山羊Zfy基因克隆分析及其敲除载体构建

doi:10.11843/j.issn.0366-6964.2022.03.005

摘要/Abstract

摘要： 旨在对西农萨能奶山羊Zfy基因进行克隆和生物信息学分析,并利用CRISPR/Cas9技术成功获得敲除Zfy基因的奶山羊成纤维细胞株,为进一步探索Zfy基因功能及性别调控提供基础数据。本研究以健康的西农萨能奶山羊为对象,采集3只3月龄公山羊睾丸组织并提取总RNA。根据NCBI中预测的山羊Zfy基因mRNA序列信息(GenBank登录号:XM_018044893)设计引物,利用RT-PCR技术分段克隆获得山羊Zfy基因序列,使用相关生物信息学软件分析Zfy基因CDS区核苷酸序列并预测其蛋白质的结构和功能。针对山羊Zfy基因CDS区共设计了4条sgRNA,构建4个打靶载体并转染山羊胎儿成纤维细胞(GFFs),利用T7E1酶切检验打靶载体的切割效率,通过嘌呤霉素筛选敲除Zfy基因的GFFs阳性单克隆细胞,经PCR扩增、测序检测突变率。结果显示,成功克隆得到了长度为2 406 bp的西农萨能奶山羊Zfy基因CDS区序列。同源性以及系统进化树分析表明,山羊Zfy基因序列与马鹿、牛、绵羊的同源性高,亲缘关系较近,与小鼠的同源性最低,亲缘关系最远。生物信息学软件分析显示,山羊Zfy基因共编码801个氨基酸,分子质量为90.37 ku,Zfy蛋白含66个磷酸化位点,等电点为5.66,亲水性较高,无信号肽,是一个结构不稳定的非分泌蛋白。T7E1酶切发现,4个打靶载体均可发挥敲除活性,切割效率分别为12.31%、40.86%、31.52%、26.37%。选择切割效率最高的打靶质粒转染山羊胎儿成纤维细胞(GFFs),经嘌呤霉素筛选获得能稳定靶向敲除Zfy基因的GFFs阳性细胞株,PCR测序检测突变率为23.91%。综上所述,本研究成功克隆了西农萨能奶山羊Zfy基因并揭示了该基因所编码蛋白的理化性质;成功构建了山羊Zfy基因CRISPR/Cas9敲除载体,并筛选出最佳敲除位点,获得了Zfy基因敲除的亚克隆细胞,为深入研究山羊Zfy基因功能及开发性别控制技术奠定了基础。

关键词: Zfy基因克隆, 西农萨能奶山羊, 生物信息学分析, 基因敲除

Abstract: The aim of this study was to clone and analyze the Zfy gene by bioinformatics in Xinong Saanen dairy goats, and to successfully establish goat fibroblasts cell lines with Zfy gene knockout by CRISPR/Cas9 technique, and provide basic data for further exploring the function and sex regulation of Zfy gene. In this study, the samples of testicles were collected from 3 healthy Xinong Saanen male goats during 3-month-old and total RNA were extracted, respectively. Primers were designed according to the predicted mRNA sequence (GenBank accession No.:XM_018044893) of Capra hircus Zfy gene published in NCBI, and the Zfy gene was segmental amplified and cloned by RT-PCR. The nucleotide sequence and protein structure and function of the CDS region of the Zfy gene were analyzed by related bioinformatics softwares. Four pairs of sgRNAs were designed according to the CDS region of the Zfy gene to construct 4 targeting vectors later transfected into goat fetal fibroblasts (GFFs). The knockout efficiency of the targeting vectors was confirmed by T7E1 digestion method. Then the Zfy gene knockout positive monoclonal GFFs cells were isolated through puromycin screening, and subsequently, PCR amplification and sequencing were used to detect the mutation rate. The results showed that the sequence of the CDS region of the Zfy gene of Xinong Saanen dairy goats with a length of 2 406 bp was successfully cloned. The homology and phylogenetic tree analysis showed that the Zfy gene sequence of goat had high homology with Cervus elaphus, Bos taurus and Ovis aries, and their genetic distance was closer, the homology with Mus musculus was the lowest and the genetic distance was the farthest. Bioinformatics softwares analysis showed that the goat Zfy gene encoded 801 amino acids, and the molecular mass was 90.37 ku. The Zfy protein contained 66 phosphorylation sites, had an isoelectric point of 5.66, was highly hydrophilic, had no signal peptide, and was a structurally unstable non-secretory protein. T7E1 digestion revealed that all 4 targeting vectors could knock out the gene effectively and the knockout efficiency was 12.31%, 40.86%, 31.52%, and 26.37%, respectively. The targeting plasmid with the highest knockout efficiency was selected to transfect into GFFs cells, and a GFFs-positive cell line that could stably target the knockout of Zfy gene was sorted out by puromycin screening, and the mutation rate was 23.91% by PCR sequencing. In conclusion, this study successfully cloned the Zfy gene of Xinong Saanen dairy goats and revealed the physicochemical properties of the protein of Zfy gene, successfully constructed a CRISPR/Cas9 knockout vector for the Zfy gene of goats and screened the best knockout site, and also obtained subcloned cells for Zfy gene knockout, which provided the foundation for in-depth research on the function of Zfy gene of goats and the development of sex control technology.

