用于细胞水平家蚕转录调控元件研究的简单基础启动子的构建
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摘要
【目的】本研究致力于构建一种能够在家蚕Bombyx mori细胞水平稳定表达的简单基础启动子,从而更准确地反映单一转录调控元件对基因启动子活性的影响,为研究家蚕乃至其他昆虫的基因转录调控奠定基础。【方法】本研究在本课题组已报道的能在家蚕细胞中稳定表达且基本不含上游转录调控元件的BmVgP78M启动子的基础上,通过PCR技术在其上游添加一定长度的间隔序列和能够应答20-羟基蜕皮激素(20E)且增强启动子活性的BrC-Z2转录因子结合基序(BrC-Z2 element, BrC-Z2E);通过基因克隆技术构建细胞转染载体;通过细胞转染技术和双荧光素酶报告基因系统检测启动子活性的变化。【结果】通过在BmVgP78M启动子上游添加28 bp间隔序列,成功构建了一个简单基础启动子,命名为VgP78ML,并证明其为可用于研究目标转录调控元件的简单基础启动子。经实验验证表明,该简单基础启动子不仅可以在家蚕细胞中稳定表达,且其本身活性不受20E及转录因子BrC-Z2的影响;当该启动子上游连接BrC-Z2E时,可以显著地应答20E及BrC-Z2转录因子,从而调控报告基因的表达。【结论】VgP78ML能够作为简单基础启动子应用于细胞水平对家蚕基因转录调控进行研究。同时,其构建方法也为其他物种构建研究转录调控的简单基础启动子提供了参考。
【Aim】 This study aims to construct a simple basic promoter, which can be stably expressed in the silkworm(Bombyx mori) cells, so as to reflect the influence of single transcriptional regulatory element on gene promoter activity more accurately and lay a foundation for studying the transcriptional regulation of the silkworm and even other insects. 【Methods】 Based on the BmVgP78 M promoter previously reported in our research group, which can be stably expressed in silkworm cells and hardly contains upstream transcriptional regulatory elements, interval sequences of a certain length and BrC-Z2 transcription factor binding motif(BrC-Z2 element, BrC-Z2 E) that can respond to 20-hydroxyecdysone(20 E) and enhance promoter activity were added to the upstream of BmVgP78 M by PCR technique. The cell transfection vectors were constructed by gene cloning technique, and the changes of promoter activity were detected by cell transfection technique and dual luciferase reporter gene system. 【Results】 A simple basal promoter named VgP78 ML was constructed successfully with a 28 bp interval sequence added to the upstream of the BmVgP78 M promoter, and proved to be a simple basic promoter to study target transcriptional regulatory elements. The verification test results showed that this simple basal promoter not only could be stably expressed in silkworm cells, but also was not affected by 20 E and transcription factor BrC-Z2. When BrC-Z2 E was linked to the upstream of this promoter, it could significantly respond to 20 E and BrC-Z2 transcription factor, thereby regulating the expression of reporter gene. 【Conclusion】 VgP78 ML can be used as a simple basic promoter to study the transcriptional regulation of genes in silkworm at the cell level. At the same time, its construction method also provides a reference for constructing simple basic promoter to study the transcriptional regulation in other species.
【Aim】 This study aims to construct a simple basic promoter, which can be stably expressed in the silkworm(Bombyx mori) cells, so as to reflect the influence of single transcriptional regulatory element on gene promoter activity more accurately and lay a foundation for studying the transcriptional regulation of the silkworm and even other insects. 【Methods】 Based on the BmVgP78 M promoter previously reported in our research group, which can be stably expressed in silkworm cells and hardly contains upstream transcriptional regulatory elements, interval sequences of a certain length and BrC-Z2 transcription factor binding motif(BrC-Z2 element, BrC-Z2 E) that can respond to 20-hydroxyecdysone(20 E) and enhance promoter activity were added to the upstream of BmVgP78 M by PCR technique. The cell transfection vectors were constructed by gene cloning technique, and the changes of promoter activity were detected by cell transfection technique and dual luciferase reporter gene system. 【Results】 A simple basal promoter named VgP78 ML was constructed successfully with a 28 bp interval sequence added to the upstream of the BmVgP78 M promoter, and proved to be a simple basic promoter to study target transcriptional regulatory elements. The verification test results showed that this simple basal promoter not only could be stably expressed in silkworm cells, but also was not affected by 20 E and transcription factor BrC-Z2. When BrC-Z2 E was linked to the upstream of this promoter, it could significantly respond to 20 E and BrC-Z2 transcription factor, thereby regulating the expression of reporter gene. 【Conclusion】 VgP78 ML can be used as a simple basic promoter to study the transcriptional regulation of genes in silkworm at the cell level. At the same time, its construction method also provides a reference for constructing simple basic promoter to study the transcriptional regulation in other species.
