‘红地球’葡萄VvARF18功能分析  

Functional Analysis of VvARF18 Gene in Red Globe Grape

作　　者：袁苗 周娟[1] 党仕卓 汤学燊 张亚红[1] YUAN Miao;ZHOU Juan;DANG ShiZhuo;TANG XueShen;ZHANG YaHong(College of Enology and Horticulture,Ningxia University,Yinchuan 750021)

机构地区：[1]宁夏大学葡萄酒与园艺学院,银川750021

出　　处：《中国农业科学》2024年第7期1363-1376,共14页Scientia Agricultura Sinica

基　　金：宁夏回族自治区重点研发计划(2021BEF02016)。

摘　　要：【目的】生长素响应因子ARF是生长素信号转导途径中的重要调控因子,在植物生长发育和各类生理过程中发挥着重要作用。分析‘红地球’葡萄VvARF18启动子、异源表达、内源激素含量及其对激素响应的表达,以探究VvARF18在‘红地球’葡萄生长素(IAA)信号转导途径及花芽分化进程中的作用机理。【方法】以设施‘红地球’葡萄花芽为试验材料,通过同源克隆获得VvARF18序列,利用在线数据库PLACE分析启动子的顺式作用元件。以pCAMBIAI2300植物表达载体为基础,通过双酶切和同源重组法构建植物超表达载体pC2300-VvARF18。采用电击法将重组载体pC2300-VvARF18转化至根癌农杆菌GV3101菌株中,以本氏烟草叶片为外植体,通过农杆菌介导的愈伤组织转化法转入烟草中,经PCR检测获得阳性转基因幼苗。利用实时荧光定量PCR(qRT-PCR)对转VvARF18烟草株系表达水平进行分析,筛选出高表达量的转基因株系培养至T_(3)代,并分别进行IAA和GA_(3)处理,以分析VvARF18的表达情况。通过酶联免疫法测定转基因烟草花芽和叶片中的IAA、GA、ABA、CTK含量。【结果】‘红地球’葡萄VvARF18位于第13条染色体,含有3个外显子和2个内含子。VvARF18启动子区域存在多种光响应、植物激素响应和逆境响应的顺式作用元件。表型分析发现,转基因烟草的花芽分化进程快于野生型烟草。qRT-PCR结果显示,VvARF18在转基因烟草花芽发育的4个时期中呈先上升后下降的表达趋势,且S3时期表达量达到最高。转基因烟草植株花芽和叶片中IAA、CTK、GA和ABA测定结果表明,转基因烟草花芽和叶片中4种植物激素的含量均高于野生型植株,其中GA/IAA在转基因烟草花芽发育的4个时期中变化趋势与VvARF18表达趋势相一致。转基因烟草植株经IAA和GA_(3)处理后,VvARF18的表达量随IAA处理浓度的增高而降低,也随GA_(3)处理时间的延长而降低。【结论】【Objective】Auxin response factor(ARF)is a significant regulatory factor in the auxin signaling pathway and plays an important role in plant growth and development as well as various physiological processes.Analysis of the Red Globe grape VvARF18 promoter,heterologous expression,endogenous hormone content and its expression in response to hormones was made in order to explore the mechanism of VvARF18 gene in the auxin(IAA)signaling pathway and flower bud differentiation process in Red Globe grapes.【Method】The VvARF18 gene sequence was obtained by homologous cloning by using facility Red Globe grape flower buds as experimental materials.The cis-acting elements of the promoter were analyzed using the online database PLACE.The plant overexpression vector pC2300-VvARF18 was constructed based on the pCAMBIAI2300 plant expression vector by double enzyme digestion and homologous recombination method.The recombinant vector pC2300-VvARF18 was transformed into Agrobacterium tumefaciens strain GV3101 by using electrical shock method.The tobacco leaves were used as explants and transferred into tobacco by Agrobacterium-mediated callus transformation method,and positive transgenic seedlings were obtained by PCR.The quantitative real-time PCR(qRT-PCR)was used to analyze the expression level of VvARF18 transgenic tobacco lines,and the transgenic lines with high expression level were screened and cultured to T_(3) generation,and treated with IAA and GA_(3) to analyze the expression level of VvARF18.The content of IAA,GA,ABA and CTK in flower buds and leaves of transgenic tobacco were determined by enzyme-linked immunosorbent assay.【Result】VvARF18 of Red Globe grape located on chromosome 13,and contained 3 exons and 2 introns.There are multiple cis-acting elements in the VvARF18 promoter region that respond to light,plant hormones,and stress.The phenotypic analysis found that the process of flower bud differentiation was faster in transgenic tobacco than in wild-type tobacco.The qRT-PCR results showed that the expressi

关 键 词：‘红地球’葡萄 VvARF18 转基因植株 花芽分化 植物激素 

分 类 号：S663.1[农业科学—果树学]