一个更加高效的组培筛选系统

在植物细胞的遗传转化系统中，筛选标记和筛选剂的选择至关重要。磺胺类除草剂作用于叶酸生物合成途径，能够抑制细胞的生长。而二氢蝶酸合酶（Dihydropteroate Synthase）对磺胺类物质不敏感，其编码的基因可以作为磺胺类除草剂的抗性基因使用。因此二氢蝶酸合酶基因和磺胺类除草剂也构成了一对转化系统中的筛选标记和筛选剂。不过，这一对筛选标记和筛选剂构建的转化系统的转化效率很低。最近，德国马普研究所的科研人员找到了导致转化效率低的原因。

通常，转化载体上的二氢蝶酸合酶基因sul会连接叶绿体定位信号肽序列，这主要是基于叶酸生物合成途径定位于叶绿体中的假设。而当研究人员将原来的RbcS叶绿体转运肽替换为一段酵母中的线粒体转运肽时，包含表达载体的细胞对磺胺类除草剂的抗性明显增强，且转化效率也得到了提升。研究人员利用这一策略构建了一系列改进的载体，这些载体可以很高效地在种子植物烟草和绿藻植物莱茵衣藻中生产转基因事件。烟草共转化实验结果表明，sul 基因比当前最高效的筛选标记nptII的筛选效率还要高。

这一结果为植物转化体系的优化提供了新的方向。

Plant Biotechnology Journal 25 August 2018

A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae

Author

Iman Tabatabaei Cristina Dal Bosco Marta Bednarska Stephanie Ruf Jörg Meurer Ralph Bock* *: Max-Planck-Institut für Molekulare Pflanzenphysiologie Germany

Abstract

The genetic transformation of plant cells is critically dependent on the availability of efficient selectable marker gene. Sulfonamides are herbicides that by inhibiting the folic acid biosynthetic pathway suppress the growth of untransformed cells. Sulfonamide resistance genes that were previously developed as selectable markers for plant transformation were baxxxxsed on the assumption that in plants the folic acid biosynthetic pathway resides in the chloroplast compartment. Consequently the Sul resistance protein a herbicide‐insensitive dihydropteroate synthase was targeted to the chloroplast. Although these vectors produce transgenic plants the transformation efficiencies are low compared to other markers. Here we show that this inefficiency is due to the erroneous assumption that the folic acid pathway is located in chloroplasts. When the RbcS transit peptide was replaced by a transit peptide for protein import into mitochondria the compartment where folic acid biosynthesis takes place in yeast much higher resistance to sulfonamide and much higher transformation efficiencies are obtained suggesting that current sul vectors are likely to function due to low‐level mistargeting of the resistance protein to mitochondria. We constructed a series of optimized transformation vectors and demonstrate that they produce transgenic events at very high frequency in both the seed plant tobacco and the green alga Chlamydomonas reinhardtii. Co‐transformation experiments in tobacco revealed that sul is even superior to nptII the currently most efficient selectable marker gene and thus provides an attractive marker for the

high‐throughput genetic transformation of plants and algae.