引文
Bulger M,Groudine M,2010.Enhancers:the abundance and function of regulatory sequences beyond promoters.Dev.Biol.,339(2):250-257.
Carroll SB,2000.Endless forms:the evolution of gene regulation and morphological diversity.Cell,101(6):577-580.
Carroll SB,2008.Evo-devo and an expanding evolutionary synthesis:a genetic theory of morphological evolution.Cell,134(1):25-36.
Han JL,Li ZJ,Wang K,2017.Progress on genome-wide identification and analysis of transcriptional regulatory elements based on open-chromatin signature.J.Fujian Agric.For.Univ.(Nat.Sci.Ed.),46(1):1-8.[韩金磊,李占杰,王凯,2017.基于开放染色质的全基因组水平转录调控元件的研究方法与进展.福建农林大学学报(自然科学版),46(1):1-8]
Horak CE,Snyder M,2002.ChIP-chip:a genomic approach for identifying transcription factor binding sites.Methods Enzymol.,350:469-483.
Kuo MH,Allis CD,1999.In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment.Methods,19(3):425-433.
Lauderdale JD,Wilensky JS,Oliver ER,Walton DS,Glaser T,2000.3′ deletions cause aniridia by preventing PAX6 gene expression.Proc.Natl.Acad.Sci.USA,97(25):13755-13759.
Lettice LA,Heaney SJ,Purdie LA,Li L,de Beer P,Oostra BA,Goode D,Elgar G,Hill RE,de Graaff E,2003.A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly.Hum.Mol.Genet.,12(14):1725-1735.
Li Y,Wang D,He L,Wang YM,2006.Long-range transcriptional regulator and genetic disease.J.Chengdu Med.Coll.,1(2):150-154.[李亚,王丹,何浪,王玉明,2006.远距离调控元件与遗传性疾病.成都医学院学报,1(2):150-154]
Liao MX,Fang FD,2000.Yeast one-hybrid system-one effective method studying DNA-protein interaction.Acta Acad.Med.Sin.,22(4):388-391.[廖名湘,方福德,2000.酵母单杂交体系——一种研究DNA-蛋白质相互作用的有效方法.中国医学科学院学报,22(4):388-391]
Lin Y,Liu HL,Yang CW,Gu JJ,Shen GW,Zhang HY,Chen EX,Han CS,Zhang YD,Xu YY,Wu JX,Xia QY,2017.The POU homeodomain transcription factor POUM2 and broad complex isoform 2 transcription factor induced by 20-hydroxyecdysone collaboratively regulate vitellogenin gene expression and egg formation in the silkworm Bombyx mori.Insect Mol.Biol.,26(5):496-506.
Liu HL,Lin Y,Shen GW,Gu JJ,Zhang HY,Wu JX,Xu YY,Long W,Xia QY,2018.Establishment of a suitable control reporter plasmid of a dual luciferase reporter gene system for hormone research in silkworm cell lines.Chin.J.Biotech.,34(11):1-10.[刘红玲,林英,沈关望,顾健健,张海燕,吴金鑫,徐银莹,龙威,夏庆友,2018.一种适于家蚕细胞激素研究的双荧光素酶报告基因系统的内参质粒的构建.生物工程学报,34(11):1-10]
Liu XL,Qiao F,Huang DY,Yuan ZH,Wang X,2017.Progress of transcription factor research methods.Prog.Physiol.Sci.,48(1):73-76.[刘相莲,乔芳,黄德玉,袁宗辉,王旭,2017.转录因子研究方法进展.生理科学进展,48(1):73-76]
Niu YD,Xu LY,Han M,Fang WK,Shen ZY,Li EM,2006.Some keys of screening cDNA library with one hybrid system.Chin.J.Lab.Diagn.,10(7):715-717.[牛永东,许丽艳,韩溟,方王楷,沈忠英,李恩民,2006.酵母单杂交筛选实验中的关键问题.中国实验诊断学,10(7):715-717]
Wang QY,He SY,Ma Y,Li LJ,Li BY,2015.Advances in analytical methods of promoter.Prog.Mod.Biomed.,15(14):2794-2800.[王秋岩,何淑雅,马云,李俐娟,李斌元,2015.启动子分析方法的研究进展.现代生物医学进展,15(14):2794-2800]
Yang CW,Lin Y,Liu HL,Shen GW,Luo J,Zhang HY,Peng ZX,Chen EX,Xing RM,Han CS,Xia QY,2014.The Broad Complex isoform 2 (BrC-Z2) transcriptional factor plays a critical role in vitellogenin transcription in the silkworm Bombyx mori.Biochim.Biophys.Acta,1840(9):2674-2684.
