黄瓜单链DNA结合蛋白基因CsWHY1的克隆及表达分析
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摘要
[目的]单链DNA结合蛋白Whirly(WHY)是植物体内广泛存在的一类转录因子,在植物叶片衰老等方面具有重要作用。本文旨在克隆黄瓜单链DNA结合蛋白基因CsWHY1,初步分析其表达特征,为黄瓜叶片衰老的功能研究提供参考。[方法]以华北型黄瓜‘9930’‘长春密刺’和‘新泰密刺’为试验材料,克隆CsWHY1的c DNA全长,应用生物信息学方法对该基因进行生物信息学分析,并利用洋葱表皮细胞瞬时表达系统对CsWHY1蛋白进行亚细胞定位,同时通过RT-qPCR技术分析CsWHY1基因在3个黄瓜品种不同组织中的表达模式。[结果]CsWHY1基因包含1个长度为831 bp的开放阅读框(ORF),共编码276个氨基酸,蛋白理论相对分子质量为30.729×10~3,理论等电点(p I)为9.00,属于亲水蛋白。氨基酸序列分析表明,CsWHY1蛋白具有保守的Whirly结构域,在双子叶植物和单子叶植物中高度同源,且与甜瓜亲缘关系最近。亚细胞定位发现CsWHY1蛋白在洋葱表皮的质体上表达。荧光定量PCR分析其表达模式,发现CsWHY1在不同黄瓜品种不同组织中均有表达,叶片中表达量最高,根和茎中表达量较低,其中衰老叶片中的表达量显著高于成熟叶片。[结论]CsWHY1基因的表达具有组织特异性,在衰老叶片高表达,参与了黄瓜叶片衰老进程。
[Objectives]The single-stranded DNA binding protein Whirly( WHY),a transcription factor,exists in plants widely and plays an important role in plant leaf senescence. This paper aimed to clone CsWHY1,a single-stranded DNA binding protein gene in cucumber,and to analyze its expression characteristics to provide reference for its functional research on cucumber leaf senescence.[Methods]The full length c DNA of CsWHY1 was cloned with North China cucumber‘9930'‘Changchunmici'and‘Xintaimici'as experimental materials. A series of bioinformatics software were used to speculate nucleotide sequence and protein function of CsWHY1. In addition,the subcellular localization was conducted by onion epidermal cells transient expression system,and gene expression pattern of CsWHY1 was investigated by RT-qPCR in different tissues of 3 cucumber varieties. [Results]The ORF of CsWHY1 gene was 831 bp,encoding 276 amino acids. The relative molecular mass of CsWHY1,a hydrophilic protein,was 30.729×10~3 with the theoretical isoelectric point( p I) of 9.00. Amino acid sequence analysis revealed that CsWHY1 protein had a conserved Whirly domain,and it was highly homologous in dicotyledonous and monocotyledonous plants and had the closest genetic relationship with melon. Subcellular localization analysis showed that CsWHY1 was located in the plastids of onion epidermal cell. RT-qPCR analysis showed that CsWHY1 expressed in different tissues of three cucumber varieties,with the highest expression level in leaves and the lower expression in roots and stems,and the expression level of CsWHY1 in aged leaves was significantly higher than that in mature leaves. [Conclusions]The expression of CsWHY1 was tissue-specific. CsWHY1 had a high expression level in senescent leaves and participated in the senescence process of leaves in cucumber.
[Objectives]The single-stranded DNA binding protein Whirly( WHY),a transcription factor,exists in plants widely and plays an important role in plant leaf senescence. This paper aimed to clone CsWHY1,a single-stranded DNA binding protein gene in cucumber,and to analyze its expression characteristics to provide reference for its functional research on cucumber leaf senescence.[Methods]The full length c DNA of CsWHY1 was cloned with North China cucumber‘9930'‘Changchunmici'and‘Xintaimici'as experimental materials. A series of bioinformatics software were used to speculate nucleotide sequence and protein function of CsWHY1. In addition,the subcellular localization was conducted by onion epidermal cells transient expression system,and gene expression pattern of CsWHY1 was investigated by RT-qPCR in different tissues of 3 cucumber varieties. [Results]The ORF of CsWHY1 gene was 831 bp,encoding 276 amino acids. The relative molecular mass of CsWHY1,a hydrophilic protein,was 30.729×10~3 with the theoretical isoelectric point( p I) of 9.00. Amino acid sequence analysis revealed that CsWHY1 protein had a conserved Whirly domain,and it was highly homologous in dicotyledonous and monocotyledonous plants and had the closest genetic relationship with melon. Subcellular localization analysis showed that CsWHY1 was located in the plastids of onion epidermal cell. RT-qPCR analysis showed that CsWHY1 expressed in different tissues of three cucumber varieties,with the highest expression level in leaves and the lower expression in roots and stems,and the expression level of CsWHY1 in aged leaves was significantly higher than that in mature leaves. [Conclusions]The expression of CsWHY1 was tissue-specific. CsWHY1 had a high expression level in senescent leaves and participated in the senescence process of leaves in cucumber.
