摘要
RUBY报告系统是一种天然无害、观察方便、成本节约的新型筛选标记工具。为了提高黄瓜的遗传转化筛选效率,实现对阳性植株的裸眼筛查,将RUBY序列构建到表达载体,在黄瓜和烟草中进行了农杆菌介导的瞬时表达,利用蛋白质免疫印迹试验探究了该系统在黄瓜中无法工作的原因;通过优化载体、2A连接肽和RUBY序列,将优化后的载体进行黄瓜遗传转化的稳定测试。结果发现,原RUBY系统在黄瓜中无法正常使用,通过将RUBY系统中的P2A连接肽更替为F2A,优化了RUBY序列和去除GFP表达盒的抑制,并将优化载体pCambia1305.4-fRUBY-ΔGFP应用于黄瓜遗传转化后,成功诱导黄瓜中合成甜菜红素并获得了RUBY阳性植株。RUBY系统在黄瓜中的建立,为提高黄瓜的遗传转化筛选效率奠定了重要的技术基础。
The RUBY reporting system is a new type of screening marker tool that is natural,harmless,easy to observe,and cost-effective.To improve the selection efficiency of genetic transformation in cucumber and enable naked-eye screening of positive plants,the RUBY gene was constructed into an expression vector and expressed in cucumber and tobacco using Agrobacterium-mediated transient expression system.Western blot experiments were used to investigate the reasons why the system failed to work in cucumber.By optimizing the vector,2A peptide,RUBY sequence,the optimized vector was tested for stable genetic transformation in cucumber.The results showed that the original RUBY system could not be used normally in cucumber.By replacing the P2A with F2A,optimizing the RUBY sequence,and removing the suppression of the GFP expression cassette,the optimized vector pCambia1305.4-fRUBY-ΔGFP was applied to cucumber genetic transformation.This successfully induced the synthesis of betalain in cucumber and obtained RUBY transgenic positive plants.The establishment of the RUBY system in cucumber lays an important technical foundation for improving the selection efficiency of genetic transformation in cucumber.
作者
李雯静
李旭彤
辛同旭
柴森
王帅
杨学勇
LI Wenjing;LI Xutong;XIN Tongxu;CHAI Sen;WANG Shuai;YANG Xueyong(College of Horticulture,Qingdao Agricultural University,Qingdao 266109,Shandong,China;Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,State Key Laboratory of Vegetable Biobreeding,Beijing 100081,China)
出处
《中国蔬菜》
北大核心
2025年第3期46-54,共9页
China Vegetables
基金
中国农业科学院科技创新工程项目(CAAS-ASTIP)。
关键词
RUBY
黄瓜
遗传转化
无创筛选
甜菜红素
RUBY
cucumber
genetic transformation
noninvasive selection
betalain
作者简介
李雯静,女,硕士研究生,专业方向:农艺与种业,E-mail:liwenjing927@163.com;通信作者:杨学勇,男,博士,研究员,博士生导师,专业方向:蔬菜发育生物学,E-mail:yangxueyong@caas.cn;通信作者:王帅,女,博士,助理研究员,专业方向:蔬菜发育生物学,E-mail:757294336@qq.com。
