Apt K E, Kroth-Pancic P G, Grossman A R
Carnegie Institution of Washington, Department of Plant Biology, Stanford, CA 94305, USA.
Mol Gen Genet. 1996 Oct 16;252(5):572-9. doi: 10.1007/BF02172403.
A nuclear transformation system has been developed for the diatom Phaeodactylum tricornutum using microparticle bombardment to introduce the sh ble gene from Streptoalloteichus hindustanus into cells. The sh ble gene encodes a protein that confers resistance to the antibiotics Zeocin and phleomycin. Chimeric genes containing promoter and terminator sequences from the P. tricornutum fcp genes were used to drive expression of sh ble. Between 10-100 transformants were recovered/10(8) cells. Transformants were able to grow on at least 500 micrograms/ml of Zeocin, which is 10 times the amount necessary to kill wild-type cells. Based on Southern hybridizations the sh ble gene was present in 1-3 copies/transformant. Relative levels of correctly processed transcripts were correlated with the abundance of the Sh ble protein (present at 0.1-2.0 micrograms/mg total protein). The cat reporter gene fused to a fcp promoter could also be introduced by microparticle bombardment and was found to be highly expressed (average of 7.1 U/mg total protein). This work demonstrates that heterologous genes can be readily expressed in P. tricornutum. The development of selectable marker and reporter gene constructs provides the tools necessary for dissecting gene structure and regulation, and introducing novel functions into diatoms.
利用微粒轰击法将印度斯坦链霉菌的sh ble基因导入三角褐指藻细胞，从而建立了一种三角褐指藻的核转化系统。sh ble基因编码一种赋予对博来霉素和腐草霉素抗生素抗性的蛋白质。含有三角褐指藻fcp基因启动子和终止子序列的嵌合基因被用于驱动sh ble的表达。每10⁸个细胞可获得10 - 100个转化体。转化体能够在至少500微克/毫升的博来霉素上生长，这是杀死野生型细胞所需量的10倍。基于Southern杂交，每个转化体中存在1 - 3个拷贝的sh ble基因。正确加工的转录本的相对水平与Sh ble蛋白的丰度相关（含量为0.1 - 2.0微克/毫克总蛋白）。与fcp启动子融合的cat报告基因也可通过微粒轰击导入，并发现其高度表达（平均为7.1单位/毫克总蛋白）。这项工作表明异源基因能够在三角褐指藻中轻易表达。可选择标记和报告基因构建体的开发为剖析基因结构和调控以及将新功能引入硅藻提供了必要的工具。
