| 摘要: |
| 【目的】克隆钩藤(Uncaria rhynchophylla)中马钱苷酸甲基转移酶(loganic acid methyltransferase,LAMT)基因及其启动子，对UrLAMT基因的组织表达及其对生物和非生物胁迫的响应进行分析，并对UrLAMT及其启动子的生物信息学进行分析，为进一步研究UrLAMT转录调控奠定基础。【方法】基于钩藤的转录组数据设计引物，采用PCR从钩藤cDNA中克隆UrLAMT序列，并对其进行生物信息学分析；采用实时荧光定量PCR，分析UrLAMT在钩藤不同组织(根、茎、叶、花、果实、钩)中的表达，以及对外源茉莉酸甲酯(MeJA)、遮光/复光胁迫的响应；利用FPNI-PCR技术克隆UrLAMT上游启动子序列，并验证其活性，结合酵母单杂交试验，分析相应转录因子与UrLAMT启动子的调控关系。【结果】克隆得到钩藤UrLAMT序列，其长度为1 137 bp，共编码378个氨基酸。UrLAMT蛋白质相对分子质量为42.64 ku，理论等电点为5.76，为亲水性蛋白，无信号肽，不含跨膜结构，定位在细胞质中；UrLAMT基因在钩藤叶中表达量最高，其次为根；UrLAMT均能响应MeJA和光；其与短小蛇根草和长春花的进化关系较近。UrLAMT启动子序列长度为1 141 bp，除核心响应元件外还含有多个光响应元件，UrLAMT启动子在酵母中与光调控转录因子HY5存在相互作用。【结论】获得了UrLAMT基因及其启动子序列，其在钩藤叶中表达量最高，可能受光诱导。 |
| 关键词: 钩藤 马钱苷酸甲基转移酶 基因克隆 启动子克隆 酵母单杂交 |
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| 基金项目:贵州省科技计划重大专项(黔科合平台人才［2017］5411-06)；国家喀斯特石漠化防治工程技术研究中心建设项目(2012FU125X13)；贵州省中药材现代产业技术体系建设项目(GZCYTX-02)；贵州省生物学一流学科建设项目(GNYL［2017］009FX1KT02) |
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| Cloning and analysis of UrLAMT gene and its promoter in Uncaria rhynchophylla |
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LI Yongquan,LIU Miao,SHANGGUAN Liyang,et al
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| Abstract: |
| 【Objective】This study cloned Uncaria rhynchophylla horse money nucleoside acid methyltransferase (loganic acid methyltransferase,LAMT) gene and promoter,analyzed UrLAMT gene expression and its biological and abiotic stress response,and conducted bioinformatics analysis on UrLAMT and its promoter to improve further study of UrLAMT transcriptional regulation.【Method】Primers were designed based on the transcriptome data of Uncaria rhynchophylla,UrLAMT sequences were cloned by PCR from cDNA,and bioinformatics analysis was performed.Quantitative real-time fluorescence PCR (qRT-PCR) was used to analyze its expression in different tissues (root,stem,leaf,flower,fruit and hook) and its response to exogenous methyl jasmonate (MeJA) and shading/relighting stress.The upstream promoter sequence of UrLAMT was cloned by FPNI-PCR and its activity was verified.The regulatory relationship between the corresponding transcription factors and the UrLAMT promoter was analyzed by yeast single hybridization test.【Result】The sequence length of UrLAMT was 1 137 bp,encoding 378 amino acids.The relative molecular weight of UrLAMT protein was 42.64 ku,and the theoretical isoelectric point was 5.76.It was a hydrophilic protein,without signal peptide and transmembrane structure,and it was located in cells.The expression of UrLAMT gene was the highest in leaves,followed by roots.UrLAMT was responsive to MeJA and light.It was closely related to the evolution of Ophiorrhiza pumila and Catharanthus roseus.The sequence length of UrLAMT promoter was 1 141 bp,containing multiple photo responsive elements in addition to core response elements.UrLAMT promoter interacted with photo-regulated transcription factor HY5 in yeast.【Conclusion】The UrLAMT gene and its promoter sequence were obtained.The expression level of UrLAMT gene was the highest leaves,which was possibly induced by light. |
| Key words: Uncaria rhynchophylla loganic acid methyltransferase gene cloning promoter cloning yeast one hybrid |