Key words: Zfy gene cloning, Xinong Saanen dairy goat, bioinformatics analysis, gene knockout

中图分类号:

S827.2

黄敏, 谢晓刚, 何琪富, 曹旭阳, 董翔宸, 康健, 刘军, 权富生. 山羊Zfy基因克隆分析及其敲除载体构建[J]. 畜牧兽医学报, 2022, 53(3): 711-721.

HUANG Min, XIE Xiaogang, HE Qifu, CAO Xuyang, DONG Xiangchen, KANG Jian, LIU Jun, QUAN Fusheng. Cloning Analysis and Construction of Knockout Vector of Zfy Gene in Goat[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 711-721.

参考文献 41

[1]	ARNAL M,LARROQUE H,LECLERC H,et al.Genetic parameters for first lactation dairy traits in the Alpine and Saanen goat breeds using a random regression test-day model[J].Genet Sel Evol,2019,51(1):43.
[2]	常卫华,王娟红,崔子龙.萨能奶山羊胎儿成纤维细胞miRNA文库的构建及生物信息学分析[J].中国畜牧兽医, 2020, 47(11):3436-3444.CHANG W H,WANG J H,CUI Z L.Construction of miRNA library and bioinformatics analysis about fetal fibroblasts of Saanen Dairy Goat[J].China Animal Husbandry&Veterinary Medicine,2020,47(11):3436-3444.(in Chinese)
[3]	耿亚楠,翁玉楠,罗军,等.西农萨能奶山羊泌乳期产奶量、乳成分及血液生理生化指标变化规律研究[J].畜牧兽医学报,2021,52(1):28-41.GENG Y N,WENG Y N,LUO J,et al.Study on change regularity of milk yield,milk composition and blood physiological and biochemical indexes in Xinong Saanen dairy goat during lactation period[J].Acta Veterinaria et Zootechnica Sinica,2021,52(1):28-41.(in Chinese)
[4]	BIFFANI S,TIEZZI F,FRESI P,et al.Genetic parameters of weeping teats in Italian Saanen and Alpine dairy goats and their relationship with milk production and somatic cell score[J].J Dairy Sci,2020,103(10):9167-9176.
[5]	SUN Y X,WANG C N,SUN X M,et al.Characterization of the milk fat globule membrane proteome in colostrum and mature milk of Xinong Saanen goats[J].J Dairy Sci,2020,103(4):3017-3024.
[6]	SUN Y X,WANG C N,SUN X M,et al.Proteomic analysis of whey proteins in the colostrum and mature milk of Xinong Saanen goats[J].J Dairy Sci,2020,103(2):1164-1174.
[7]	SEOK S H,KANG S Y,IM Y B,et al.Sex identification using ZFX and ZFY genes in leopard cats (Prionailurus bengalensis euptilurus) in Korea[J].J Vet Med Sci,2019,81(5):793-798.
[8]	WATERS P D,RUIZ-HERRERA A.Meiotic executioner genes protect the Y from extinction[J].Trends Genet,2020,36(10):728-738.
[9]	KAM J,GRESSHOFF P M,SHORTER R,et al.The Q-type C2H2 zinc finger subfamily of transcription factors in Triticum aestivum is predominantly expressed in roots and enriched with members containing an EAR repressor motif and responsive to drought stress[J].Plant Mol Biol,2008,67(3):305-322.
[10]	GRANT J,FLANAGAN E,YEE A,et al.Characterization of the DNA binding activity of the ZFY zinc finger domain[J]. Biochemistry, 2010,49(4):679-686.
[11]	MARDON G,LUOH S W,SIMPSON E M,et al.Mouse Zfx protein is similar to Zfy-2:each contains an acidic activating domain and 13 zinc fingers[J].Mol Cell Biol,1990,10(2):681-688.
[12]	MARDON G,PAGE D C.The sex-determining region of the mouse Y chromosome encodes a protein with a highly acidic domain and 13 zinc fingers[J].Cell,1989,56(5):765-770.
