靶向鸡Stra8基因的miRNA预测及鉴定

doi:10.3969/j.issn.1004-1524.2017.05.07

浙江农业学报 ›› 2017, Vol. 29 ›› Issue (5): 729-736.DOI: 10.3969/j.issn.1004-1524.2017.05.07

靶向鸡Stra8基因的miRNA预测及鉴定

王颖洁, 左其生, 张良良, 张文慧, 金晶, 王飞, 纪艳芹, 靳锴, 何娜娜, 李碧春^*, 张亚妮^*

扬州大学动物科学技术学院江苏省动物遗传繁育与分子设计重点实验室,江苏扬州 225009

收稿日期:2016-12-08 出版日期:2017-05-20 发布日期:2017-06-06
通讯作者: 张亚妮,E-mail: ynzhang@yzu.edu.cn;李碧春,E-mail: yubcli@yzu.edu.cn
作者简介:王颖洁(1992-),女,江苏镇江人,博士研究生,主要从事动物胚胎工程与遗传工程研究。E-mail: maggieyingjie@foxmail.com
基金资助:
国家自然科学基金(31301959); 江苏省自然科学基金(BK20161331); 校大学生学术科技创新基金资助项目(x20160665); 江苏高校优势学科建设工程资助项目

Prediction and validation of miRNA targeting chicken Stra8 gene

WANG Yingjie, ZUO Qisheng, ZHANG Liangliang, ZHANG Wenhui, JIN Jing, WANG Fei, JI Yanqin, JIN Kai, HE Nana, LI Bichun^*, ZHANG Yani^*

College of Animal Science and Technology, Yangzhou University, Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, Yangzhou 225009, China

Received:2016-12-08 Online:2017-05-20 Published:2017-06-06

摘要/Abstract

摘要： 为克隆如皋黄鸡Stra8基因的3’UTR,寻找靶向Stra8基因的microRNA(miRNA),以鸡精原干细胞的cDNA为模板,根据NCBI数据库Stra8的CDS序列设计引物,通过3’RACE技术克隆Stra8基因的3’UTR,并构建相应的荧光素酶表达载体和突变载体;利用生物学预测软件对靶向Stra8 3’UTR的miRNA进行预测,选择评分最高的miRNA进行慢病毒载体构建;以pRL-TK为内参,分别将miRNA与Stra8 3’UTR荧光素酶表达载体和突变载体共转染DF-1细胞,利用双荧光素酶基因报告系统对miRNA进行活性检测。结果表明,采用3’RACE技术成功克隆Stra8 基因的3’UTR;Targetscan生物在线软件预测到靶向Stra8基因的4个特异性较高的miRNA,并成功构建其相应的慢病毒载体;双荧光素酶活性检测结果显示,gga-miR-1a、gga-miR-31、gga-miR-218均可通过3’UTR序列抑制Stra8基因的表达,其中gga-miR-31抑制效果最佳。该结果可为后续深入探讨gga-miR-31介导调控的Stra8基因在雄性生殖细胞分化中的调控网络提供依据。

关键词: 鸡, miRNA, Stra8, gga-miR-31, 双荧光素酶活性检测系统

Abstract: This paper was aimed to clone 3’UTR of Stra8 gene of Rugao yellow chicken, and predicted the microRNAs (miRNA) targeting the gene to provide theoretical basis for further function study of Stra8 in the process of embryonic stem cells differentiation into the male germ cell mediated by miRNAs. 3'UTR of Stra8 was cloned by 3'RACE based on primers designed from cDNA of Gallus gallus in NCBI, then the vectors of Luciferase expression and mutation was constructed. Targetscan bioinformatics database was used to predict the prospective miRNAs targeting to Stra8 and synthesize miRNA mimics. The candidate miRNA mimics and the vectors of Stra8-3’UTR were transiently co-transfected with into DF-1 cells, and pRL-TK vector was used as an internal control reporter. The dual-luciferase reporter assay system was used to evaluate the activity of luciferase. The results showed that 3'UTR of Stra8 was cloned. Four miRNAs has been predicted that targeting to Stra8 by using the online bioinformatics database and their lentiviral vectors were constructed successfully. Dual luciferase activity test results showed that gga-miR-1a,gga-miR-31,gga-miR-218 can inhibit gene expression of Stra8, but inhibitor of gga-miR-31 was the best. The gga-miR-31 could significantly inhibit expression of Stra8, the results will provide references for the investigation on Stra8 gene regulatory networks in male germ cell differentiation.

