Daniell H, Vivekananda J, Nielsen B L, Ye G N, Tewari K K, Sanford J C
Department of Biological Sciences, University of Idaho, Moscow 83843.
Proc Natl Acad Sci U S A. 1990 Jan;87(1):88-92. doi: 10.1073/pnas.87.1.88.
Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.
本文报道了通过高速微弹将合适的载体导入培养的烟草细胞叶绿体中，使氯霉素乙酰转移酶（cat）得以表达。本研究使用了几种含有细菌cat基因的叶绿体表达载体，这些基因置于豌豆psbA启动子区域（pHD系列）或玉米rbcL启动子区域（pAC系列）的控制之下。此外，还测试了含有豌豆、烟草或玉米叶绿体DNA复制子片段的叶绿体表达载体在烟草细胞叶绿体中cat表达的效率和持续时间。将收集在滤纸上的培养NT1烟草细胞用涂有pUC118（阴性对照）、35S-CAT（核表达载体）、pHD312（无复制子叶绿体表达载体）以及pHD407、pACp18和pACp19（含复制子的叶绿体表达载体）的钨颗粒进行轰击。用pUC118轰击的细胞的超声提取物在放射自显影图中未显示出可检测到的cat活性。cat的核表达在轰击后48小时达到最大值的三分之二，在72小时达到最大值。用叶绿体表达载体轰击的细胞在培养48小时之前表达水平较低。在用pHD407轰击的样品中，向培养细胞中添加新鲜培养基24小时后，观察到cat表达急剧增加；无复制子载体pHD312显示出约为该最大活性50%的活性。核cat和无复制子叶绿体载体的表达在72小时后下降，但在用pHD407轰击的细胞中维持了高水平的叶绿体cat表达。通过电穿孔将各种质粒构建体导入烟草原生质体，检查了cat在适当区室中的细胞器特异性表达。尽管核表达载体35S-CAT显示出cat的表达，但任何叶绿体载体均未观察到活性。
