Construction of Plastid Expression Vector and Development of Genetic Transformation System for the Seaweed Pyropia yezoensis
Construction of Plastid Expression Vector and Development of Genetic Transformation System for the Seaweed Pyropia yezoensis
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海藻条斑紫菜质体表达载体的构建及遗传转化体系的开发
DOI：
10.1007/s10126-017-9736-x
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发表时间：
2017-02
影响因子：
3
通讯作者：
Mao YX
中科院分区：
文献类型：
--
作者：
Kong Fanna;Zhao Hailong;Liu Weixun;Li Na;Mao Yunxiang;Kong Fanna;Zhao Hailong;Liu Weixun;Li Na;Mao Yunxiang;Kong FN;Mao YX;Kong FN;Mao YX
条斑紫菜属于红藻门，是一种重要的经济海藻。在本研究中，我们开发了条斑紫菜高效质体转化平台。在质体转化载体中，使用条斑鱼的psbA UTR，包括启动子和3'UTR，用于表达外源基因。整合位点是 rrsB 和 trnI 基因之间的转录活性基因间区域，位于质体基因组的反向重复区域。 CAT 和 eGFP 基因在该位点整合到质体基因组中。转化体中CAT的表达通过氯霉素乙酰转移酶的作用赋予氯霉素抗性，从而使药物失活，从而使植物在选择压力下良好生长。在转化细胞和转化子中也观察到了eGFP荧光信号。经处理的细胞的平均存活率估计约为 4.2‰（每 1000 个配子体细胞 4 个转质体集落）。多重 PCR 分析证实 CAT 和 eGFP 基因成功整合到 rrsB 和 trnI 之间的位点。 Western blot 还显示转化子细胞中 eGFP 表达。因此，本研究提出了第一个便捷的条斑紫菜质体基因表达系统，为研究条斑紫菜基因功能提供了重要的平台。
Pyropia yezoensis, belonging to the Rhodophyta, is an economically important seaweed. In this study, we developed a high-efficiency plastid transformation platform for P. yezoensis. In the plastid transformation vector, psbA UTR of P. yezoensis, including the promoter and 3′ UTR, was used to express foreign genes. The integration site was a transcriptionally active intergenic region between the rrsB and trnI genes, located in the inverted repeat regions of the plastid genome. The CAT and eGFP genes were integrated into the plastid genome at this site. The expression of CAT in the transformants confers resistance to chloramphenicol through the action of chloramphenicol acetyltransferase, which inactivates the drug, thereby allowing the plant to grow well under selective pressure. The eGFP fluorescence signal was also observed in transformed cells and the transformants. The average survival rate of treated cells was estimated to be approximately 4.2‰ (4 transplastomic colonies per 1000 gametophyte cells). Multiple-PCR analyses confirmed that the CAT and eGFP genes were successfully integrated in the site between rrsB and trnI. Western blot also showed eGFP expression in the cells of transformants. Thus, this study presents the first convenient plastid gene expression system for P. yezoensis and provides an important platform for studying gene function in P. yezoensis.
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影响因子：
3
作者：
Hirata, Ryo;Takahashi, Megumu;Mikami, Koji
通讯作者：
Mikami, Koji
影响因子：
3.5
作者：
Muto M;Henry RE;Mayfield SP
通讯作者：
Mayfield SP
影响因子：
5.5
作者：
J. Watson;V. Koya;S. Leppla;H. Daniell
通讯作者：
J. Watson;V. Koya;S. Leppla;H. Daniell
影响因子：
3
作者：
Kanamoto, H;Yamashita, A;Tomizawa, K
通讯作者：
Tomizawa, K
影响因子：
14.9
作者：
O. Zoubenko;Lori A. Allison;Z. Sváb;P. Maliga
通讯作者：
O. Zoubenko;Lori A. Allison;Z. Sváb;P. Maliga