bioinformatics. RBM3 relative expression levels of gene and protein in 9 groups of experimental samples were detected by quantitative real-time PCR(qRT-PCR) and Western blot(WB). The distribution of RBM3 protein in samples was detected by immunohistochemistry (IHC). RBM3 gene(GenBank No.:MF142258.1) of yak was cloned in this experiment, it had the closest relationship with wild yak(Bos mutus). Bioinformatics analysis showed that nucleotide No.51 and No.98 were different from several common mammals, and its secondary and tertiary structure was constructed. After prediction, the encoded protein was stable non-transmembrane protein. WB results showed that RBM3 was expressed in fallopian tube, ovary and uterus of yak, but the expression level at luteal phase was significantly higher than that at follicular and pregnancy phases (P<0.05). The expression in fallopian tube at follicular phase was significantly lower than that at luteal and pregnancy phases (P<0.05). The expression in uterus at follicular phase was significantly higher than that at luteal and pregnancy phases(P<0.05). Immunohistochemical results showed that the main expression sites of RBM3 in the ovary were theca follicle, granular layer and luteal cells, while the expression sites in the fallopian tube were epithelium mucosae. For the uterus, the expression sites was mainly in the endometrium and uterine glands. The results of this study suggest that RBM3 may be involved in the regulation of individual estrous cycle and pregnancy process in yak, as well as pregnancy recognition. It provides a reference for the research of RBM3, a cold stress regulatory factor, in the physiological mechanism of yak in cold and hypoxic environment at high altitude, and other related aspects.

Key words: yak, reproductive regulation, RNA-binding motif protein 3, cold stress

中图分类号:

S823.85

张晖, 潘阳阳, 王靖雷, 张瑞, 黄嘉馨, 余四九, 崔燕. 牦牛RBM3的基因克隆及其在不同繁殖时期卵巢、输卵管、子宫中的表达定位[J]. 畜牧兽医学报, 2022, 53(7): 2202-2214.

ZHANG Hui, PAN Yangyang, WANG Jinglei, ZHANG Rui, HUANG Jiaxin, YU Sijiu, CUI Yan. RBM3 Gene Cloning and Its Expression, Localization in Ovary, Fallopian Tube and Uterus at Different Reproductive Stages of Yak (Bos grunniens)[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2202-2214.

