YUAN Gaoya,SHEN Jia,ZHAO Yu,et al.Cloning and expression analysis of the single-stranded DNA binding protein gene CsWHY1 in cucumber[J].Journal of Nanjing Agricultural University,2019,42(2):246-252.[doi:10.7685/jnau.201804038]





Cloning and expression analysis of the single-stranded DNA binding protein gene CsWHY1 in cucumber
袁高雅 沈佳 赵宇 王星 张璐 李季 陈劲枫
南京农业大学园艺学院/作物遗传与种质创新国家重点实验室, 江苏 南京 210095
College of Horticulture/State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China
cucumberCsWHY1 geneleaf senescencegene cloneexpression analysis
[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cDNA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30.729×103,理论等电点(pI)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 cDNA 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 30.729×103 with the theoretical isoelectric point(pI) 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.


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