绵羊CYP19基因卵巢启动子的克隆及其表达活性研究

doi:10.11843/j.issn.0366-6964.2015.09.008

摘要/Abstract

摘要：

本研究旨在克隆绵羊CYP19基因卵巢启动子序列片段，构建真核表达载体，并在细胞水平检测其组织特异性表达情况。参考已知序列设计特异性引物，PCR扩增绵羊CYP19基因卵巢启动子1.1和0.5 kb两个片段，并与已公布的序列进行同源性比较；将测序正确的两个片段分别定向克隆到去除CMV的pEGFP-N₂载体骨架中，构建真核表达载体pCYP19-1.1-EGFP-N₂和 pCYP19-0.5-EGFP-N₂，重组质粒在脂质体Lipofectamine^TMLTX+PLUS介导下，分别转染绵羊的颗粒细胞和胎儿成纤维细胞，并于转染后24、48 和72 h观察EGFP表达情况。结果表明，扩增获得片段与已公布序列高度同源；应用转录因子结合位点预测软件对所得序列分析表明，该扩增片段含有类似于TATA-box核心启动顺式元件，并含有多个潜在转录因子的结合位点。转染24 h后，发现pCYP19-1.1-EGFP-N₂在颗粒细胞可观测到绿色荧光表达，48 h荧光细胞数量有所增加；转染72 h时荧光细胞数最多；在胎儿成纤维细胞也有少量EGFP基因表达。pCYP19-0.5-EGFP-N₂ 在颗粒细胞和成纤维细胞中均未检测到荧光。结果表明，扩增所得的绵羊CYP19基因卵巢启动子1.1 kb片段可引导外源基因在颗粒细胞中的表达，可用于与繁殖相关的基因功能及转基因动物研究，但并非卵巢特异性启动子。

Abstract:

The research was conducted to clone the sheep CYP19 gene ovarian promoter fragments and detect the tissue-specific expression in cells level.According to the known sequence the specific prmiers were designed，1.1 and 0.5 kb of sheep CYP19 gene ovarian promoter fragment were amplified by PCR，then the sequences were analyzed by software with published sequences.After eukaryotic expression vector pCYP19-1.1-EGFP-N₂ and pCYP19-0.5-EGFP-N₂were constructed by cloning promoter fragments into the pEGFP-N₂ vector without CMV，then the recombinant plasmids were transfected into sheep granular cells and fetal fibroblast cells by liposome Lipofectamine ^TM LTX+PLUS.The EGFP fluorescence expression was observed under the microscope after transfection for 24，48 and 72 h.The sequenced results showed that sheep CYP19 gene promoter fragments which were cloned were 1.1 and 0.5 kb length and had highly homologous with the published sequences.The sequence analyzing with transcription factor binding sites prediction software indicated that the promoter fragment contained a core promoter cis-element smilar with TATA box and transcription factor binding sites.24 h after transferring with pCYP19-1.1-EGFP-N₂，the EGFP-expressing positive granulosa cells could be observed，48 h after transferring，the EGFP-expressing positive granulosa was increased，72 h after transfection the EGFP-expressing positive granulosa increased to the most.But after transfection the EGFP which was expressed in sheep fetal fibroblast cells was little.The results also showed that no EGFP-expressing positive granulosa cells and fetal fibroblast cells were observed at 24，48 and 72 h after transfection with pCYP19-0.5-EGFP-N₂.The sheep CYP19 promoter 1.1 kb can drive foreign gene expressing in sheep granulosa cells and it can be used for the functional studies of fecundity-related genes and transgenic animal research，but it is not ovarian specific expression promoter.

中图分类号:

S826.2

孙洪新，张英杰，刘月琴，陈晓勇，敦伟涛. 绵羊CYP19基因卵巢启动子的克隆及其表达活性研究[J]. 畜牧兽医学报, 2015, 46(9): 1540-1548.

