中国农业科学 ›› 2012, Vol. 45 ›› Issue (13): 2690-2703.doi: 10.3864/j.issn.0578-1752.2012.13.013

• 园艺 • 上一篇    下一篇

菊花节律钟输出基因CmGI(GIGANTEA)的cDNA全长克隆、序列信息及定量表达分析

 孙霞, 王秀峰, 郑成淑, 邢世岩, 束怀瑞   

  1. 1.山东农业大学园艺科学与工程学院,山东泰安 271018
    2.山东农业大学林学院,山东泰安 271018
    3.作物生物学国家重点实验室,山东泰安 271018
    4.国家苹果工程技术中心,山东泰安 271018
  • 收稿日期:2011-12-18 出版日期:2012-07-01 发布日期:2012-03-08
  • 通讯作者: 通信作者王秀峰,Tel:0538-82402456;E-mail:xfwang@sdau.edu.cn
  • 作者简介:孙 霞,E-mail:sunxia65@sina.com
  • 基金资助:

    国家自然科学基金项目(30872040)、山东省自然科学基金项目(ZR2009DM003)

The cDNA Cloning and Analysis of Sequence Information and Quantitative Express of Chrysanthemum Rhythms Clock Output Gene CmGI (GIGANTEA)

 SUN  Xia, WANG  Xiu-Feng, ZHENG  Cheng-Shu, XING  Shi-Yan, SHU  Huai-Rui   

  1. 1.山东农业大学园艺科学与工程学院,山东泰安 271018
    2.山东农业大学林学院,山东泰安 271018
    3.作物生物学国家重点实验室,山东泰安 271018
    4.国家苹果工程技术中心,山东泰安 271018
  • Received:2011-12-18 Online:2012-07-01 Published:2012-03-08

摘要: 【目的】克隆菊花节律钟输出基因GIGANTEA的cDNA全长序列,进行序列信息学分析,研究其mRNA的相对定量表达。【方法】利用多聚酶链式反应(PCR)结合5′RACE、3′RACE技术,克隆节律钟输出基因GIGANTEA的cDNA全长序列,应用生物信息学软件对获得的基因核苷酸序列及编码的蛋白质序列进行分析;通过在线建模软件对蛋白质的三维结构进行建模预测;利用实时荧光定量PCR技术,用2-△△Ct法进行GIGANTEA的mRNA相对定量表达分析。【结果】从菊花品种‘Jinba’中克隆得到节律钟输出基因GIGANTEA的cDNA全长序列,核苷酸序列长度3 461 bp,开放阅读框3 453 bp,编码1 150个氨基酸。氨基酸序列分析显示,该基因编码的蛋白与植物节律钟输出基因GIGANTEA同源,命名为CmGI基因,序列提交到GenBank,登录号为JQ043439。序列比对显示与葡萄、蓖麻等的GI的相似度依次为76%、75%。构建类似蛋白系统进化树显示,菊花CmGI与拟南芥(Arabidopsis thaliana GIGANTEA,ABP96482.1)分子进化距离最近,其次是白菜(Brassica rapa GIGANTEA,AEB33730.1);预测CmGI蛋白有6个跨膜螺旋多次跨膜;为转录因子,定位在细胞核中,为非分泌性蛋白质;不具备信号肽;对CmGI三级结构建模预测表明,蛋白核心结构符合转录因子与DNA结合常见的功能域HTH、HLH;采用荧光相对定量分析,菊花CmGI的表达呈昼夜节律表达模式;不同花芽分化阶段叶片中CmGI基因mRNA水平差异大,两个高峰值分别出现在花芽分化启动期和小花原基分化中期;营养生长的组培苗、长日照条件下的叶、芽、花蕾期均是痕量表达;盛花期表达量依次为叶片>舌状花>筒状花。【结论】从菊花中克隆得到节律钟输出基因CmGI,对该基因的进一步深入研究有助于探索光周期途径菊花成花的分子调控机制,可作为切花菊花期调控分子育种的目标基因。

关键词: 菊花, 花芽分化, 节律钟输出基因CmGI, 定量表达

Abstract: 【Objective】The cDNA sequence of chrysanthemum rhythms clock output gene GIGANTEA was cloned, and the bioinformatics of the sequence and the relative quantitative expression of mRNA were analyzed.【Method】 Polymerase chain reaction (PCR) combined with 5′RACE, and 3′RACE technology were used to clone the full length cDNA of chrysanthemum rhythms clock output gene CmGI,analysis of sequence of nucleotides and code of protein was made by using the software of bioinformatics. Protein structure prediction of 3D modeling was made by using the online modeling software. The relative quantitative expression analysis of CmGI was conducted by real-time quantitative fluorescence PCR technology and 2-△△Ct method. 【Result】The cDNA sequence of GIGANTEA was cloned from chrysanthemum ‘Jniba’, the full-length cDNA was 3 461 bp, open reading frame (ORF ) was 3 453 bp, and encoded 1 150 amino acids. Sequence analysis showed that the genetic code of protein was homologous with plant rhythms clock output gene GIGANTEA, named CmGI gene. The sequence was submitted to GenBank, and the registration number is JQ043439. Sequence alignment displayed that it was a similarity of 76% and 75% with GIGANTEA of Vitis vinifera,Ricinus communis, respectively. The phylogenetic tree showed that chrysanthemum CmGI and Arabidopsis thaliana GIGANTEA are closest in molecular evolution distance, followed by Brassica rapa GIGANTEA. It was speculated that CmGI protein has six transmembrane spiral across a cell membrane many times. They are transcription factors, located in the nucleus and it is a non-secretory protein. They do not have a signal peptide. CmGI 3D structure modeling projections show that the protein core structure accords with the transcription factors and the function of the common DNA combining domain HTH and HLH. Fluorescent relative quantitative analysis shows that the expression patterns of chrysanthemum CmGI are circadian rhythms expression. At different flower bud differentiation stage, the CmGI gene in the leaf blade mRNA level is different, two peak values were appeared in the flower bud differentiation start-up and floret primordia middle differentiation periods. The tissue culture plantlets leaves and buds under long-day conditions, during alabastrum period are all trace expression and was in the order of leaves>tongue shape flower>tubular flowers during flowers blooming period. 【Conclusion】Rhythms clock output gene CmGI was cloned from chrysanthemum, further research on this gene will help exploration of photoperiod pathway of the flower of chrysanthemum molecular control mechanism and could be used as the target gene of molecular breeding of flowering phase.

Key words: Chrysanthemum morifolium, flower bud differentiation, rhythm clock output gene CmGI, quantitative expression