Transformation of nonselectable reporter genes in marine diatoms
Transformation of nonselectable reporter genes in marine diatoms
复制标题
DOI：
10.1007/pl00011773
复制
发表时间：
1999-05-01
影响因子：
3
通讯作者：
Bowler, C
中科院分区：
文献类型：
--
作者：
Falciatore, A;Casotti, R;Bowler, C
我们报告了两种海洋硅藻的遗传转化微粒轰击。用来自印度斯坦链霉菌的细菌基因Sh ble转化羽状硅藻三角褐指藻，该基因赋予对抗生素腐草霉素和博莱霉素的抗性。转化体含有1至10个拷贝的外源DNA，通过在明显随机的位置处的非法重组整合到基因组中。转化效率约为10（-6），并且单个细胞系在冷冻保存后可以维持在-80 ° C。此外，三角褐指藻可以用两种不同的质粒同时转化，一种含有Sh ble基因，另一种含有萤火虫荧光素酶基因（LUC），该基因在来自岩藻黄质叶绿素a/c结合蛋白基因（FCP）的启动子的控制下。在这些共转化体中，LUC活性是光诱导的。用细菌β-葡萄糖醛酸酶（GUS）基因瞬时转化中心硅藻Weissflorescent海链藻（Thalassiosira weissflorescent）也已经使用类似的转化技术实现。海洋硅藻的基因转移协议的可用性，连同一系列功能报告基因和调控表达系统，将允许分子解剖其生物学，并允许评估这些生物的生物技术潜力。
We report the genetic transformation of two marine diatoms by microparticle bombardment. The pennate diatom Phaeodactylum tricornutum was transformed with the bacterial gene Sh ble from Streptoalloteichus hindustanus, which confers resistance to the antibiotics phleomycin and zeocin. Transformants contained between 1 and 10 copies of the exogenous DNA integrated into the genome by illegitimate recombination at apparently random locations. Transformation efficiencies were around 10(-6), and individual cell lines could be maintained at -80 degrees C following cryopreservation. Also, P. tricornutum could be transformed simultaneously with two different plasmids, one containing the Sh ble gene and another containing the firefly luciferase gene (LUC) under control of a promoter derived from a fucoxanthin, chlorophyll a/c-binding protein gene (FCP). In these cotransformants, LUC activity was light inducible. The transient transformation of the centric diatom Thalassiosira weissflogii with the bacterial beta-glucuronidase (GUS) gene has also been achieved using similar transformation technology. The availability of gene transfer protocols for marine diatoms, together with a range of functional reporter genes and regulated expression systems, will permit molecular dissection of their biology and allow an assessment of the biotechnological potential of these organisms.