DeGray G, Rajasekaran K, Smith F, Sanford J, Daniell H
Department of Molecular Biology and Microbiology, 12722 Research Parkway, University of Central Florida, Orlando, FL 32826-3227, USA.
Plant Physiol. 2001 Nov;127(3):852-62.
The antimicrobial peptide MSI-99, an analog of magainin 2, was expressed via the chloroplast genome to obtain high levels of expression in transgenic tobacco (Nicotiana tabacum var. Petit Havana) plants. Polymerase chain reaction products and Southern blots confirmed integration of MSI-99 into the chloroplast genome and achievement of homoplasmy, whereas northern blots confirmed transcription. Contrary to previous predictions, accumulation of MSI-99 in transgenic chloroplasts did not affect normal growth and development of the transgenic plants. This may be due to differences in the lipid composition of plastid membranes compared with the membranes of susceptible target microbes. In vitro assays with protein extracts from T(1) and T(2) plants confirmed that MSI-99 was expressed at high levels to provide 88% (T(1)) and 96% (T(2)) inhibition of growth against Pseudomonas syringae pv tabaci, a major plant pathogen. When germinated in the absence of spectinomycin selection, leaf extracts from T(2) generation plants showed 96% inhibition of growth against P. syringae pv tabaci. In addition, leaf extracts from transgenic plants (T(1)) inhibited the growth of pregerminated spores of three fungal species, Aspergillus flavus, Fusarium moniliforme, and Verticillium dahliae, by more than 95% compared with non-transformed control plant extracts. In planta assays with the bacterial pathogen P. syringae pv tabaci resulted in areas of necrosis around the point of inoculation in control leaves, whereas transformed leaves showed no signs of necrosis, demonstrating high-dose release of the peptide at the site of infection by chloroplast lysis. In planta assays with the fungal pathogen, Colletotrichum destructivum, showed necrotic anthracnose lesions in non-transformed control leaves, whereas transformed leaves showed no lesions. Genetically engineering crop plants for disease resistance via the chloroplast genome instead of the nuclear genome is desirable to achieve high levels of expression and to prevent pollen-mediated escape of transgenes.
抗菌肽MSI-99是蛙皮素2的类似物，通过叶绿体基因组进行表达，以便在转基因烟草（烟草品种 Petit Havana）植株中获得高水平表达。聚合酶链反应产物和Southern杂交证实了MSI-99已整合到叶绿体基因组中并实现了同质化，而Northern杂交则证实了转录。与先前的预测相反，MSI-99在转基因叶绿体中的积累并未影响转基因植株的正常生长和发育。这可能是由于质体膜与易感靶微生物的膜相比，脂质组成存在差异。对T(1)和T(2)植株的蛋白质提取物进行的体外试验证实，MSI-99高水平表达，对主要植物病原菌烟草丁香假单胞菌pv tabaci的生长抑制率分别为88%（T(1)）和96%（T(2)）。在无壮观霉素选择的情况下萌发时，T(2)代植株的叶片提取物对烟草丁香假单胞菌pv tabaci的生长抑制率为96%。此外，与未转化的对照植株提取物相比，转基因植株（T(1)）的叶片提取物对三种真菌——黄曲霉、串珠镰刀菌和大丽轮枝菌的预萌发孢子的生长抑制率超过95%。用细菌病原菌烟草丁香假单胞菌pv tabaci进行的植物体内试验导致对照叶片接种点周围出现坏死区域，而转化叶片未出现坏死迹象，这表明通过叶绿体裂解在感染部位高剂量释放了该肽。用真菌病原菌毁灭炭疽菌进行的植物体内试验表明，未转化的对照叶片出现坏死炭疽病斑，而转化叶片未出现病斑。通过叶绿体基因组而非核基因组对作物进行抗病基因工程改造，有利于实现高水平表达并防止转基因通过花粉传播。
