利用种子红色荧光标记鉴定转基因番茄后代
Identification of Tomato Transgenic Offspring with Red Fluorescence Marker of Seeds
章力 1魏凯 1李珊珊 2宁宇 2路菲菲 2王孝宣 2国艳梅 2刘磊 2李鑫 2杜永臣 2李君明 2黄泽军2
作者信息
- 1. 中国农业科学院蔬菜花卉研究所/蔬菜生物育种全国重点实验室,北京 100081;中国农业大学园艺学院,北京 100081
- 2. 中国农业科学院蔬菜花卉研究所/蔬菜生物育种全国重点实验室,北京 100081
- 折叠
摘要
转基因技术有助于番茄基因功能研究和遗传改良,其中转基因后代的筛选是一个非常重要但工作量大的环节.本研究基于番茄油体蛋白基因家族序列分析和番茄基因表达数据库SGN-TEA搜索结果,克隆了一个种子特异且高水平表达的油体蛋白基因SlOLE1(Solyc06g034040).利用无缝克隆的方法将红色荧光蛋白基因TagRFP的编码区序列插入到SlOLE1 基因的终止密码子前,形成一个嵌合基因SlOLE1-TagRFP.将嵌合基因插入到pBI121 双元载体,构建植物表达载体pSlOLE1-TagRFP,利用农杆菌介导法转化番茄品种'Money Maker',T0代植株的自交种子在荧光显微镜下呈现出明亮红色荧光或无荧光.PCR分子标记进一步验证发现,红色荧光种子萌发的幼苗均存在TagRFP序列,表明在种子阶段检测红色荧光筛选转基因番茄后代的准确率为 100%.由此,本研究建立了一种利用SlOLE1-TagRFP嵌合基因,可以通过种子红色荧光可视化分析,简单快速、低成本地鉴定转基因番茄后代的方法.
Abstract
Transgenic technology has contributed to gene functional studies and genetic improvement of tomato.The identification of transgenic offspring is a cost/labor-intensive and time-consuming process.We cloned a seed-specific and highly expressed oleosin gene SlOLE1(Solyc06g034040)according to the results of sequence analysis of tomato oleosin gene family and a search in the tomato gene expression database SGN-TEA.The coding region of the red fluorescent protein gene TagRFP was inserted into the upstream of the stop codon of SlOLE1 gene to produce a chimeric gene SlOLE1-TagRFP,which was then inserted into binary vector pBI121 to construct a plant expression vector pSlOLE1-TagRFP.The expression vector was introduced into to-mato variety Money Maker by Agrobacterium-mediated method.The seeds from self-crossed T0 generation plants displayed bright red fluorescence or not under fluorescence microscope.Further verification with PCR molecu-lar marker showed that TagRFP sequence was present in the seedlings arising from fluorescent seeds,indica-ting that the accuracy of identifying transgenic tomato progeny by red fluorescence at seed stage was 100%.Therefore,this study established a simple,fast and low-cost method for identifying transgenic tomato offspring through seed red fluorescence visualization analysis using SlOLE1-TagRFP chimeric gene.
关键词
番茄/种子/红色荧光蛋白/油体蛋白/转基因鉴定Key words
Tomato/Seed/Red fluorescence protein/Oleosin protein/Transgenic identification引用本文复制引用
基金项目
国家重点研发计划(2022YFD1200500)
国家大宗蔬菜产业技术体系建设项目(CARS-23-A06)
国家自然科学基金(31872949)
出版年
2024
国家科技期刊平台
NSTL
万方数据