SUN Hong-xin，ZHANG Ying-jie，LIU Yue-qin，CHEN Xiao-yong,DUN Wei-tao. Cloning and Expression Assay of Sheep CYPl9 Gene Ovarian Promoter[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(9): 1540-1548.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/ 10.11843/j.issn.0366-6964.2015.09.008

https://www.xmsyxb.com/CN/Y2015/V46/I9/1540

参考文献

［1］GUIGUEN Y，FOSTIER A，PIFERRER F，et al.Ovarian aromatase and estrogens：A pivotal role for gonadal sex differentiation and sex change in fish［J］.Gen Comp Endocrinol，2010，165(3)：352-366.
［2］KELLER J M,MCCLELLAN-GREEN P.Effects of organochlorine compounds on cytochrome P450 aromatase activity in an immortal sea turtle cell line［J］.Mar Environ Res，2004（58）：347-351.
［3］GYLLENHAMMAR I，ERIKSSON H，SODERQVIST A，et al.Clotrimazole exposure modulates aromatase activity ingonads brain during gonadal differentiation in Xenopus tropicalis frogs［J］.Aquatic Toxicol，2009，91（2）：102-109.
［4］CHEN S A，BESMAN M J，SPARKES R S，et a1．Human aromatase：cDNA cloning，Southern blot analysis，and assignment to the gene to chromosomel 5［J］.DNA，1988，7（1）：27-38.
［5］MAHENDROO M S，MENDELSON C R，SIMPSON E R．Tissue-specific and hormonally-controlled alternative promoters regulate aromatase cytochrome P450 gene expression in human adipose tissue［J］.Biol Chem，1993（268）：19463-19470．
［6］SEBASTIAN S，TAKAYAMA K，SHOZU M，et al.Cloning and characterization of a novel endothelial promoter of the human CYP19(Aromatase P450) gene that is up-regulated in breast cancer tissue［J］.Mol Endocrinol， 2002，16(10)：2243-2254.
［7］BULUN S E，SEBASTIAN S，TAKAYAMA K，et al.The human CYP19(aromatase P450) gene：update on physiologic roles and genomic organization of promoters［J］.Steroid Biochem Mol Biol，2003，86（3-5）：219-224.
［8］HINSHELWOOD M M，LIU Z，CONLEY A J，et al.Demonstration of tissue-specific promoters in nonprimate species that express aromatase P450 in placentae［J］.Biol Reprod，1995，53（5）：1151-1159.
［9］FüRBASS R，KALBE C，VANSELOW J.Tissue-specific expression of the bovine aromatase encoding gene uses multiple transcriptional start sites and alternative first exons［J］.Endocrinology，1997，138（7）：2813-2819.
［10］VANSELOW J，ZSOLNAI A，FESüS L，et al.Placenta-specific transcripts of the aromatase encoding gene include different untranslated first exons in sheep and cattle［J］.Eur J Biochem，1999（265）：318-324.
［11］王金玲，王永，郑玉才，等.草地藏系绵羊CYP19基因第9内含子和启动子2多态性分析［J］.绵阳师范学院学报，2009，28（5）：64-66.
WANG J L，WANG Y，ZHENG Y C，et al.Polymorphism of the intron 9 and promoter 2 of CYP19 gene in Tibetan sheep［J］.Journal of Mianyang Normal University(Natural Science Edition)，2009，28（5）：64-66.(in Chinese)
［12］SIMPSON E R，ZHAO Y，AGARWAL V L，et al．Aromatase expression in health and disease［J］.Rec Prog Horm Res，1997（52）：185-214.
［13］SIMPSON E R，CLYNE C，RUBIN G，et al.Aromatase——a brief overview［J］.Annu Rev Physiol，2002（64）：93-127.
［14］MEANS G D，KILGORE M W，MAHENDROO M S，et al.Tissue-specific promoters regulate aromatase cytochrome P450 gene expression in human ovary and fetal tissues［J］.Mol Endocrinol，1991（5）：2005-2013.
