牦牛FHL2基因的克隆、组织表达与蛋白定位分析

doi:10.11843/j.issn.0366-6964.2023.01.014

摘要/Abstract

摘要： 旨在获得牦牛FHL2基因序列，阐明该基因序列、蛋白质结构与性质、mRNA的组织表达模式、在雌性生殖器官及颗粒细胞的蛋白表达特性。本研究以5头健康空怀成年母牦牛的心、肝、脾、肺、肾、卵巢、子宫、输卵管组织及5头妊娠2个月左右母牦牛的子宫、输卵管和卵巢为研究材料，利用逆转录PCR(RT-PCR)技术克隆FHL2基因，并使用在线分子生物学软件分析其生物信息学特性；利用实时荧光定量PCR(RT-qPCR)技术分析FHL2基因的组织表达特性；应用免疫组化(IHC)和免疫荧光(IF)技术检测FHL2蛋白在雌性生殖器官的分布和颗粒细胞上的亚细胞定位。结果发现，FHL2基因CDS区全长840bp(ON456866)，编码279个氨基酸，FHL2蛋白属于弱碱性亲水不稳定蛋白，没有跨膜结构。不同物种的FHL2氨基酸序列比对和进化树分析表明，FHL2基因编码区在物种进化中比较保守。RT-qPCR结果显示，FHL2基因在检测的组织都有表达，在心的表达量极显著高于其他检测组织(P＜0.01)；在肺、卵巢、输卵管和子宫中的表达量极显著高于肝、脾和肾(P＜0.01)。FHL2基因在妊娠期子宫中的表达量极显著高于空怀期(P＜0.01)，在卵巢中妊娠期的表达量极显著低于空怀期(P＜0.01)。IHC结果表明，在生殖器官中FHL2蛋白主要在卵泡颗粒细胞、子宫内膜和输卵管黏膜表达；IF定位结果显示，FHL2蛋白主要分布在卵泡颗粒细胞的细胞核中。综上所述，牦牛FHL2基因在动物进化过程中比较保守，在心、肺、卵巢、输卵管和子宫中表达量高，可能在参与牦牛低氧适应性、卵泡发育和妊娠维持等生理功能的调控中发挥重要作用。

关键词: 牦牛, FHL2基因, 表达特性, 实时荧光定量PCR, 免疫组化

Abstract: The objectives of the study were to obtain the sequence of yak FHL2 gene and to elucidate its sequence, protein structure and properties, mRNA expression in tissues, protein localization in the reproductive organs and granulosa cells of female yaks (Bos grunniens). Tissue samples of heart, liver, spleen, lung, kidney, ovaries, uterus and oviducts of 5 healthy non-pregnant adult female yaks and ovaries, uterus and oviducts of 5 pregnant female yaks were used as study materials. The FHL2 gene was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and its bioinformatics properties were analyzed using online softwares. mRNA expression levels in tissues were detected by a real-time quantitative PCR (RT-qPCR). The distribution of FHL2 protein in female reproductive organs and localization in granulosa cells was detected by immunohistochemistry (IHC) and immunofluorescence (IF) techniques. The results showed that the CDS region of FHL2 gene was 840 bp (ON456866) in length and encoded 279 amino acids. The FHL2 protein was a weakly basic hydrophilic unstable protein with no transmembrane structure. Based on amino acid sequence comparison and evolutionary tree results among multiple species, the coding region of the FHL2 gene was relatively conserved during evolution. RT-qPCR results showed that the FHL2 gene was expressed in all detected tissues, and the expression in the heart was significantly higher than in other tissues(P<0.01). Its expression levels in lung, ovary, oviducts and uterus were significantly higher than in liver, spleen and kidney (P<0.01). Its expression in the uterus during pregnancy was significantly higher than that in the non-pregnant period (P<0.01), but in the ovary its expression was significantly lower during pregnancy than in the non-pregnant period (P<0.01). IHC result showed that FHL2 protein was mainly expressed in follicular granulosa cells, endometrium and oviductal mucosa. IF result showed that FLH2 protein was mainly localized in nuclear of granulosa cells. In conclusion, the sequence of yak FHL2 gene is relatively conserved during animal evolution. It is highly expressed in heart, lung, ovaries, oviducts and uterus, indicating that it may be involved in the regulation of physiological functions such as hypoxia adaptation, follicular development and pregnancy maintenance in yak.

Key words: Bos grunniens, FHL2 gene, expression characteristics, real-time quantitative PCR, immunohistochemistry

中图分类号:

S823.85

龚三你, 蒋旭东, 马瑶, 卢建远, 字向东. 牦牛FHL2基因的克隆、组织表达与蛋白定位分析[J]. 畜牧兽医学报, 2023, 54(1): 146-156.

GONG Sanni, JIANG Xudong, MA Yao, LU Jianyuan, ZI Xiangdong. Cloning, Tissue Expression and Protein Localization of FHL2 Gene in Yaks[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 146-156.

