关键词: Gateway技术, TA克隆, 入门克隆, 原核表达, 拟南芥原生质体瞬时表达

Abstract:

Gateway technology is a universal cloning approach that enables rapid cloning of DNA fragments into multiple Gatewaycompatible destination vectors using λ phage sitespecific recombination, eliminating the requirement to work with restriction enzymes and ligase. But a problem using this system for making entry clone is the expensiveness and long time to buy the enzyme. To solve this problem, we created the TA cloning entry vector that contained a Ttail in each 3’end through modification of pDONR207. The TA cloning approach can construct entry clones simply, economically and rapidly. Using Gateway T vectors prepared by this improved method, prokaryotic expression vector and eukaryotic expression vector for SOS2 gene were constructed. Through the methods of prokaryotic expression and transient gene expression in Arabidopsis protoplasts, it proved that the SOS2 gene expressed well in both prokaryotic cells and eukaryotic cells.

Key words: Gateway technology, TA cloning, Entry clone, Prokaryotic expression, Transient gene expression in Arabidopsis protoplasts