水牛SFRP1基因序列分析、真核表达载体构建及组织表达分析

doi:10.16431/j.cnki.1671-7236.2024.05.002

Abstract

Abstract: 【Objective】 The aim of this study was to obtain the CDS region sequence of the secreted fizzled-related protein 1 (SFRP1) gene in buffalo,predict the structure and function of the encoded protein,construct the eukaryotic expression vector of SFRP1 gene,and detect the expression of SFRP1 gene in different tissues of buffalo,laying a foundation for exploring the role of SFRP1 gene in the growth and development of buffalo.【Method】 Using the cDNA of ovarian tissue as template,the CDS region sequence of SFRP1 gene was amplified by RT-PCR and sequenced,and the SFRP1 gene of buffalo was compared with different species by bioinformatics online analysis softwares, the phylogenetic tree was constructed,and the physicochemical properties,signal peptide,and transmembrane structure of SFRP1 protein were predicted.The obtained target gene was connected to pCMV-HAhyPBase-mcheery vector and transfected into buffalo granulosa cells. The fluorescence and gene expression were detected after transfection.The expression of SFRP1 gene in different tissues of buffalo was detected by Real-time quantitative PCR.【Result】 The CDS region of SFRP1 gene in buffalo was 927 bp,encoding 308 amino acids.Multiple sequence comparison results showed that the amino acid sequence of SFRP1 gene similarity between buffalo and Bos taurus,Bos mutus,Capra hircus,Orcinus orca,Pan troglodytes,Vulpes lagopus, Felis catuswas,Homo sapiens and Mus musculus were 100%,100%,99.65%,98.58%,98.23%,98.23%,98.58%,98.23% and 96.45%,respectively. There were CRD_FZ,NTR_like and DUF3367 domains.The nucleotide sequence of SFRP1 gene similarity between buffalo and Bos taurus,Ovis aries, Capra hircus,Bos mutus,Bison bison bison,Cervus elaphus,Camelus ferus,Sus scrofa and Homo sapiens were 97.3%,95.9%,95.8%,94.5%,94.2%,93.7%,80.4%,78.5% and 77.3%,respectively.Phylogenetic tree analysis showed that buffalo,Bos taurus,Bos mutus,and Bison bison bison converged into one group.Bioinformatics analysis showed that SFRP1 protein was alkaline and unstable.Signal peptide existed at amino acids 1-15, which was a secreted protein with a transmembrane structure and was mainly located outside the cell.There were 21 phosphorylation sites and 8 O-glycosylation modification sites.The secondary structure was mainly alpha-helix,extended chain and random coil.The tertiary structure of SFRP1 protein in buffalo,Bos taurus and Homo sapiens was highly similar.pCMV-mcheery-SFRP1 eukaryotic expression vector was successfully constructed and transfected into buffalo granulosa cells.After transfection 72 h,the expression of SFRP1 gene in pCMV-mcheery-SFRP1 recombinant plasmid group was extremely significantly higher than that in control group (P＜0.01).Real-time quantitative PCR result showed that SFRP1 gene was expressed in different tissues of buffalo,and the expression level in spleen was the highest,which was extremely significantly higher than that in other tissues (P＜0.01).【Conclusion】 SFRP1 gene was highly conserved in different species and in the process of genetic evolution,and was widely expressed in different tissue of buffalo.The results laid a foundation for future research for the function and molecular mechanism of SFRP1 gene in the growth and development of buffalo.

Key words: buffalo; SFRP1 gene; sequence analysis; eukaryotic expression vector; tissue expression

CLC Number:

S823.8⁺3

HUANG Liqing, DUAN Anqin, ZHENG Haiying, YANG Chunyan, SHANG Jianghua. Sequence Analysis,Eukaryotic Expression Vector Construction and Tissue Expression of SFRP1 Gene in Buffalo[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 1807-1818.