参考文献 36

[1]	NISHIKAWA M,SATO K,NAKANO S,et al.Specific activation of PLEKHG2-induced serum response element-dependent gene transcription by four-and-a-half LIM domains (FHL) 1,but not FHL2 or FHL3[J].Small GTPases,2019,10(5):361-366.
[2]	TRAN M K,KURAKULA K,KOENIS D S,et al.Protein-protein interactions of the LIM-only protein FHL2 and functional implication of the interactions relevant in cardiovascular disease[J].Biochim Biophys Acta,2016,1863(2):219-228.
[3]	LI H Y,KOTAKA M,KOSTIN S,et al.Translocation of a human focal adhesion LIM-only protein,FHL2,during myofibrillogenesis and identification of LIM2 as the principal determinants of FHL2 focal adhesion localization[J].Cell Motil Cytoskeleton,2001,48(1):11-23.
[4]	HABIBE J J,CLEMENTE-OLIVO M P,DE VRIES C J.How (Epi)genetic regulation of the LIM-domain protein FHL2 impacts multifactorial disease[J].Cells,2021,10(10):2611.
[5]	JOHANNESSEN M,MØLLER S,HANSEN T,et al.The multifunctional roles of the four-and-a-half-LIM only protein FHL2[J]. Cell Mol Life Sci,2006,63(3):268-284.
[6]	VERSET L,FEYS L,TREPANT A L,et al.FHL2:a scaffold protein of carcinogenesis,tumour-stroma interactions and treatment response[J].Histol Histopathol,2016,31(5):469-478.
[7]	KROONE C,VOS M,RADEMAKERS T,et al.LIM-only protein FHL2 attenuates vascular tissue factor activity,inhibits thrombus formation in mice and FHL2 genetic variation associates with human venous thrombosis[J].Haematologica, 2020, 105(6):1677-1685.
[8]	KIRFEL J,PANTELIS D,KABBA M,et al.Impaired intestinal wound healing in Fhl2-deficient mice is due to disturbed collagen metabolism[J].Exp Cell Res,2008,314(20):3684-3691.
[9]	WIXLER V.The role of FHL2 in wound healing and inflammation[J].FASEB J,2019,33(7):7799-7809.
[10]	VAN DE POL V,VOS M,DERUITER M C,et al.LIM-only protein FHL2 attenuates inflammation in vascular smooth muscle cells through inhibition of the NFκB pathway[J].Vascul Pharmacol,2020,125-126:106634.
[11]	GOLTZ D,HITTETIYA K,GEVENSLEBEN H,et al.Loss of the LIM-only protein Fhl2 impairs inflammatory reaction and scar formation after cardiac ischemia leading to better hemodynamic performance[J].Life Sci,2016,151:348-358.
[12]	李秋芳,魏来,吕晶,等.SCAR分子标记对水貂食毛症遗传病因的初步研究[J].畜牧兽医学报,2011,42(7):939-945.LI Q F,WEI L,LV J,et al.Preliminary studies on genetic etiology of mink trichophagia by SCAR[J].Acta Veterinaria et Zootechnica Sinica,2011,42(7):939-945.(in Chinese)
[13]	LI N,XU L,ZHANG J,et al.High level of FHL2 exacerbates the outcome of non-small cell lung cancer (NSCLC) patients and the malignant phenotype in NSCLC cells[J].Int J Exp Pathol,2022,103(3):90-101.
[14]	BRUN J,DIEUDONNÉ F X,MARTY C,et al.FHL2 silencing reduces Wnt signaling and osteosarcoma tumorigenesis in vitro and in vivo[J].PLoS One,2013,8(1):e55034.
[15]	孙晖.FHL2基因对小鼠卵泡发育及繁殖力的调控作用及机制研究[D].武汉:华中农业大学,2021.SUN H.Regulation of FHL2 gene in mouse follicular development and fertility and its molecular mechanism[D]. Wuhan:Huazhong Agricultural University,2021.(in Chinese)
[16]	张丽萌.FHL2对绵羊卵巢颗粒细胞功能的影响及机制研究[D].保定:河北农业大学,2020.ZHANG L M.Effect and mechanism of FHL2 on the function of ovary granulosa cells in sheep[D].Baoding:Hebei Agricultural University,2020.(in Chinese)
[17]	张天留,高雪,徐凌洋,等.高原家养动物环境适应性的研究进展[J].畜牧兽医学报,2020,51(7):1475-1487.ZHANG T L,GAO X,XU L Y,et al.Research progress on environment adaptation of plateau domestic animals[J].Acta Veterinaria et Zootechnica Sinica,2020,51(7):1475-1487.(in Chinese)
[18]	WIENER G,HAN J L,LONG R J.The yak[M].2nd ed.Bangkok:FAO,2003.