参考文献 40

[1]	CUTHBERT R L,SHUTE R J,SLIVKA D R.Skeletal muscle cold shock and heat shock protein mRNA response to aerobic exercise in different environmental temperatures[J].Temperature (Austin),2019,6(1):77-84.
[2]	JACKSON T C,MANOLE M D,KOTERMANSKI S E,et al.Cold stress protein RBM3 responds to temperature change in an ultra-sensitive manner in young neurons[J].Neuroscience,2015,305:268-278.
[3]	KIM D Y,KIM K M,KIM E J,et al.Hypothermia-induced RNA-binding motif protein 3(RBM3) stimulates osteoblast differentiation via the ERK signaling pathway[J].Biochem Biophys Res Commun,2018,498(3):459-465.
[4]	潘阳阳,王萌,芮弦,等.IGF-1调控RBM3表达抑制低温应激诱导牦牛卵丘细胞凋亡[J].中国农业科学,2020,53(11):2285-2296.PAN Y Y,WANG M,RUI X,et al.RNA-binding motif protein 3(RBM3) expression is regulated by insulin-like growth factor (IGF-1) for protecting yak (Bos grunniens) cumulus cells from apoptosis during hypothermia stress[J].Scientia Agricultura Sinica,2020,53(11):2285-2296.(in Chinese)
[5]	李云龙,李昌盛,李静辉,等.RNA结合基序蛋白3的生物学功能[J].生理科学进展,2014,45(6):429-433.LI Y L,LI C S,LI J H,et al.Biological function of RNA binding motif protein 3[J].Progress in Physiological Sciences,2014,45(6):429-433.(in Chinese)
[6]	DRESIOS J,ASCHRAFI A,OWENS G C,et al.Cold stress-induced protein Rbm3 binds 60S ribosomal subunits,alters MicroRNA levels,and enhances global protein synthesis[J].Proc Natl Acad Sci U S A,2005,102(6):1865-1870.
[7]	WELLMANN S,TRUSS M,BRUDER E,et al.The RNA-binding protein RBM3 is required for cell proliferation and protects against serum deprivation-induced cell death[J].Pediatr Res,2010,67(1):35-41.
[8]	YAN J Y,GOERNE T,ZELMER A,et al.The RNA-binding protein RBM3 promotes neural stem cell (NSC) proliferation under hypoxia[J].Front Cell Dev Biol,2019,7:288.
[9]	FEDOROV V B,GOROPASHNAYA A V,TØIEN Ø,et al.Modulation of gene expression in heart and liver of hibernating black bears (Ursus americanus)[J].BMC Genomics,2011,12(1):171.
[10]	FEDOROV V B,GOROPASHNAYA A V,TØIEN Ø,et al.Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus)[J].Physiol Genomics,2009,37(2):108-118.
[11]	付东海.牦牛部分生长性状与血常规指标的全基因组关联分析和基因组选择研究[D].北京:中国农业科学院,2020.FU D H.Genome-wide association study and genomic selection of partial index of growth performance and hematology in yak[D].Beijing:Chinese Academy of Agricultural Sciences,2020.(in Chinese)
[12]	阎萍,潘和平.环境因素对牦牛繁殖性能的影响[J].畜牧与兽医,2004,36(5):15-16.YAN P,PAN H P.Influence of environmental factors on reproductive performance of yak[J].Animal Husbandry&Veterinary Medicine,2004,36(5):15-16.(in Chinese)
[13]	张君,余四九.高原型牦牛繁育状况及繁殖母牛体况调查[J].畜牧与兽医,2005,37(8):21-22.ZHANG J,YU S J.Investigation on the breeding status of plateau yaks and the body status of breeding cows[J].Animal Husbandry&Veterinary Medicine,2005,37(8):21-22.(in Chinese)
[14]	程华琴,袁钰洁,罗文,等.褪黑素对牦牛黄体细胞生长及功能影响的研究[J].畜牧兽医学报,2022,53(2):459-469.CHENG H Q,YUAN Y J,LUO W,et al.Research on the effect of melatonin on the growth and function of yak luteal cells[J].Acta Veterinaria et Zootechnica Sinica,2022,53(2):459-469.(in Chinese)
[15]	袁昕.亚低温环境中冷休克蛋白RBM3基因转录的分子调控机制[D].新乡:新乡医学院,2021.YUAN X.Molecular mechanism of cold shock protein RBM3 gene transcription under mild hypothermia[D].Xinxiang:Xinxiang Medical University,2021.(in Chinese)
[16]	YANG K,ZHANG Q,WEN Z,et al.Cloning and expression of cold-inducible RNA binding protein in domestic yak (Bos grunniens)[J].Folia Morphol,2016,75(4):460-466.
[17]	DANNO S,NISHIYAMA H,HIGASHITSUJI H,et al.Increased transcript level of RBM3,a member of the glycine-rich RNA-binding protein family,in human cells in response to cold stress[J].Biochem Biophys Res Commun,1997,236(3):804-807.
[18]	LLEONART M E.A new generation of proto-oncogenes:cold-inducible RNA binding proteins[J].Biochim et Biophys Acta-Rev Cancer,2010,1805(1):43-52.
[19]	陈秦俊杰.环状RNA ACTN4促进肝内胆管癌进展的机制研究[D].上海:中国人民解放军海军军医大学,2021.CHEN Q J J.CircRNA ACTN4 facilitates the progression of intrahepatic cholangiocarcinoma and its molecular mechanisms[D].Shanghai:Naval Medical University,2021.(in Chinese)
[20]	WANG X H,CHE H L,ZHANG W B,et al.Effects of mild chronic intermittent cold exposure on rat organs[J].Int J Biol Sci,2015,11(10):1171-1180.
[21]	汪南.RBM3结合Raptor调控自噬保护缺血复灌诱导的心肌细胞损伤[D].昆明:昆明医科大学,2021.WANG N.RBM3 interacts with Raptor to regulate autophagy and protect cardiomyocytes from ischemia-reperfusion-induced injury[D].Kunming:Kunming Medical University,2021.(in Chinese)