Key words: Gallus gallus, miRNA, Stra8, gga-miR-31, dual luciferase activity detection

中图分类号:

S831.2

王颖洁, 左其生, 张良良, 张文慧, 金晶, 王飞, 纪艳芹, 靳锴, 何娜娜, 李碧春, 张亚妮. 靶向鸡Stra8基因的miRNA预测及鉴定[J]. 浙江农业学报, 2017, 29(5): 729-736.

WANG Yingjie, ZUO Qisheng, ZHANG Liangliang, ZHANG Wenhui, JIN Jing, WANG Fei, JI Yanqin, JIN Kai, HE Nana, LI Bichun, ZHANG Yani. Prediction and validation of miRNA targeting chicken Stra8 gene[J]. , 2017, 29(5): 729-736.

参考文献

[1] BOUILLET P, OULAD-ABDELGHANI M, VICAIRE S, et al. Efficient cloning of cDNAs of retinoic acid-responsive genes in P19 embryonal carcinoma cells and characterization of a novel mouse gene, Stra1 (mouse LERK-2/Eplg2)[J]. Developmental Biology , 1995, 170(2):420-433.
[2] STARK A, BRENNECKE J, BUSHATI N, et al. Animal microRNAs confer robustness to gene expression and have a significant impact on 3' UTR evolution[J]. Cell , 2005, 123(6): 1133-1146.
[3] WILKERSON D C, WOLFE S A, GRIMES S R. H1t/GC-box and H1t/TE1 element are essential for promoter activity of the testis-specific histone H1t gene[J]. Biology of Reproduction , 2002, 67(4): 1157-1164.
[4] TAKADA S, BEREZIKOV E, CHOI Y L, et al. Potential role of miR-29b in modulation of Dnmt3a and Dnmt3b expression in primordial germ cells of female mouse embryos[J]. RNA , 2009, 15(8): 1507-1514.
[5] BJORK J K, SANDQVIST A, ELSING A N, et al. mi R-18, a member of Oncomir-1, targets heat shock transcription factor 2 in spermatogenesis[J]. Development , 2010, 137(19): 3177-3184
[6] YU M, MU H, NIU Z, et al. miR-34c enhances mouse spermatogonial stem cells differentiation by targeting Nanos2[J]. Journal of Cellular Biochemistry , 2014, 115(2): 232-242.
[7] MENKE D B, KOUBOVA J, PAGE D C. Sexual differentiation of germ cells in XX mouse gonads occurs in an anterior to-posterior wave[J]. Developmental Biology , 2003, 262(2): 303-312.
[8] CHEUNG A M S, TAM C K H, CHOW H C H, et al. All-trans retinoic acid induces proliferation of an irradiated stem cell supporting stromal cell line AFT024[J]. Experimental Hematology ,2007,35(1):56-63.
[9] VOLLE D H, DUGGAVATH I R, MAGNIER B C, et al. The small heterodiner partner is a gonadal gatekeeper of sexual maturation in male mice[J]. Genes & Development , 2007, 21(3) :303-315.
[10] Lambrot R, Coffigny H, Pairault C, et al. Use of organ culture to study the human fetal testis development: effect of retinoic acid[J]. Journal of Clinical Endocrinology & Metabolism , 2006, 91(7):2696-703.
[11] ANDERSON E L, BALTUS A E, ROEPERS-GAJADIEN H L, et al. Stra8 and its inducer,retinoic acid,regulate meiotic initiationinboth spermatogenesis and oogenesis in mice[J]. Proceedings of the National Academy of Sciences of the United States of America , 2008, 105(39):14976-14980.
[12] MARK M,JACOBS H,OULAD-ABDELGHANI M,et al. STRA8-deficient spermatocytes initiate,but fail to complete,meiosis and undergo premature chromosome condensation[J]. Journal of Cell Science , 2008, 121(19):3233-3242.
