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| 铁皮石斛DobHLH33转录因子启动子的克隆及表达分析 |
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杨莎1,2，黄园1,2,3，陈春伶1,2,3，王健1,2，陈晓凯1,2，陈宏宇1,2，孙庆文1,2,3
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1.贵州中医药大学 药学院，贵州 贵阳 550025;2.贵州省中药民族药材种质资源保存及评价工程研究中心，贵州 贵阳 550025;3.贵州中医药大学 药用植物抗逆种质研究中心，贵州 贵阳 550025
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| 摘要: |
| 为初步探究碱性螺旋-环-螺旋（basic helix-loop-helix， bHLH）家族成员DobHLH33基因在铁皮石斛（Dendrobium officinale Kimura et Migo）中的生物学功能，本研究从铁皮石斛中克隆了DobHLH33基因的启动子序列。结果显示，克隆出DobHLH33基因起始密码子上游启动子序列1 821 bp，经PlantCARE预测分析该序列包含逆境胁迫顺式作用元件、光响应元件、激素响应元件、TATA-box、CAAT-box和CAAT-box核心启动子元件等。构建了DobHLH33启动子的β-葡萄糖苷酸酶（β-glucuronidase， GUS）融合表达载体并通过花序浸染获得拟南芥转基因植株。GUS染色结果显示，该启动子能够驱动GUS基因在幼苗及各器官部位叶、花、果、柱头、花粉和叶脉中表达，其中在幼苗中表达最强；转基因幼苗在甘露醇和氯化钠胁迫下，DobHLH33启动子活性受诱导，转基因幼苗GUS染色明显加深。综上所述，DobHLH33可能参与铁皮石斛生长发育以及高盐和渗透胁迫响应，为铁皮石斛bHLH家族的功能研究提供基础和参考依据。 |
| 关键词: 铁皮石斛 启动子 DobHLH33基因 组织化学染色 逆境胁迫 |
| DOI：10.14188/j.ajsh.2024.01.010 |
| 分类号:Q81 |
| 基金项目:国家自然科学基金项目（82260740）；贵州省科技计划项目（黔科合基础-ZK［2021］522）；2019年博士启动基金项目（贵中医博士启动［2019］12号）；贵州省梵净山地区生物多样性保护与利用重点实验室开放课题基金资助（［2020］2003）；2020年博士启动基金项目（贵中医博士启动［2020］08号）；2023年省级财政种业发展项目“贵州药用植物种质资源库建设” |
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| Analysis on cloning and expression of DobHLH33 transcription factor promoter from Dendrobium officinale |
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YANG Sha1,2, HUANG Yuan1,2,3, CHEN Chunling1,2,3, WANG Jian1,2, CHEN Xiaokai1,2, CHEN Hongyu1,2, SUN Qingwen1,2,3
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1.School of Pharmacy， Guizhou University of Traditional Chinese Medicine， Guiyang 550025， Guizhou， China;2.Guizhou Engineering Research Center for Conservation and Evaluation of Germplasm Resources of Traditional Chinese Medicine， Guiyang 550025， Guizhou， China;3.Research Center for Medicinal Plant Stress Resistance Germplasm， Guizhou University of Traditional Chinese Medicine， Guiyang 550025， Guizhou， China
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| Abstract: |
| To investigate the biological function of DobHLH33 gene in Dendrobium officinale， a member of the basic helix-loop-helix （bHLH） family， the promoter sequence of DobHLH33 gene was cloned from Dendrobium officinale.The results showed that the 1 821 bp upstream promoter sequence of the start codon of DobHLH33 gene was cloned， which was predicted by PlantCARE to contain stress cis-acting elements， light responsive elements， hormone responsive elements， TATA-box， CAAT-box and CAAT-box core promoter elements. The GUS fusion expression vector of DobHLH33 promoter was constructed and the transgenic Arabidopsis thaliana plants were obtained by inflorescence impregnation. GUS staining results showed that the promoter could activate GUS gene expressed in seedlings and various organ parts such as leaves， flowers， fruits， stigma， pollen and leaf veins， with the strongest expression in seedlings. Under the stress of mannitol and NaCl， the promoter activity of DobHLH33 was induced and GUS staining of transgenic seedlings was significantly enhanced. In conclusion， DobHLH33 may be involved in the growth and development of Dendrobium officinale， as well as its response to high salt and osmotic stress. This study provides the basis and reference for the functional research of the bHLH family of Dendrobium officinale. |
| Key words: Dendrobium officinale promoter DobHLH33 histochemical staining adversity stress |
