植物基因操作系统研究组

基于重组酶的植物基因叠加开放系统

 

转基因作物产品的创制已经由传统的单一性状转变为多基因性状。传统方法通过杂交进行性状基因(转基因或非转基因)聚合,因转化基因数目的增多而费时费力。为减轻育种过程中这一巨大负担,可以将多个基因构建到一个载体上,减少分离位点。但是外源基因能否正常表达并稳定遗传,且顺利通过监管审批,是多基因同时转化面临的挑战。所以,我们提出一种新的策略通过位点特异性重组,把新DNA附加到已有DNA位点上从而减少分离位点的数目,且新转基因产品安全审批可在原有产品的基础上进行。我们在烟草中实现了:1)Bxb1-att重组酶系统介导基因叠加,和2)Cre-lox系统介导筛选标记的删除。大约有3% 的转化个体成功并准确地叠加了三个目的基因,且基因表达正常。总之,我们发展了一个在效率上可行的,且没有专利限制的开放系统,可用于多性状转基因作物创制。

An Open-Source System for In Planta Gene Stacking by Bxb1 and Cre Recombinases

The genetics modified crops with multiple transgenic traits have taken the place of single transgene trait rapidly. Traditional method stacking gene by crossing is a time and labor consuming work for plant breeder when multiple transgenes and non-transgenic elite traits are involved. To solve this problem, multiple transgenes are packed into a same construct to reduce the number of segregating loci. However, we are still facing the challenge to find a good integration event with ideal gene expression and genetically stable. Particularly, the old traits would likely have to undergo regulatory scrutiny again because of new integrations. We proposed a strategy by reducing the number of segregating loci through the site-specific appending of new DNA next to previous DNA. We have achieved the Bxb1 recombinase-mediated gene stacking and removed unneeded DNA and selectable marker by Cre-lox. The overall rate with precise integration and ideal gene expression is about 3% of total transformation events, which is reasonable for the development of commercial cultivars. Given that the system we proposed is not under patent, there is freedom for implementation.

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