［15］KATSUMI T，SATOSHI N，YUTAKAS S.Identification and characterization of transcriptional regulatory elements of the human aromatase cytochrome P450 gene［J］.Steroid Biochem Mol Biol， 1996，56(1)：151-159.
［16］STOCCO C.In vivo and in vitro inhibition of cyp19 gene expression by prostagland in F2a in murine luteal cells：implication of GATA-4［J］.Endocrinology ，2004，145（11）：4957-4966.
［17］LENZ S，P HLAND R，BECKER F，et al.Expression of the bovine aromatase cytochrome P450 gene(CYP19) is primarily regulated by promoter 2 in bovine follicles and by promoter 1.1 in corpora lutea［J］.Mol Reprod，2004，67（4）：406-413.
［18］MONGA R，GHAI S，DATTA T K，et al.Tissue-specific promoter methylation and histone modification regulate CYP19 gene expression during folliculogenesis and luteinization in buffalo ovary［J］.Gen Comp Endocrinol，2011，173(1)：205-215.
［19］ZHOU J，GURATES B，YANG S，et al.Malignant breast epithelial cells stimulate aromatase expression via promoterⅡin human adipose fibroblasts：an epithelial-stromal interaction in breast tumors mediated by CCAAT/Enhancer binding proteinbeta［J］. Cancer Res， 2001，61(5)：2328-2334.
［20］PANNETIER M，FABRE S，BATISTA F，et al.FOXL2 activates P450 aromatase gene transcription：towards a better characterization of the early steps of mammalian ovarian development［J］.Mol Endocrinol， 2006，36（3）：399-413.
［21］于今非.CYP19和OSP-1卵巢特异表达启动子克隆与检测［D］.南宁：广西大学，2010.
YU J F.Cloning and expression assay of ovarian specific expressing promoter：OSP-1 and CYP19［D］.Nanning：Guangxi University，2010.(in Chinese)
［22］斯佩克特D L，戈德曼R D，苯因万德L A.细胞实验指南［M］.黄培堂，译.北京：科学出版社，2003.
SPECTOR D L，GOLDMAN R D，LEINWAND L A.Cells：A laboratory manual［M］.HUANG P T，Translation.Beijing：Science Press，2003.(in Chinese)
［23］FITZPATRICK S L，RICHARDS J S.Regulation of cytochrome P450 aromatase messenger ribonucleic acid and activity by steroids and gonadotropins in rat granulosa cells［J］.Endocrinology，1991，129（3）：1452-1462.
［24］MICHAEL M D，MICHAEL L F，SIMPSON E R.A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450(CYP19) gene［J］.Mol Cell Endocrinol，1997，134（2）：147-156.
［25］STOCCO C.Aromatase expression in the ovary：hormonal and molecular regulation［J］.Steroids，2008，73（5）：473-487.
［26］KUO F T，BENTSI-BARNES I K，BARLOW G M，et al.Mutant Forkhead L2(FOXL2) proteins associated with premature ovarian failure(POF) dimerize with wild-type FOXL2，leading to altered regulation of genes associated with granulosa cell differentiation［J］. Endocrinology ，2011，152（10）：3917-3929.
［27］KUO FT，FAN K，BENTSI-BARNES I，et al.Mouse forkhead L2 maintains repression of FSH-dependent genes in the granulosa cell［J］.Reproduction，2012，144（14）：485-494.
［28］TU C H，LIU W P，HUANG L L.Cloning and transcriptional activity of a novel ovarian-specific promoter from rat retrovirus-like elements［J］.Arch Biochem Biophys，2009，485(1)：24-29.
［29］MEANS G D，MAHENDROO M.Structural analysis of the gene encoding human aromatase cytochrome P450，the enzyme responsible for estrogen biosynthesis［J］.Biol Chem，1989，264（32）：19385-19391．
［30］HINSHELWOOD M M，MENDELSON C R.Tissue-specific expression of the human CYP19(aromatase) gene in ovary and adipose tissue of transgenic mice［J］.JSteroid Biochem Mol Biol，2001（79）：193-201.