引文
[1] Gan S,Amasino R M. Making sense of senescence(molecular genetic regulation and manipulation of leaf senescence)[J]. Plant Physiology,1997,113(2):313-319.
[2] Martin C,Thimann K V. The role of protein synthesis in the senescence of leaves:I. the formation of protease[J]. Plant Physiology,1972,49(1):64-71.
[3] Pastori G M,del Rio L A. Natural senescence of pea leaves(an activated oxygen-mediated function for peroxisomes)[J]. Plant Physiology,1997,113(2):411-418.
[4] Navabpour S,Morris K,Allen R,et al. Expression of senescence-enhanced genes in response to oxidative stress[J]. Journal of Experimental Botany,2003,54(391):2285-2292.
[5] Grabowski E,Miao Y,Mulisch M,et al. Single-stranded DNA-binding protein Whirly1 in barley leaves is located in plastids and the nucleus of the same cell[J]. Plant Physiology,2008,147(4):1800-1804.
[6] Desveaux D,Després C,Joyeux A,et al. PBF-2 is a novel single-stranded DNA binding factor implicated in PR-10a gene activation in potato[J]. The Plant Cell,2000,12(8):1477-1489.
[7] Krause K,Kilbienski I,Mulisch M,et al. DNA-binding proteins of the Whirly family in Arabidopsis thaliana are targeted to the organelles[J].FEBS Letters,2005,579(17):3707-3712.
[8] Desveaux D,Subramaniam R,Després C,et al. A“Whirly”transcription factor is required for salicylic acid-dependent disease resistance in Arabidopsis[J]. Developmental Cell,2004,6(2):229-240.
[9]姚沁涛,张文蔚,刘莉,等. Whirly转录因子对非寄主菌诱导水稻HR反应的负调控作用[J].中国农业科技导报,2008,10(5):53-58.Yao Q T,Zhang W W,Liu L,et al. Negative regulation of rice Whirly transcription factor for the hypersensitive response induced by non-host pathogen bacterium[J]. Journal of Agricultural Science and Technology,2008,10(5):53-58(in Chinese with English abstract).
[10] Maréchal A,Parent J S,Sabar M,et al. Overexpression of mt DNA-associated At Why2 compromises mitochondrial function[J]. BMC Plant Biology,2008,8(1):42.
[11] Maréchal A,Parent J S,Véronneau-Lafortune F,et al. Whirly proteins maintain plastid genome stability in Arabidopsis[J]. Proc Natl Acad Sci USA,2009,106(34):14693-14698.
[12] Yoo H H,Kwon C,Lee M M,et al. Single-stranded DNA binding factor At WHY1 modulates telomere length homeostasis in Arabidopsis[J]. The Plant Journal,2007,49(3):442-451.
[13] Desveaux D,Maréchal A,Brisson N. Whirly transcription factors:defense gene regulation and beyond[J]. Trends in Plant Science,2005,10(2):95-102.
[14] Desveaux D,Allard J,Brisson N,et al. A new family of plant transcription factors displays a novel ss DNA-binding surface[J]. Nature Structural Biology,2002,9(7):512-517.
[15] Miao Y,Jiang J,Ren Y,et al. The single-stranded DNA-binding protein WHIRLY1 represses WRKY53 expression and delays leaf senescence in a developmental stage-dependent manner in Arabidopsis[J]. Plant Physiology,2013,163(2):746-756.
[16] Huang S,Li R,Zhang Z,et al. The genome of the cucumber,Cucumis sativus L.[J]. Nature Genetics,2009,41(12):1275.
[17] Wan H,Zhao Z,Qian C,et al. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber[J]. Analytical Biochemistry,2010,399(2):257-261.
[18]王洁如.番茄Le Why1低温胁迫下的功能分析及转录组分析[D].泰安:山东农业大学,2015.Wang J R. Functional analysis and RNA-sequencing of LeW hy1 in tomato under chilling stress[D]. Tai’an:Shandong Agricultural University,2015(in Chinese with English abstract).
[19]杨洋.三明野生蕉和天宝蕉WHIRLY基因克隆及其定量表达分析[D].福州:福建农林大学,2014.Yang Y. Cloning and expression of WHIRLY in the wild banana from Sanming City and Musa spp. cv. Tianbaojiao[D]. Fuzhou:Fujian Agriculture and Forestry University,2014(in Chinese with English abstract).
[20] Miao Y,Laun T,Zimmermann P,et al. Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis[J]. Plant Molecular Biology,2004,55(6):853-867.
[21] Janack B,Sosoi P,Krupinska K,et al. Knockdown of WHIRLY1 affects drought stress-induced leaf senescence and histone modifications of the senescence-associated gene HvS 40[J]. Plants,2016,5(3):37.
[22]梁秋霞,曹刚强,苏明杰,等.植物叶片衰老研究进展[J].中国农学通报,2006,22(8):282-285.Liang Q X,Cao G Q,Su M J,et al. Research progress on plant leaf senescence[J]. Chinese Agricultural Science Bulletin,2006,22(8):282-285(in Chinese with English abstract).
