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光作为重要的环境因子控制着植物一系列生长发育过程,其调控作用是通过光受体介导的信号传导的途径来完成的[1]。目前,在拟南芥Arabidopsis thaliana中共发现了3种CRY(CRYPTOCHROME):CRY1,CRY2和CRY3,其中CRY1和CRY2通过负调控光形态建成的负调控因子COP1(COnstitutive Photomorphogenic 1)来实现其调控光形态建成的功能[2-3]。COP1作为一种重要的调控因子,广泛存在于动植物体内[4]。在植物中,COP1作为一种E3泛素连接酶,具有E3泛素连接酶活性使转录调控因子LAF1,HY5,HFR1等泛素化,并调控其降解,从而起到抑制光信号调节的作用,进而在光信号转导及植物光形态建成中起重要作用[5-7]。COP1在细胞核内充当一个特异转录因子的总的调节因子[8],通过与其互作因子相互结合而使这些光形态建成发育的正向调节子失活[9-11]。CO是光周期开花转换途径中一个最重要的基因[12],通过激活下游基因FT,SOC1等表达,使植物由营养生长向生殖发育转变[13-14]。COP1与CO存在直接的蛋白相互作用,并在体外作为泛素E3连接酶使CO蛋白发生泛素化,在植物体内促进CO蛋白的降解,而CRY介导的信号可能通过负调控COP1对CO蛋白的降解作用,使CO蛋白稳定并发生积累,从而激活下游基因FT的表达,促进开花[15-17]。山核桃Carya cathayensis是中国著名的特色干果,因其种仁中的不饱和脂肪酸成分对人体具有滋补、健脑等保健效果,得到了广大消费者的青睐[18]。成花是决定山核桃产量的关键因素,而木本植物需要较长时间的营养生长(童期)才转向生殖生长的[19]。在广泛的研究中,已通过利用分子生物学、遗传学等技术研究花的发育,极大地促进了成花机制的研究[20-21]。如何运用分子机制促进木本植物山核桃提早开花,为将来进一步阐释山核桃成花的分子机制提供了重要的基因平台,又为促进山核桃早花、早实、丰产等的实际生产应用推广提供必需的理论支持。山核桃全基因组测序工作的完成(数据未发表)得到CcCOP1 E3连接酶片段,利用cDNA末端快速扩增技术(RACE)技术克隆获得了CcCOP1 E3连接酶的全长;利用生物信息学手段分析了它的蛋白特性和不同物种的同源基因之间的亲缘关系;利用实时荧光定量聚合酶链式反应(real-time PCR)技术进一步分析了在CcCOP1 E3连接酶雌雄花发育过程中的表达变化。该研究旨在为研究该基因的生物学功能,为进一步阐明山核桃成花调控的分子机制打下良好的基础。
Cloning and expression analysis of COP1 E3 ligase in Carya cathayensis
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摘要: COP1 E3连接酶是一个光形态建成的抑制子和光调控植物发育的分子开关。对山核桃Carya cathayensis花芽454测序获得CcCOP1 E3连接酶的片段, 通过cDNA末端快速扩增技术(RACE), 分别获得该基因的全长, 大小为2 331 bp, 它由2个特殊的结构域组成即环形锌指结合域和WD-40重复序列, 其编码的蛋白质有较强的亲水性, 在氨基端主要是亲水性氨基酸, 而羧基端主要是疏水性氨基酸。CcCOP1 E3连接酶与毛果杨Populus trichocarpa等的COP1 E3连接酶同源基因相似度较高, 总体高达77.80%。实时荧光定量聚合酶链式反应(real-timePCR)结果显示:CcCOP1 E3连接酶的表达贯穿于在山核桃雌雄花的发育过程, CcCOP1 E3连接酶在山核桃的茎、叶、果实、花芽中均有表达, 但在花芽中表达量最高, 3月中旬表达量最高, 在5月中旬雄花表达量相对较高。CcCOP1 E3连接酶与山核桃雌雄花分化有关。Abstract: COP1 E3 ligase is a photomorphogenic repressor, a molecular switch for light control in plant development. A fragment of CcCOP1 E3 ligase for Carya cathayensis (hickory) was obtained with 454 sequencing. Then, the rapid amplification of cDNA ends (RACE) method was used to obtain the full length of the COP1 E3 ligase gene. Analysis was conducted with reverse transcription polymerase chain reaction (RT-PCR) and included a comparison between CcCOP1 E3 ligase and COP1 E3 ligase. Results showed that the full-length of COP1 E3 ligase was 2 331 bp and consisted of two special structural domains:an annular zinc finger domain and WD-40 repeat sequences with the coding protein being strongly hydrophilic. The end of the amino was mainly hydrophilic amino acids; whereas, the end of the carboxyl was mostly hydrophobic amino acids. The identity between CcCOP1 E3 ligase and COP1 E3 ligase in Populus trichocarpa reached 77.8%. Results of the RT-PCR illustrated that expression of CcCOP1 E3 ligase in hickory occurred throughout the development of staminate and pistillate flowers. The gene expressed in stems, leaves, fruits, and flower buds had the highest expression in the flower bud with a peak in the middle of March; however, the male flower bud had its peak in the middle of May. Expression for the staminate flower peaked on March 12th and for the pistillate flower in the middle of May. Thus, it was supposed that COP1 E3 ligase was relevant to the differentiation of pistillate and staminate flowers in hickory.
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Key words:
- cash forestry /
- Carya cathayensis /
- CcCOP1 E3 ligase /
- RACE /
- sequence analysis /
- expression analysis
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链接本文:
https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2014.06.002