[1]	王海波, 占今舜, 谷志勇, 陈新锋, 潘月, 贾浩滨, 钟小军, 李开嵘, 赵生国, 霍俊宏. 湖羊与三元杂交绵羊夏杜湖、夏澳湖肉质特性比较研究[J]. 畜牧兽医学报, 2024, 55(1): 110-119.
[2]	韩浩园, 李世凯, 杨瑞巧, 李曼曼, 李君, 哈斯, 赵金艳, 魏红芳, 权凯. 基于转录组测序挖掘槐山羊高繁关键候选基因[J]. 畜牧兽医学报, 2023, 54(12): 5077-5090.
[3]	栗登攀, 马克岩, 韩金涛, 白雅琴, 李讨讨, 马友记. 永登七山羊全基因组选择信号检测分析[J]. 畜牧兽医学报, 2023, 54(11): 4577-4588.
[4]	赵雪洋, 李丹妮, 王钰晨, 郭磊, 王丽, 黄杰, 焦仪强, 安小鹏, 张希云, 张磊, 宋宇轩. 东湖杂交羊产羔性状的GWAS分析及候选基因GRID2的验证[J]. 畜牧兽医学报, 2023, 54(11): 4625-4635.
[5]	张寒月, 赵丹, 梁煜, 赵弼时, 范蒙丹, 乔利英, 刘建华, 杨凯捷, 潘洋洋, 刘文忠. miR-150靶向AOC3调控绵羊前体脂肪细胞分化的研究[J]. 畜牧兽医学报, 2023, 54(8): 3262-3274.
[6]	刘林丽, 彭雪兰, 李博, 程乐凡, 次仁拉姆, 张恩平. 过表达UCP3基因对萨湖羊前体脂肪细胞分化的影响[J]. 畜牧兽医学报, 2023, 54(8): 3275-3285.
[7]	王万年, 陈思佳, 郜金荣, 温中豪, 袁梦娇, 张洪志, 庞志旭, 乔利英, 刘文忠. 基于多层感知机的绵羊限性性状基因组选择模拟研究[J]. 畜牧兽医学报, 2023, 54(7): 2824-2835.
[8]	董亚洁, 郝晓静, 吴晋强, 王荣, 张鹏翔, 王海东, 赫晓燕. 基于绵羊角质形成细胞探究SHH通过Krox20调节IGFBP5的表达影响羊毛弯曲[J]. 畜牧兽医学报, 2023, 54(6): 2365-2375.
[9]	毕亚珍, 尚明玉, 胡文萍, 张莉. 绵羊生长性状间的相关和回归分析及TRHDE基因多态性与生长性状的关联分析[J]. 畜牧兽医学报, 2023, 54(4): 1415-1428.
[10]	李隐侠, 牙生江·那斯尔, 赛里克·都曼, 钱勇, 曹少先, 王伟列, 孟春花, 张俊, 张建丽. SNP芯片评估柯尔克孜羊群体遗传多样性和遗传结构[J]. 畜牧兽医学报, 2023, 54(2): 572-583.
[11]	李玲, 李业芳, 梁奔梦, 孙玉江, 马月辉, 马青, 蒋琳, 刘书琴. 基于SNP标记的滩羊亲子鉴定研究[J]. 畜牧兽医学报, 2022, 53(9): 2912-2919.
[12]	周世卫, 吴庭杰, 王倩, 蔡蓓, 韩赛铮, 吴彦虎, 王小龙, 陈玉林. 绵羊FecB突变检测方法的建立及高繁滩羊核心群的构建[J]. 畜牧兽医学报, 2022, 53(9): 2920-2929.
[13]	丁自强, 葛闻博, 段宏伟, 吕建树, 曾建林, 王文娟, 牛甜, 张勇, 赵兴绪, 胡俊杰. 双氢睾酮通过调控孕酮、雌二醇和细胞凋亡参与绵羊子宫功能[J]. 畜牧兽医学报, 2022, 53(9): 2993-3005.
[14]	李亭亭, 刘秋月, 李向臣, 王海涛. 绵羊经济性状相关基因研究进展及其应用[J]. 畜牧兽医学报, 2022, 53(8): 2417-2434.
[15]	徐颂为, 向华, 张焕容, 杨璐, 王宪军, 撒朗文朱, 朱江江. 干扰SERBP1表达对山羊鼻甲骨原代细胞增殖、周期及凋亡的作用[J]. 畜牧兽医学报, 2022, 53(8): 2708-2720.