References

[1] FINCH P W,HE X,KELLEY M J,et al.Purification and molecular cloning of a secreted,Frizzled-related antagonist of Wnt action[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(13):6770-6775.
[2] XU Q,D'AMORE P A,SOKOL S Y.Functional and biochemical interactions of Wnts with FrzA,asecreted Wnt antagonist[J].Development (Cambridge,England),1998,125(23):4767-4776.
[3] MELKONYAN H S,CHANG W C,SHAPIRO J P,et al.SARPs:A family of secreted apoptosis-related proteins[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(25):13636-13641.
[4] FAGERBERG L,HALLSTRÖM B M,OKSVOLD P,et al.Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics[J]. Molecular & Cellular Proteomics,2014,13(2):397-406.
[5] ZHAO C H,BU X M,ZHANG N.Hypermethylation and aberrant expression of Wnt antagonist secreted frizzled-related protein 1 in gastric cancer[J].World Journal of Gastroenterology,2007,3(15):2214-2217.
[6] TAKADA T,YAGI Y,MAEKITA T,et al.Methylation-associated silencing of the Wnt antagonist SFRP1 gene in human ovarian cancers[J].Cancer Science,2004,95(9):741-744.
[7] 徐媛媛,莫霞,黄晨茜,等.水牛TRAIL基因克隆及生物信息学分析[J].中国畜牧兽医,2022,49(10):3693-3703. XU Y Y,MO X,HUANG C Q,et al.Cloning and bioinformatics analysis of TRAIL gene in buffalo[J].China Animal Husbandry & Veterinary Medicine,2022,49(10):3693-3703.(in Chinese)
[8] 段安琴,庞春英,邓廷贤,等.水牛LPIN1基因克隆及序列分析[J].中国畜牧兽医,2018,45(10):2653-2660. DUAN A Q,PANG C Y,DENG T X,et al.Cloning and sequence analysis of buffalo LPIN1 gene[J].China Animal Husbandry & Veterinary Medicine,2018,45(10):2653-2660.(in Chinese)
[9] 段安琴,庞春英,朱鹏,等.腺病毒载体转染水牛不同原代体细胞的效果比较[J].中国畜牧兽医,2016,43(10):2661-2665. DUAN A Q,PANG C Y,ZHU P,et al.Effect comparison of Adenovirus vector transfer into different buffalo cells[J].China Animal Husbandry & Veterinary Medicine,2016,43(10):2661-2665.(in Chinese)
[10] 颜君.含Fz-CRD结构域的蛋白家族及两类bHLH转录因子家族的系统进化分析[D].武汉:华中科技大学,2012. YAN J.Phylogenetic and evolutionary analysis on proteinfamilies with Fz-CRD domain and two families of bHLH transcription factors[D].Wuhan:Huazhong University of Science and Technology,2012.(in Chinese)
[11] BU Q,LI Z,ZHANG J,et al.The crystal structure of full-length Sizzled from Xenopus laevis yields insights into Wnt-antagonistic function of secreted Frizzled-related proteins[J].The Journal of Biological Chemistry,2017,292(39):16055-16069.
[12] CRUCIAT C M,NIEHRS C.Secreted and transmembrane Wnt inhibitors and activators[J].Cold Spring Harbor Perspectives in Biology,2013,5(3):a015081.
[13] SKOVIEROVÁ H,LARROUY-MAUMUS G,ZHANG J,et al.AftD,a novel essential arabinofuranosyltransferase from mycobacteria[J].Glycobiology,2009,19(11):1235-1247.
[14] YANG Y,SUN W,WANG R,et al.Wnt antagonist,secreted frizzled-related protein 1,is involved in prenatal skeletal muscle development and is a target of miRNA-1/206 in pigs[J].BMC Molecular Biology,2015,16:4.
[15] DUPLÀA C,JASPARD B,MOREAU C,et al.Identification and cloning of a secreted protein related to the cysteine-rich domain of frizzled.Evidence for a role in endothelial cell growth control[J].Circulation Research,1999,84(12):1433-1445.
[16] AGOSTINO M,POHL S Ö,DHARMARAJAN A.Structure-based prediction of Wnt binding affinities for Frizzled-type cysteine-rich domains[J].The Journal of Biological Chemistry,2017,291(27):11218-11229.
[17] 颜静,王文鹏,李宣,等.SFRP1基因甲基化在儿童急性淋巴细胞白血病中的临床意义[J].中国实验血液学杂志,2023,31(2):377-382. YAN J,WANG W P,LI X,et al.Clinical significance of SFRP1 gene methylation in patients with childhood acute lymphoblastic leukemia[J].Journal of Experimental Hematology,2023,31(2):377-382.(in Chinese)
[18] 王胜东,叶凯山.SFRP1-Wnt信号通路在骨肉瘤中的研究进展[J].重庆医科大学学报,2022,47(2):222-225. WANG S D,YE K S.Research progress of SFRP1-Wnt signaling pathway in osteosarcoma[J].Journal of Chongqing Medical University,2022,47(2):222-225.(in Chinese)
[19] 吴琳,张科,祖珍玉,等.SFRP1/Wnt/β-catenin通路在宫颈癌干细胞中的作用[J].实用医学杂志,2021,37(11):1403-1408. WU L,ZHANG K,ZU Z Y,et al.The role of SFRP1/Wnt/β-catenin signaling pathway in cervical cancer stem cells[J].The Journal of Practical Medicine,2021,37(11):1403-1408.(in Chinese)
[20] CHEN C,LI S,HU C,et al.Protective effects of puerarin on premature ovarian failure via regulation of Wnt/β-catenin signaling pathway and oxidative stress[J].Reproductive Sciences,2021,28:982-990.
[21] LAPOINTE E,BOERBOOM D.Wnt signaling and the regulation of ovarian steroidogenesis[J].Frontiers in Bioscience (Scholar Edition),2011,3:276-285.
[22] FAN H Y,O'CONNOR A,SHITANAKA M,et al.Beta-catenin (CTNNB1) promotes preovulatory follicular development but represses LH-mediated ovulation and luteinization[J].Molecular Endocrinology,2010,24:1529-1542.
[23] HERNANDEZ GIFFORD J A.The role of WNT signaling in adult ovarian folliculogenesis[J].Reproduction (Cambridge,England),2015,150(4):R137-R148.