[13] BALTUS A E,MENKE D B,HU Y C,et al. In germ cells of mouse embryonic ovaries,the decision to enter meiosis precedes premeiotic DNA replication[J]. Nature Genetics , 2006, 38(12):1430-1434.
[14] 王丹. Stra8基因克隆表达及其功能的初步研究[D].扬州:扬州大学,2013.
WANG D. Study on gene cloning, expression of Stra8 and its function[D]Yangzhou: Yangzhou University, 2013.(in Chinese with English abstract)
[15] 刘志永,左其生,肖天荣,等. Am80和TSA对鸡Stra8基因启动子活性的调控作用[J]. 畜牧兽医学报,2015,46(6):896-902.
LIU Z Y,ZUO Q S,XIAO T R, et al. Regulatory effects of Am80 and TSA on chicken Stra8 gene promoter activity.[J] Chinese Journal of Animal and Veterinary Sciences , 2015,46(6):896-902. (in Chinese with English abstract)
[16] BJORK J K, SANDQVIST A, ELSING A N, et al. miR-18, amember of Oncomir-1, targets heat shock transcription factor 2 inspermatogenesis[J]. Development , 2010, 137(19):3177-3184.
[17] LUO L, YE L, LIU G, et al. Microarray-based approach identifies differentially expressed micro RNAs in porcine sexually immature and mature testes[J]. PLoS One , 2010, 5(8):e11744.
[18] MCLVER S C, ROMAN S D, NIXON B, et al. miRNA and mammalian male germ cells[J]. Human Reproduction Update , 2012,18(1):44-59.
[19] TONG M H, MITCHELL D A, MC GOWAN S D, et al. Two miRNA clusters, Mir-17-92 (Mirc1) and Mir-106b-25 (Mirc3), are involved in the regulation of spermatogonial differentiation in mice[J]. Biology of Reprod uction,2012, 86(3):72.
[20] RO S, SONG R, PARK C, et al. Cloning and expression profiling of small RNAs expressed in the mouse ovary[J]. RNA , 2007. 13(12):2366-2380.
[21] 宋文聪. miR-544靶向PLZF调控奶山羊雄性生殖干细胞的自我更新[D].杨凌:西北农林科技大学,2014.
SONG W C. Mir-544 Regulates the dairy goat male-germ line stem cells' selerenewal by targeting to PLZF [D].Yangling: Northwest A&F University, 2014. (in Chinese with English abstract)
[22] HUSZAR J M, PAYNE C J. Micro RNA 146 (Mir146) modulates spermatogonial differentiation by retinoic acid in mice[J]. Biology of Reproduction ,2013,88(1):15.
[23] LIU T, HUANG Y, LIU J, et al. MicroRNA-122 influences the development of sperm abnormalities from human induced pluripotent stem cells by regulating TNP2 expression[J]. Stem Cells & Development ,2013, 22(12):1839-1850.
[24] KIMURA T, SUZUKI A, FUJITA Y, et al. Conditional loss of PTEN leads to testicular teratoma and enhances embryonic germ cell production[J]. Development , 2003, 130(8): 1691-1700.
[25] ZHONG Z, DONG Z, YANG L, et al. Micro RNA-31-5pmodulates cell cycle by targeting human mutL homolog 1 in human cancer cells[J] .Tumor Biology , 2013, 34( 3) : 1959-1965.
[26] 刘慰华, 黎佼, 刘彬,等. miRNA-31在高糖诱导内皮细胞功能障碍中的作用[J]. 广东医学, 2014, 35(22):3449-3452.
LIU W H, LI J, LIU B, et al. The role of miRNA-31 in high glucose-induced endothelial cell dysfunction[J]. Guangdong Medical Journal , 2014, 35(22):3449-3452. (in Chinese with English abstract)
[27] 陈涛. MicroRNA-31/FIH-1调控关系在结直肠癌发生发展中的作用及其机制研究[D]. 上海:复旦大学, 2014.
CHEN T. The role of MiRNA-31/FIH-1 nexus in the progression of colorectal cancer and the mechanism study[D].Shanghai: Fudan University,2014. (in Chinese with English abstract)

