Isolation and regeneration of transiently transformed protoplasts from gametophytic blades of the marine red alga Porphyra yezoensis
Isolation and regeneration of transiently transformed protoplasts from gametophytic blades of the marine red alga Porphyra yezoensis
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DOI：
10.2225/vol13-issue2-fulltext-7
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发表时间：
2010-03
影响因子：
2.7
通讯作者：
Megumu Takahashi;T. Uji;N. Saga;K. Mikami
中科院分区：
文献类型：
--
作者：
Megumu Takahashi;T. Uji;N. Saga;K. Mikami
尽管近年来利用基因枪技术在条斑紫菜（Porphyrayezoensis）中建立了瞬时基因表达系统，但在海洋红色植物中还没有建立稳定的转化系统。红藻遗传转化困难的原因之一是缺乏从配子体叶片中选择和分离转化细胞的系统。因此，为了建立稳定的条斑紫菜转化体系，我们开发了一种方法，通过该方法从粒子轰击配子体叶片制备瞬时转化的配子体细胞作为可再生原生质体。使用海洋细菌酶的混合物，原生质体的产量很高，如其他地方报道的，但是，这些原生质体没有发展。相反，从用尿囊素处理的配子体制备的原生质体正常发育，其中β-葡萄糖醛酸酶报告基因的过表达对原生质体的再生没有影响。因此，尿囊素在原生质体制备中的应用为实现配子体叶片转化细胞的高效分离和筛选提供了新的思路。
Despite the recent progress of transient gene expression systems in a red alga Porphyra yezoensis by particle bombardment, a stable transformation system has yet to establish in any marine red macrophytes. One of the reasons of the difficulty in genetic transformation in red algae is the lack of systems to select and isolate transformed cells from gametophytic blades. Thus, toward the establishment of the stable transformation system in P. yezoensis , we have developed a procedure by which transiently transformed gametophytic cells were prepared from particle bombarded-gametophytic blade as regeneratable protoplasts. Using mixture of marine bacterial enzymes, yield of protoplasts was high as reported elsewhere; however, these protoplasts did not develop. In contrast, protoplasts prepared from gametophytes treated with allantoin were normally developed, in which the overexpression of a β-glucuronidase reporter gene had no effect on the regeneration of protoplasts. Therefore, the use of allantoin in protoplast preparation sheds a new light on the realization of an efficient isolation and selection of study transformed cells from gametophytic blades.