[13]	VERNET N,MAHADEVAIAH S K,YAMAUCHI Y,et al.Mouse Y-linked Zfy1 and Zfy2 are expressed during the male-specific interphase between meiosis I and meiosis II and promote the 2nd meiotic division[J].PLoS Genet,2014,10(6):e1004444.
[14]	YAMAUCHI Y,RIEL J M,STOYTCHEVA Z,et al.Two Y genes can replace the entire Y chromosome for assisted reproduction in the mouse[J].Science,2013,343(6166):69-72.
[15]	MATSUBARA Y,KATO T,KASHIMADA K,et al.TALEN-mediated gene disruption on Y chromosome reveals critical role of EIF2S3Y in mouse spermatogenesis[J].Stem Cells Dev,2015,24(10):1164-1170.
[16]	YAMAUCHI Y,RIEL J M,RUTHIG V,et al.Mouse Y-encoded transcription factor Zfy2 is essential for sperm formation and function in assisted fertilization[J].PLoS Genet,2015,11(12):e1005476.
[17]	NAKASUJI T,OGONUKI N,CHIBA T,et al.Complementary critical functions of Zfy1 and Zfy2 in mouse spermatogenesis and reproduction[J].PLoS Genet,2017,13(1):e1006578.
[18]	PENG Q,LI R Y,JIA B,et al.Sex control by Zfy siRNA in the mouse[J].Theriogenology,2011,76(3):507-511.
[19]	MANI M,KANDAVELOU K,DY F J,et al.Design,engineering,and characterization of zinc finger nucleases[J].Biochem Biophys Res Commun,2005,335(2):447-457.
[20]	KONG N R,BASSAL M A,TAN H K,et al.Zinc finger protein SALL4 functions through an AT-Rich motif to regulate gene expression[J].Cell Rep,2021,34(1):108574.
[21]	NOMAN A,AQEEL M,KHALID N,et al.Zinc finger protein transcription factors:integrated line of action for plant antimicrobial activity[J].Microb Pathog,2019,132:141-149.
[22]	SU J,MIAO X,ARCHAMBAULT D,et al.ZC3H4-a novel Cys-Cys-Cys-His-type zinc finger protein-is essential for early embryogenesis in mice[J].Biol Reprod,2021,104(2):325-335.
[23]	HUNING L,KUNKEL G R.The ubiquitous transcriptional protein ZNF143 activates a diversity of genes while assisting to organize chromatin structure[J].Gene,2021,769:145205.
[24]	PAGE D C,MOSHER R,SIMPSON E M,et al.The sex-determining region of the human Y chromosome encodes a finger protein[J].Cell,1987,51(6):1091-1104.
[25]	BERTA P,HAWKINS J R,SINCLAIR A H,et al.Genetic evidence equating SRY and the testis-determining factor[J].Nature, 1990,348(6300):448-450.
[26]	AASEN E,MEDRANO J F.Amplification of the ZFY and ZFX genes for sex identification in humans,cattle,sheep and goats[J].Bio/Technology,1990,8(12):1279-1281.
[27]	ZHANG Y S,DU Y C,SUN L R,et al.A genetic method for sex determination in Ovis spp. by interruption of the zinc finger protein,Y-linked (ZFY) gene on the Y chromosome[J].Reprod Fertil Dev,2018,30(9):1161-1168.
[28]	SEOK S H,KANG S Y,IM Y B,et al.Sex identification using ZFX and ZFY genes in leopard cats (Prionailurus bengalensis euptilurus) in Korea[J].J Vet Med Sci,2019,81(5):793-798.