靶向鸡Stra8基因的miRNA预测及鉴定

Prediction and validation of miRNA targeting chicken Stra8 gene

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	桂雪儿, 王志, 李思婷, 贺濛初, 朱杰, 冯士彬, 吴金节. 鸡源复合益生菌对青年白羽肉杂鸡免疫球蛋白和Toll样受体通路的影响[J]. 浙江农业学报, 2020, 32(9): 1609-1614.
[2]	喻宗岗, 蒋隽, 姚亚铃, 郭英, 李闯, 燕海峰. 雪峰乌骨鸡HSP70基因多态性与精液品质关联分析[J]. 浙江农业学报, 2020, 32(8): 1378-1384.
[3]	盛中伟, 姬改革, 刘一帆, 巨晓军, 单艳菊, 邹剑敏, 章明, 屠云洁, 束婧婷. 鸡生长发育早期骨骼肌IGF1R mRNA表达规律研究[J]. 浙江农业学报, 2020, 32(7): 1160-1165.
[4]	刘均, 沈佳敏, 沈建良, 刘雅丽. 不同包装方式对货架期冷鲜鸡微生物菌相变化的影响[J]. 浙江农业学报, 2020, 32(7): 1274-1280.
[5]	卢祎, 高有领, 王水涛, 何盛盛. MicroRNA-499对中华鳖脂类代谢相关基因表达的影响[J]. 浙江农业学报, 2020, 32(5): 798-803.
[6]	李仙春, 芦艳, 毛耀芳, 杨海峰, 余海山, 马永华, 万学瑞. 鸡Prnp基因原核表达载体的构建及其在大肠埃希菌中的表达[J]. 浙江农业学报, 2020, 32(12): 2138-2146.
[7]	吉小凤, 吕文涛, 汪建妹, 廖琳慧, 徐杰, 钱鸣蓉. 超高效液相色谱-串联质谱法测定鲜鸡蛋中氟虫腈及其代谢物残留[J]. 浙江农业学报, 2020, 32(10): 1849-1854.
[8]	云涛, 华炯钢, 叶伟成, 倪征, 陈柳, 张存. 新型鸭呼肠孤病毒在DF-1细胞系中的增殖特性[J]. 浙江农业学报, 2019, 31(5): 716-721.
[9]	张娟, 母童, 赵平, 陈佳萍, 冯小芳, 郭鹏, 武泽文, 刘丽元, 蒋秋斐, 顾亚玲. 静原鸡ELOVL5基因遗传多样性研究[J]. 浙江农业学报, 2019, 31(2): 200-206.
[10]	刘江英, 朱建津, 蒋蓉, 刘子微. 多菌种联合发酵饲料对肉鸡抗沙门氏菌感染能力的影响[J]. 浙江农业学报, 2019, 31(2): 229-234.
[11]	倪黎纲, 赵旭庭, 王宵燕, 宋成义, 吴信生, 甘源. 基于高通量测序分析肺炎支原体感染姜曲海猪肺组织miRNA表达谱[J]. 浙江农业学报, 2019, 31(12): 1979-1986.
[12]	周会明, 张焱珍, 柴红梅, 杨荣情, 谭莹, 张萍萍, 白玉英, 赵一莲, 金玉洁. 一株临沧野生黄鸡?的鉴定与生长条件研究[J]. 浙江农业学报, 2019, 31(10): 1655-1662.
[13]	辛世杰, 王晓慧, 戴国俊, 安婷婷, 张涛, 张跟喜, 谢恺舟, 王金玉, 王宏胜. 京海黄鸡柔嫩艾美尔球虫感染对脾脏和盲肠IL-6、IL-8、CCLi2基因表达量的影响及其相关性[J]. 浙江农业学报, 2019, 31(1): 39-46.
[14]	桂国弘, 杨华, 朱江群, 朱建芬, 肖英平, 徐娥. 冷鲜鸡冷藏保存过程中菌群结构变化分析[J]. 浙江农业学报, 2019, 31(1): 47-55.
[15]	夏魏, 李祖光, 杨华, 吉小凤, 汪建妹, 钱鸣蓉. 鸡蛋磺胺间甲氧嘧啶残留研究[J]. 浙江农业学报, 2019, 31(1): 155-160.