Carroll SB,2000.Endless forms:the evolution of gene regulation and morphological diversity.Cell,101(6):577-580.
Carroll SB,2008.Evo-devo and an expanding evolutionary synthesis:a genetic theory of morphological evolution.Cell,134(1):25-36.
Han JL,Li ZJ,Wang K,2017.Progress on genome-wide identification and analysis of transcriptional regulatory elements based on open-chromatin signature.J.Fujian Agric.For.Univ.(Nat.Sci.Ed.),46(1):1-8.[韩金磊,李占杰,王凯,2017.基于开放染色质的全基因组水平转录调控元件的研究方法与进展.福建农林大学学报(自然科学版),46(1):1-8]
Horak CE,Snyder M,2002.ChIP-chip:a genomic approach for identifying transcription factor binding sites.Methods Enzymol.,350:469-483.
Kuo MH,Allis CD,1999.In vivo cross-linking and immunoprecipitation for studying dynamic protein:DNA associations in a chromatin environment.Methods,19(3):425-433.
Lauderdale JD,Wilensky JS,Oliver ER,Walton DS,Glaser T,2000.3′ deletions cause aniridia by preventing PAX6 gene expression.Proc.Natl.Acad.Sci.USA,97(25):13755-13759.
Lettice LA,Heaney SJ,Purdie LA,Li L,de Beer P,Oostra BA,Goode D,Elgar G,Hill RE,de Graaff E,2003.A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly.Hum.Mol.Genet.,12(14):1725-1735.
Li Y,Wang D,He L,Wang YM,2006.Long-range transcriptional regulator and genetic disease.J.Chengdu Med.Coll.,1(2):150-154.[李亚,王丹,何浪,王玉明,2006.远距离调控元件与遗传性疾病.成都医学院学报,1(2):150-154]
Liao MX,Fang FD,2000.Yeast one-hybrid system-one effective method studying DNA-protein interaction.Acta Acad.Med.Sin.,22(4):388-391.[廖名湘,方福德,2000.酵母单杂交体系——一种研究DNA-蛋白质相互作用的有效方法.中国医学科学院学报,22(4):388-391]
Lin Y,Liu HL,Yang CW,Gu JJ,Shen GW,Zhang HY,Chen EX,Han CS,Zhang YD,Xu YY,Wu JX,Xia QY,2017.The POU homeodomain transcription factor POUM2 and broad complex isoform 2 transcription factor induced by 20-hydroxyecdysone collaboratively regulate vitellogenin gene expression and egg formation in the silkworm Bombyx mori.Insect Mol.Biol.,26(5):496-506.
Liu HL,Lin Y,Shen GW,Gu JJ,Zhang HY,Wu JX,Xu YY,Long W,Xia QY,2018.Establishment of a suitable control reporter plasmid of a dual luciferase reporter gene system for hormone research in silkworm cell lines.Chin.J.Biotech.,34(11):1-10.[刘红玲,林英,沈关望,顾健健,张海燕,吴金鑫,徐银莹,龙威,夏庆友,2018.一种适于家蚕细胞激素研究的双荧光素酶报告基因系统的内参质粒的构建.生物工程学报,34(11):1-10]
Liu XL,Qiao F,Huang DY,Yuan ZH,Wang X,2017.Progress of transcription factor research methods.Prog.Physiol.Sci.,48(1):73-76.[刘相莲,乔芳,黄德玉,袁宗辉,王旭,2017.转录因子研究方法进展.生理科学进展,48(1):73-76]
Niu YD,Xu LY,Han M,Fang WK,Shen ZY,Li EM,2006.Some keys of screening cDNA library with one hybrid system.Chin.J.Lab.Diagn.,10(7):715-717.[牛永东,许丽艳,韩溟,方王楷,沈忠英,李恩民,2006.酵母单杂交筛选实验中的关键问题.中国实验诊断学,10(7):715-717]
Wang QY,He SY,Ma Y,Li LJ,Li BY,2015.Advances in analytical methods of promoter.Prog.Mod.Biomed.,15(14):2794-2800.[王秋岩,何淑雅,马云,李俐娟,李斌元,2015.启动子分析方法的研究进展.现代生物医学进展,15(14):2794-2800]
Yang CW,Lin Y,Liu HL,Shen GW,Luo J,Zhang HY,Peng ZX,Chen EX,Xing RM,Han CS,Xia QY,2014.The Broad Complex isoform 2 (BrC-Z2) transcriptional factor plays a critical role in vitellogenin transcription in the silkworm Bombyx mori.Biochim.Biophys.Acta,1840(9):2674-2684.