Wakasa Yuhya, Takaiwa Fumio
Transgenic Crop Research and Development Center, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
Methods Mol Biol. 2012;847:467-79. doi: 10.1007/978-1-61779-558-9_36.
A mutated rice acetolactate synthase (mALS) gene expressed under the control of the rice callus-specific promoter (CSP) (CSP:mALS) becomes a useful selectable marker for producing transgenic rice seed with higher accumulation of recombinant protein. When amounts of transgene products in mature seeds are compared between transgenic rice lines containing the CSP:mALS selection gene cassette and those with the hygromycin phosphotransferase (HPT) gene under the control of the CaMV 35S promoter (35S:HPT), the former transgenic rice seeds usually resulted in 1.2- to 2-fold higher accumulation of transgene products than in the latter. It is considered that specific expression of a selection marker gene at the selection stage may allow enhanced transgene products in seeds.This chapter represents a highly efficient Agrobacterium-mediated rice transformation system using the CSP:mALS gene cassette in place of the conventional constitutive selection using bacterial antibiotics. This selection stage specific expression using a rice-derived selection marker mALS will be especially beneficial for developing transgenic rice seeds accumulating bioactive proteins or peptides contributing to human health promotion; pharmaceutical products such as vaccine, antibodies, and biopharmaceuticals; and industrial enzymes.
在水稻愈伤组织特异性启动子（CSP）控制下表达的突变型水稻乙酰乳酸合酶（mALS）基因（CSP:mALS），成为生产重组蛋白积累量更高的转基因水稻种子的有用选择标记。当比较含有CSP:mALS选择基因盒的转基因水稻品系与在花椰菜花叶病毒35S启动子（35S:HPT）控制下含有潮霉素磷酸转移酶（HPT）基因的转基因水稻品系成熟种子中转基因产物的量时，前者转基因水稻种子中转基因产物的积累量通常比后者高1.2至2倍。据认为，选择阶段选择标记基因的特异性表达可能会使种子中转基因产物增加。本章介绍了一种高效的农杆菌介导的水稻转化系统，该系统使用CSP:mALS基因盒代替传统的使用细菌抗生素的组成型选择。使用源自水稻的选择标记mALS进行这种选择阶段特异性表达，对于开发积累有助于促进人类健康的生物活性蛋白或肽的转基因水稻种子、疫苗、抗体和生物制药等药品以及工业酶将特别有益。
