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’,T_(0)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。
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 tomato variety Money Maker by Agrobacterium-mediated method.The seeds from self-crossed T_(0) generation plants displayed bright red fluorescence or not under fluorescence microscope.Further verification with PCR molecular marker showed that TagRFP sequence was present in the seedlings arising from fluorescent seeds,indicating 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.
作者
章力
魏凯
李珊珊
宁宇
路菲菲
王孝宣
国艳梅
刘磊
李鑫
杜永臣
李君明
黄泽军
Zhang Li;Wei Kai;Li Shanshan;Ning Yu;Lu Feifei;Wang Xiaoxuan;Guo Yanmei;Liu Lei;Li Xin;Du Yongchen;Li Junming;Huang Zejun(Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences/State Key Laboratory of Vegetable Biobreeding,Beijing 100081,China;College of Horticulture,China Agricultural University,Beijing 100081,China)
出处
《山东农业科学》
北大核心
2024年第4期1-8,共8页
Shandong Agricultural Sciences
基金
国家重点研发计划项目(2022YFD1200500)
国家大宗蔬菜产业技术体系项目(CARS-23-A06)
国家自然科学基金项目(31872949)。
关键词
番茄
种子
红色荧光蛋白
油体蛋白
转基因鉴定
Tomato
Seed
Red fluorescence protein
Oleosin protein
Transgenic identification