Sequence Analysis,Eukaryotic Expression Vector Construction and Tissue Expression of SFRP1 Gene in Buffalo

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	ZOU Juhong, MO Zhihua, LIU Yufan, ZOU Jianwei, LU Jun, HE Haien, WANG Fan, HUANG Yanna, JIANG Qinyang. Cloning,Bioinformatics Analysis and Eukaryotic Expression Vector Construction of Rheb Gene in Nubian Goats [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 1819-1826.
[2]	PAN Shiyu, YIN Na, WANG Jiaxing, WANG Qiangzhou, CHEN Shihao, BAI Hao, CHANG Guobin. Bioinformatic Analysis of Chicken DCAF1 and Its Impact on the Replication of ALV-J [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(5): 1837-1845.
[3]	YAN Zunqiang, LIANG Yuhao, SONG Kelin, GUN Shuangbao, WANG Pengfei. Cloning,Bioinformatics Analysis and Tissue Expression of SIRT3 Gene in Hezuo Pigs [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1390-1399.
[4]	MA Rong, LA Yongfu, BAO Pengjia, GUO Xian, LU Jianwei, ZHA Lao, ZHAO Xue, LIANG Chunnian, CHENG Shuru. Cloning,Bioinformatics Analysis and Tissue Expression of MYL9 Gene in Meiren Yak [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1410-1419.
[5]	HAN Hui, GUO Yajing, YAN Guangzhi, CHEN Shengnan, LIU Mingjie, MO Meilian, HUANG Liangzong. Isolation,Identification and Genetic Evolution Analysis of a Eurasian Avian-like Swine Influenza Virus [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1642-1650.
[6]	DOU Junfeng, WANG Zui, LI Li, LU Qin, JIN Xinxin, LING Xiaochun, LUO Qingping, ZHAI Xinguo. Identification of Subgroups A, B and J Avian Leukosis Virus Co-infection and Sequence Analysis of gp85 Gene [J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(1): 302-311.
[7]	BI Yazhen, SHANG Mingyu, XIONG Jinke, HU Wenping, HE Jianning, ZHANG Li. Polymorphism of SRP68 Gene and Its Association with Growth Traits in Hu Sheep [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(8): 3189-3198.
[8]	XING Shanshan, LI Xin, YU Lei, XU Huifen, CAI Hanfang, LI Ming. Cloning, Bioinformatics Analysis and Tissue Expression of SOX6 Gene in Xianan Cattle [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2651-2660.
[9]	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.
[10]	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.
[11]	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.
[12]	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.
[13]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[14]	LI Dan, ZHANG Haisen, WANG Yiqun, GAO Dengke, ZHAO Hongcong, LI Chao, JIN Yaping, CHEN Huatao. Eukaryotic Expression Vector Construction,Bioinformatics and Tissue Expression Profiles Analysis of CRY1 Gene in Dairy Cows [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 870-881.
[15]	ZHANG Yongjie, SUI Zhiyuan, ZHANG Zhishuai, WANG Chenguang, LI Xiaojun, XING Feng. Cloning and Bioinformatics Analysis of NPTX1 Gene and Its Tissues Expression at Puberty in Dolang Sheep [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1048-1058.