[2] Martin C,Thimann K V. The role of protein synthesis in the senescence of leaves:I. the formation of protease[J]. Plant Physiology,1972,49(1):64-71.
[3] Pastori G M,del Rio L A. Natural senescence of pea leaves(an activated oxygen-mediated function for peroxisomes)[J]. Plant Physiology,1997,113(2):411-418.
[4] Navabpour S,Morris K,Allen R,et al. Expression of senescence-enhanced genes in response to oxidative stress[J]. Journal of Experimental Botany,2003,54(391):2285-2292.
[5] Grabowski E,Miao Y,Mulisch M,et al. Single-stranded DNA-binding protein Whirly1 in barley leaves is located in plastids and the nucleus of the same cell[J]. Plant Physiology,2008,147(4):1800-1804.
[6] Desveaux D,Després C,Joyeux A,et al. PBF-2 is a novel single-stranded DNA binding factor implicated in PR-10a gene activation in potato[J]. The Plant Cell,2000,12(8):1477-1489.
[7] Krause K,Kilbienski I,Mulisch M,et al. DNA-binding proteins of the Whirly family in Arabidopsis thaliana are targeted to the organelles[J].FEBS Letters,2005,579(17):3707-3712.
[8] Desveaux D,Subramaniam R,Després C,et al. A“Whirly”transcription factor is required for salicylic acid-dependent disease resistance in Arabidopsis[J]. Developmental Cell,2004,6(2):229-240.
[9]姚沁涛,张文蔚,刘莉,等. Whirly转录因子对非寄主菌诱导水稻HR反应的负调控作用[J].中国农业科技导报,2008,10(5):53-58.Yao Q T,Zhang W W,Liu L,et al. Negative regulation of rice Whirly transcription factor for the hypersensitive response induced by non-host pathogen bacterium[J]. Journal of Agricultural Science and Technology,2008,10(5):53-58(in Chinese with English abstract).
[10] Maréchal A,Parent J S,Sabar M,et al. Overexpression of mt DNA-associated At Why2 compromises mitochondrial function[J]. BMC Plant Biology,2008,8(1):42.
[11] Maréchal A,Parent J S,Véronneau-Lafortune F,et al. Whirly proteins maintain plastid genome stability in Arabidopsis[J]. Proc Natl Acad Sci USA,2009,106(34):14693-14698.
[12] Yoo H H,Kwon C,Lee M M,et al. Single-stranded DNA binding factor At WHY1 modulates telomere length homeostasis in Arabidopsis[J]. The Plant Journal,2007,49(3):442-451.
[13] Desveaux D,Maréchal A,Brisson N. Whirly transcription factors:defense gene regulation and beyond[J]. Trends in Plant Science,2005,10(2):95-102.
[14] Desveaux D,Allard J,Brisson N,et al. A new family of plant transcription factors displays a novel ss DNA-binding surface[J]. Nature Structural Biology,2002,9(7):512-517.
[15] Miao Y,Jiang J,Ren Y,et al. The single-stranded DNA-binding protein WHIRLY1 represses WRKY53 expression and delays leaf senescence in a developmental stage-dependent manner in Arabidopsis[J]. Plant Physiology,2013,163(2):746-756.
[16] Huang S,Li R,Zhang Z,et al. The genome of the cucumber,Cucumis sativus L.[J]. Nature Genetics,2009,41(12):1275.
[17] Wan H,Zhao Z,Qian C,et al. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber[J]. Analytical Biochemistry,2010,399(2):257-261.
[18]王洁如.番茄Le Why1低温胁迫下的功能分析及转录组分析[D].泰安:山东农业大学,2015.Wang J R. Functional analysis and RNA-sequencing of LeW hy1 in tomato under chilling stress[D]. Tai’an:Shandong Agricultural University,2015(in Chinese with English abstract).
[19]杨洋.三明野生蕉和天宝蕉WHIRLY基因克隆及其定量表达分析[D].福州:福建农林大学,2014.Yang Y. Cloning and expression of WHIRLY in the wild banana from Sanming City and Musa spp. cv. Tianbaojiao[D]. Fuzhou:Fujian Agriculture and Forestry University,2014(in Chinese with English abstract).
[20] Miao Y,Laun T,Zimmermann P,et al. Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis[J]. Plant Molecular Biology,2004,55(6):853-867.
[21] Janack B,Sosoi P,Krupinska K,et al. Knockdown of WHIRLY1 affects drought stress-induced leaf senescence and histone modifications of the senescence-associated gene HvS 40[J]. Plants,2016,5(3):37.
[22]梁秋霞,曹刚强,苏明杰,等.植物叶片衰老研究进展[J].中国农学通报,2006,22(8):282-285.Liang Q X,Cao G Q,Su M J,et al. Research progress on plant leaf senescence[J]. Chinese Agricultural Science Bulletin,2006,22(8):282-285(in Chinese with English abstract).