[22]	雷冰冰.RBM3分子突变对其核定位和神经保护功能的影响[D].新乡:新乡医学院,2020.LEI B B.Effect of molecular mutations of cold shock protein RBM3 on its nuclear localization and neuroprotective roles[D].Xinxiang:Xinxiang Medical University,2020.(in Chinese)
[23]	宋克芹,肖琦,肖建生,等.亚低温上调冷休克蛋白RBM3表达减轻肾脏缺血-再灌注损伤[J].器官移植,2021,12(5):571-578.SONG K Q,XIAO Q,XIAO J S,et al.Mild hypothermia alleviates renal ischemia-reperfusion injury by up-regulating the expression of cold-shock protein RBM3[J].Organ Transplantation,2021,12(5):571-578.(in Chinese)
[24]	曹勤,叶啟发,王伟.冷休克蛋白的研究进展[J].中华实验外科杂志,2019,36(10):1921-1926.CAO Q,YE Q F,WANG W.Progress knowledge of cold shock protein[J].Chinese Journal of Experimental Surgery,2019,36(10):1921-1926.(in Chinese)
[25]	王强龙,潘阳阳,闫翠霞,等.雌性牦牛主要生殖器官中DBI的表达与定位分析[J].畜牧兽医学报,2022,53(1):169-178.WANG Q L,PAN Y Y,YAN C X,et al.Expression and localization of DBI in main reproductive organs of female yaks[J].Acta Veterinaria et Zootechnica Sinica,2022,53(1):169-178.(in Chinese)
[26]	王瑶瑶.RNA结合基序蛋白3对急性脑损伤的保护作用研究[D].合肥:安徽医科大学,2019.WANG Y Y.The research of protective effect of RNA binding motif Protein3 on acute brain injury[D].Hefei:Anhui Medical University,2019.(in Chinese)
[27]	HORN G,HOFWEBER R,KREMER W,et al.Structure and function of bacterial cold shock proteins[J].Cell Mol Life Sci,2007,64(12):1457-1470.
[28]	陈静波.牛妊娠期黄体形态结构及卵母细胞体外胚胎生产的研究[D].杨凌:西北农林科技大学,2003.CHEN J B.The study on the morphology and structure of corpus luteum&the oocyte in vitro embryo production during the pregnancy period in the cow[D].Yangling:Northwest A&F University,2003.(in Chinese)
[29]	XU M,KREEGER P K,SHEA L D,et al.Tissue-engineered follicles produce live fertile,offspring[J].Tissue Eng,2006,12(10):2739-2746.
[30]	黄正洋.表皮生长因子参与鹅卵泡颗粒细胞增殖调控机理的研究[D].扬州:扬州大学,2016.HUANG Z Y.Study of epidermal growth factor on follicular granulosa cell development in laying geese[D].Yangzhou:Yangzhou University,2016.(in Chinese).
[31]	陈华丽.Wnt/PLC/Ca2+通路对猪卵泡颗粒细胞和卵母细胞凋亡及颗粒细胞激素分泌的影响机制[D].杨凌:西北农林科技大学,2020.CHEN H L.Effect of Wnt/PLC/Ca2+ pathway on apoptosis of porcine follicular granulosa cells and oocytes and hormone secretion of granulosa cells[D].Yangling:Northwest A&F University,2020.(in Chinese)
[32]	EYESTONE W H,FIRST N L.Co-culture of early cattle embryos to the blastocyst stage with oviducal tissue or in conditioned medium[J].J Reprod Fertil,1989,85(2):715-720.
[33]	谭娟.牦牛生殖周期中输卵管的组织结构观察[D].兰州:甘肃农业大学,2008.TAN J.Observation on the histological structure of the yak (Bos grunniens) oviduct during reproductive cycle[D]. Lanzhou:Gansu Agricultural University,2008.(in Chinese)
[34]	杨珊珊,何翃闳,潘阳阳,等.整合素αv在牦牛繁殖周期输卵管不同部位中的表达与定位[J].畜牧兽医学报, 2020, 51(11):2710-2719.YANG S S,HE H H,PAN Y Y,et al.Expression and localization of integrin αv in different parts of the fallopian tube in reproduction cycle of yak[J].Acta Veterinaria et Zootechnica Sinica,2020,51(11):2710-2719.(in Chinese)
[35]	李芝丰.FecBBB型和FecB++型小尾寒羊卵泡期和黄体期输卵管转录组表达分析[D].扬州:扬州大学,2021.LI Z F.Transcriptome analysis of follicular and luteal oviducts of small tail han sheep with FecBBB and FecB++ genotypes[D]. Yangzhou:Yangzhou University,2021.(in Chinese).
[36]	张蓉,赵乐,杨海丽,等.细胞外囊泡调控哺乳动物胚胎附植研究进展[J].畜牧兽医学报,2021,52(5):1154-1162.ZHANG R,ZHAO L,YANG H L,et al.Research progress of extracellular vesicles regulating mammalian embryo implantation[J]. Acta Veterinaria et Zootechnica Sinica,2021,52(5):1154-1162.(in Chinese)
[37]	张芮琪,张莹利,蒋明,等.成纤维生长因子4在妊娠早期牛胎盘组织的分布及表达特点[J].畜牧兽医学报, 2019,50(8):1685-1693.ZHANG R Q,ZHANG Y L,JIANG M,et al.Expression of fibroblast growth factor 4 in bovine placenta at early pregnancy[J]. Acta Veterinaria et Zootechnica Sinica,2019,50(8):1685-1693.(in Chinese)
[38]	郭文文.奶牛子宫内膜永生化细胞系的建立[D].杨凌:西北农林科技大学,2019.GUO W W.Establishment of cow endometrial immortalized cell lines[D].Yangling:Northwest A&F University,2019.(in Chinese)
[39]	张晖,潘阳阳,王萌,等.牦牛PTGES基因克隆及其在发情周期主要生殖器官的表达定位[J].农业生物技术学报,2021, 29(10):1949-1957.ZHANG H,PAN Y Y,WANG M,et al.Cloning of yak's (Bos grunniens) PTGES gene and its expression and location in main reproductive organs of estrus cycle[J].Journal of Agricultural Biotechnology,2021,29(10):1949-1957.(in Chinese)
[40]	喇永富.利用转录组和蛋白质组测序筛选绵羊多羔基因的研究[D].兰州:甘肃农业大学,2020.LA Y F.Study on screening polytocous genes in sheep based on transcriptome and proteomics sequencing[D].Lanzhou:Gansu Agricultural University,2020.(in Chinese)