[29]	席继锋,袁立岗,杨楠,等.奶牛性别控制试验的效果验证[J].江苏农业科学,2019,47(23):195-197.XI J F,YUAN L G,YANG N,et al.Verification on effect of sex control experiment for dairy cattle[J].Jiangsu Agricultural Sciences, 2019,47(23):195-197.(in Chinese)
[30]	DECARPENTRIE F,VERNET N,MAHADEVAIAH S K,et al.Human and mouse ZFY genes produce a conserved testis-specific transcript encoding a zinc finger protein with a short acidic domain and modified transactivation potential[J].Hum Mol Genet,2012, 21(12):2631-2645.
[31]	VERNET N,SZOT M,MAHADEVAIAH S K,et al.The expression of Y-linked Zfy2 in XY mouse oocytes leads to frequent meiosis 2 defects,a high incidence of subsequent early cleavage stage arrest and infertility[J].Development,2014,141(4):855-866.
[32]	VERNET N,MAHADEVAIAH S K,DECARPENTRIE F,et al.Mouse Y-encoded transcription factor Zfy2 is essential for sperm head remodelling and sperm tail development[J].PLoS One,2016,11(1):e0145398.
[33]	魏丽敏,孙丽荣,张永生,等.马鹿Zfx/Zfy基因的克隆及序列分析[J].中国畜牧兽医,2016,43(9):2272-2278.WEI L M,SUN L R,ZHANG Y S,et al.Cloning and sequence analysis of Zfx/Zfy gene in Cervus elaphus[J].China Animal Husbandry&Veterinary Medicine,2016,43(9):2272-2278.(in Chinese)
[34]	季文博,王会,柴志欣,等.西藏牦牛mtDNA-Cytb及ZFY基因遗传多样性与系统进化分析[J].家畜生态学报, 2019, 40(11):12-17.JI W B,WANG H,CHAI Z X,et al.Analysis on genetic diversity and phyletic evolution of mtDNA Cytb&ZFY gene in Tibetan yak[J].Acta Ecologae Animalis Domastici,2019,40(11):12-17.(in Chinese)
[35]	营瑞文,张永生,夏欢,等.驴Zfy基因cDNA克隆及生物信息学分析[J].中国畜牧兽医,2018,45(1):39-46.YING R W,ZHANG Y S,XIA H,et al.cDNA cloning and bioinformatics analysis of Zfy gene in Equus asinus[J].China Animal Husbandry&Veterinary Medicine,2018,45(1):39-46.(in Chinese)
[36]	熊勇,徐亚欧.藏系绵羊ZFX/ZFY基因片段进化分析[J].生物技术,2010,20(6):1-5.XIONG Y,XU Y O.Evolution analysis the tibetan sheep ZFXY/ZFY gene fragment[J].Biotechnology,2010,20(6):1-5.(in Chinese)
[37]	唐榕,马匆匆,毛裕民,等.奶牛ZFY、ZFX基因片段的克隆及测序[J].生物化学与生物物理进展,1995,22(1):60-63.TANG R,MA C C,MAO Y M,et al.Cloning and sequencing of ZFY and ZFX gene from cow[J].Progress in Biochemistry and Biophysics,1995,22(1):60-63.(in Chinese)
[38]	贾浩,张小白,宋晓峰.人类胞内蛋白半衰期与其亚细胞定位的相关性研究[J].计算机与应用化学,2011,28(4):411-414.JIA H,ZHANG X B,SONG X F.Relationship between intracellular protein half-life and subcellular localization in human cells[J].Computers and Applied Chemistry,2011,28(4):411-414.(in Chinese)
[39]	XI J F,WANG X Z,ZHANG Y S,et al.Sex control by Zfy siRNA in the dairy cattle[J].Anim Reprod Sci,2019,200:1-6.
[40]	袁立岗,席继峰,柳炜,等.Zfy干扰基因对荷斯坦牛性别控制的效果[J].中国奶牛,2019(3):18-21.YUAN L G,XI J F,LIU W,et al.Effects of Zfy interference genes on sex control in Holstein cows[J].China Dairy Cattle,2019(3):18-21.(in Chinese)
[41]	彭强,班谦,贾斌,等.siRNA介导小鼠Zfy基因干扰的定量分析[J].石河子大学学报:自然科学版,2011,29(4):448-451.PENG Q,BAN Q,JIA B,et al.Quantitative analysis siRNA mediated silencing Zfy gene of Mus[J].Journal of Shihezi University:Natural Science,2011,29(4):448-451.(in Chinese)