[19]	王世雷.运用B超观察母牦牛生殖系统及胎儿发育[D].兰州:甘肃农业大学,2008.WANG S L.Ultrasonographic assessment of embryonic and fetal development,and adult female genital system changes in the yak[D].Lanzhou:Gansu Agricultural University,2008.(in Chinese)
[20]	刘宇,邬建飞,李恒,等.牦牛StAR基因克隆及其生物信息学与组织表达特性的研究[J].畜牧兽医学报,2021,52(9):2452-2463.LIU Y,WU J F,LI H,et al.Cloning and analysis of bioinformatics and tissue expression characteristics of yak StAR gene[J].Acta Veterinaria et Zootechnica Sinica,2021,52(9):2452-2463.(in Chinese)
[21]	邬建飞,刘宇,马瑶,等.牦牛NGF基因克隆及其在生殖器官中的表达定位[J].畜牧兽医学报,2022,53(3):778-790.WU J F,LIU Y,MA Y,et al.Cloning of yak NGF gene and its expression and localization in reproductive organs of yaks[J].Acta Veterinaria et Zootechnica Sinica,2022,53(3):778-790.(in Chinese)
[22]	霍晓澎.FHL2在小鼠卵巢中的表达和定位及对卵泡颗粒细胞的调控作用研究[D].武汉:华中农业大学,2015.HUO X P.FHL2 expression and localization in mouse ovary and the regulation on follicle granulosa cell[D].Wuhan:Huazhong Agricultural University,2015.(in Chinese)
[23]	WANG G F,NIU X Y,LIU H N,et al.c-Abl kinase regulates cell proliferation and ionizing radiation-induced G2/M arrest via phosphorylation of FHL2[J].FEBS Openbio,2021,11(6):1731-1738.
[24]	ZHANG L M,LI R T,CHEN L X,et al.Expression,location and biological effects of four and a half LIM domain protein 2(FHL2) on granulosa cells in ovine[J].Reprod Domest Anim,2020,55(6):737-746.
[25]	RAJAN A,VARGHESE G R,YADEV I,et al.Modulation of BRCA1 mediated DNA damage repair by deregulated ER-α signaling in breast cancers[J].Am J Cancer Res,2022,12(1):17-47.
[26]	ZHANG W W,LUO J Y,YANG F,et al.BRCA1 inhibits AR-mediated proliferation of breast cancer cells through the activation of SIRT1[J].Sci Rep,2016,6(1):22034.
[27]	SALINAS I,SINHA N,SEN A.Androgen-induced epigenetic modulations in the ovary[J].J Endocrinol,2021,249(3):R53-R64.
[28]	ZHOU R Q,LI S,LIU J S,et al.Up-regulated FHL2 inhibits ovulation through interacting with androgen receptor and ERK1/2 in polycystic ovary syndrome[J].eBioMedicine,2020,52:102635.
[29]	YANG Y H,HOU H Y,HALLER E M,et al.Suppression of FOXO1 activity by FHL2 through SIRT1-mediated deacetylation[J]. EMBO J,2005,24(5):1021-1032.
[30]	韩琦.绵羊FoxO1基因的克隆及其在卵泡颗粒细胞中的功能研究[D].晋中:山西农业大学,2020.HAN Q.Molecular cloning and functional analysis of FoxO1 gene of sheep granulosa cells[D].Jinzhong:Shanxi Agricultural University,2020.(in Chinese)
[31]	贾青青,郝晓莹,郭慧慧,等.FOXO1与早期妊娠的研究进展[J].医学综述,2021,27(13):2546-2551.JIA Q Q,HAO X Y,GUO H H,et al.Research advances in FOXO1 and early pregnancy[J].Medical Recapitulate,2021, 27(13):2546-2551.(in Chinese)
[32]	卿莹.FHL2基因在鸡骨骼肌卫星细胞增殖与分化中的作用研究[D].成都:四川农业大学,2016.QING Y.Preliminary research about the functional role of FHL2 gene in the proliferation and differentiation of chicken skeletal muscle satellite cells[D].Chengdu:Sichuan Agricultural University,2016.(in Chinese)
[33]	王晨,李俊,杜超,等.FHL2调控人和动物生殖的研究进展[J].基因组学与应用生物学,2018,37(2):774-779.WANG C,LI J,DU C,et al.Research progress of FHL2 regulating human and animal reproduction[J].Genomics and Applied Biology,2018,37(2):774-779.(in Chinese)
[34]	LIANG Y,BRADFORD W H,ZHANG J,et al.Four and a half LIM domain protein signaling and cardiomyopathy[J].Biophys Rev,2018,10(4):1073-1085.
[35]	ALNAJAR A,NORDHOFF C,SCHIED T,et al.The LIM-only protein FHL2 attenuates lung inflammation during bleomycin-induced fibrosis[J].PLoS One,2013,8(11):e81356.
[36]	SUN L L,YU S Y,XU H,et al.FHL2 interacts with EGFR to promote glioblastoma growth[J].Oncogene,2018,37(10):1386-1398.