猕猴桃Ac-miR160d启动子区的克隆及表达分析  

Cloning and expression analysis of Ac-miR160d promoter region of kiwifruit

作　　者：李哲馨 祝滢 蒋娟 决登伟 李洪雷 唐建民[1,2,3] 兰建彬 LI Zhexin;ZHU Ying;JIANG Juan;JUE Dengwei;LI Honglei;TANG Jianmin;LAN Jianbin(College of Landscape Architecture and Life Science/Institute of Special Plants,Chongqing University of Arts and Sciences,Chongqing 402160,China;Chongqing Special Plant Industry Collaborative Innovation Center,Chongqing 402160,China;Chongqing Key Laboratory of Economic Plant Biotechnology,Chongqing 402160,China)

机构地区：[1]重庆文理学院园林与生命科学学院/特色植物研究院,重庆402160 [2]重庆市特色植物产业协同创新中心,重庆402160 [3]重庆市经济植物生物技术重点实验室,重庆402160

出　　处：《经济林研究》2021年第3期18-24,共7页Non-wood Forest Research

基　　金：国家自然科学基金项目“MiR160d调控猕猴桃果实对灰霉病抗性的分子机制”(32001351);重庆市教委项目“基于代谢组解析避雨栽培对红阳猕猴桃果实品质的影响”(KJQN202001305)。

摘　　要：【目的】为深入研究猕猴桃miR160d转录调控机制提供线索,也为猕猴桃分子育种及品种改良提供参考。【方法】根据‘红阳’猕猴桃基因组信息,设计引物克隆miR160d(Ac-miR160d)前体基因及其启动子序列,并对其前体序列进行二级结构验证,对启动子序列进行保守结构域和顺式作用元件分析。同时,对猕猴桃果实进行非生物(NaCl、GA_(3))和生物(灰霉菌)胁迫处理,利用qRT-PCR技术检测miR160d在不同胁迫处理后0~6 d时的表达情况。【结果】获得的Ac-miR160d前体序列具有稳定的茎环折叠的RNA二级结构以及miR/miR*二聚体结构,Ac-miR160d启动子序列不仅具有TATA-box、CAAT-box基本元件,还包含参与激素(GA3、水杨酸、脱落酸、茉莉酸等)应答、厌氧响应、干旱诱导等逆境胁迫以及光响应等多种顺式作用元件。猕猴桃果实内AcmiR160d的表达分析结果显示:当猕猴桃果实受到盐胁迫时,Ac-miR160d的水平在胁迫后1 d时即发生显著下调,胁迫后3~6 d时下调更为明显;当猕猴桃果实受到赤霉素胁迫和灰霉菌侵染时,Ac-miR160d的水平出现先升高、后降低的趋势,在处理后1~2 d时急剧升高,随后逐渐降低。【结论】Ac-miR160d启动子与猕猴桃植物激素信号及胁迫信号响应密切相关。通过非生物和生物胁迫试验,证实Ac-miR160d参与猕猴桃植物激素信号响应,并在其抗逆境胁迫过程中发挥重要调控作用。【Objective】Provide clues for in-depth study of the transcriptional regulation mechanism of miR160 d,and also lay the foundation for molecular breeding and variety improvement in kiwifruit.【Method】According to the’Hongyang’kiwifruit genome information,primers were designed to clone the miR160 d precursor gene and its promoter sequence,and the precursor sequence was verified for secondary structure,and the promoter sequence was analyzed for conserved domains and cis-acting elements.The kiwifruit was also subjected to abiotic(NaCl,GA3)and biotic(Grey mold)stress treatments,and qRT-PCR technology was used to detect the expression of miR160d during 0-6 d of different stress treatments.【Result】The obtained Ac-miR160 d precursor sequence had a stable stem-loop folded RNA secondary structure and a miR/miR*dimer structure.The Ac-miR160 d promoter sequence not only had the basic elements of TATA-box and CAAT-box,but also included various cis-acting elements such as gibberellin,salicylic acid,abscisic acid,jasmonic acid and other hormone responses,anaerobic response,drought-induced stress and light response and other cis-acting elements.The expression analysis of Ac-miR160 d in kiwifruit after salt stress and Botrytis cinerea stress showed that when kiwifruit was subjected to salt stress,the level of Ac-miR160d was significantly down-regulated after 1 d treatment,and down-regulated 3-6 d continuously.When kiwifruit was infected by B.cinerea,the level of AcmiR160d first increased and then decreased.It increased sharply 1-2 days after infection,and then gradually decreases.【Conclusion】Ac-miR160d promoter is closely related to kiwi phytohormone signal and stress signal response.It has been confirmed by abiotic stress,biotic stress and hormone treatment that Ac-miR160d participates in kiwifruit phytohormonal signal response and plays an important regulatory role in its resistance to adversity stress.

关 键 词：猕猴桃 miR160d 启动子 克隆 表达分析 

分 类 号：S663.4[农业科学—果树学]