甜樱桃PavMYC2基因克隆与表达分析  

Cloning and expression analysis of PavMYC2 gene in Prunus avium L.

作　　者：王继源 王丽 纠松涛 孙菀霞 徐岩 刘勋菊 张才喜[1] WANG Jiyuan;WANG Lia;JIU Songtao;SUN Wanxia;XU Yan;LIU Xunju;ZHANG Caixi(School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学农业与生物学院,上海200240

出　　处：《果树学报》2022年第5期701-711,共11页Journal of Fruit Science

基　　金：现代农业产业技术体系专项资金(CARS-30-5-02);国家自然科学基金青年科学基金(32102347)。

摘　　要：【目的】克隆甜樱桃PavMYC2基因并研究其表达模式,为深入研究其在花芽响应温度逆境中的功能奠定基础。【方法】从甜樱桃基因组数据库中获得PavMYC2基因的序列,对该基因进行生物信息学分析和亚细胞定位;通过qRT-PCR技术探究PavMYC2基因在甜樱桃不同组织及花芽各个时期的表达模式;运用双分子荧光互补实验(BiFC)检测其与PavJAZ蛋白的互作关系;结合前人转录组结果分析PavJAZ的季节表达模式。【结果】PavMYC2编码689个氨基酸,具有bHLH-Zip保守结构域,属于bHLH家族。亚细胞定位结果表明,该基因在细胞核中发挥功能。进化树结果表明,甜樱桃PavMYC2与中国樱桃(Prunus pseudocerasus)和樱花(Prunus yedoensis)亲缘关系最近。实时荧光定量PCR结果分析发现,PavMYC2的表达具有组织特异性,在花芽中表达量最高,依次是叶、茎、花、根中表达量的4.8、4.9、8.8、37.4倍。在花芽中,PavMYC2的表达量在夏秋季高,然后逐渐降低,在冬季维持一定的表达水平,春季时最低。顺式作用元件分析发现,该基因启动子上含有大量光响应元件、多种激素响应元件和低温胁迫等相关元件。互作蛋白结果显示,PavMYC2可以与PavJAZ1/2/3蛋白发生互作,进一步对JAZs基因的表达模式进行分析,发现PavJAZ1/2/3/5与PavMYC2的表达量变化相似。【结论】克隆得到1个PavMYC2基因,夏秋季高温时期在花芽中高表达,随后逐渐降低,在冬季维持一定的表达水平,春季开花时最低。其与PavJAZ1/2/3互作,协同响应温度胁迫。该研究为进一步探究甜樱桃花芽中MYC2在响应温度胁迫和调控开花进程中的作用奠定了基础。【Objective】Sweet cherry(Prunus avium L.)is an important economic fruit crop in temperate regions of the world.Temperature stress is closely involved in flower bud differentiation.JA-activated transcription factor MYC2,which is the most extensively studied components in the JA signaling pathway,plays a regulatory role in temperature stress.However,the MYC2 gene has been less studied in sweet cherry.In the present research,PavMYC2 gene was cloned successfully,and the functional localization of MYC2 gene was identified.Moreover,its expression pattern during organogenesis,dormancy and flowering stages was analyzed.【Methods】The sweet cherries Royal Lee and Hongdeng were selected for the current experiment,which were grafted on Chinese cherry rootstock(P.pseudocerasus Lindl.‘Daqingye’).Royal Lee is a low chilling requirement cultivar from the breeding program of low-chill sweet cherries in California,USA,and Hongdeng is a high chilling requirement cultivar from China.Both cultivars were grown in the experimental farm at Shanghai Jiao Tong University in Shanghai(121.48°E,31.25°N).Floral buds of sweet cherries were collected on 15 July,15 August,15 September,15 October,15 November and 15 December in 2019;and 15 January,5 February,5 March,10 March and 15 March in 2020.All materials were collected for three biological replicates.These buds were frozen in liquid nitrogen and stored at-80℃before RNA extraction.According to the manufacturer’s instructions,total RNA was extracted using an RNAprep purePlant Kit(TianGen,China).To isolate the full-length cDNA of these genes,1μg of total RNA was converted into cDNA using PrimeScriptTMII1st Strand cDNA Synthesis Kit(TaKaRaBiotechnology,Dalian,China)and was subsequently diluted five times with sterile water.Primers were designed using Primer 5 software.The PCR-products were cloned into the p EASY?-Blunt Cloning Vector(TransGen Biotech,Beijing,China),and then sequenced.Phylogenetic and molecular evolutionary analysis were conducted using MEGA version 6.To generate

关 键 词：甜樱桃 花芽 温度胁迫 MYC2 

分 类 号：S662.5[农业科学—果树学]