Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 10617, Taiwan.
Appl Environ Microbiol. 2011 Jul;77(13):4508-19. doi: 10.1128/AEM.02033-10. Epub 2011 May 13.
Entomopathogenic fungi have been used for biocontrol of insect pests for many decades. However, the efficacy of such fungi in field trials is often inconsistent, mainly due to environmental stresses, such as UV radiation, temperature extremes, and desiccation. To circumvent these hurdles, metabolic engineering of dihydroxynaphthalene (DHN) melanin biosynthetic genes (polyketide synthase, scytalone dehydratase, and 1,3,8-trihydroxynaphthalene reductase genes) cloned from Alternaria alternata were transformed into the amelanotic entomopathogenic fungus Metarhizium anisopliae via Agrobacterium-mediated transformation. Melanin expression in the transformant of M. anisopliae was verified by spectrophotometric methods, liquid chromatography/mass spectrometry (LC/MS), and confocal microscopy. The transformant, especially under stresses, showed notably enhanced antistress capacity and virulence, in terms of germination and survival rate, infectivity, and reduced median time to death (LT50) in killing diamondback moth (Plutella xylostella) larvae compared with the wild type. The possible mechanisms in enhancing the stress tolerance and virulence, and the significance and potential for engineering melanin biosynthesis genes in other biocontrol agents and crops to improve antistress fitness are discussed.
虫生真菌被广泛用于防治害虫已有数十年的历史。然而，此类真菌在田间试验中的效果往往不一致，主要是由于环境压力，如紫外线辐射、极端温度和干燥等。为了克服这些障碍，本研究通过农杆菌介导的转化，将从链格孢菌中克隆的二羟基萘（DHN）黑色素生物合成基因（聚酮合酶、丙烯醛脱水酶和 1,3,8-三羟基萘还原酶基因）进行代谢工程改造，转化为非黑色素型的昆虫病原真菌金龟子绿僵菌。通过分光光度法、液相色谱/质谱（LC/MS）和共聚焦显微镜验证了黑色素在金龟子绿僵菌转化体中的表达。与野生型相比，转化体，尤其是在压力条件下，在发芽率和存活率、感染性以及降低致死中时（LT50）方面表现出显著增强的抗应激能力和毒力，显著提高了对小菜蛾幼虫的毒力。讨论了增强抗应激能力和毒力的可能机制，以及在其他生物防治剂和作物中工程黑色素生物合成基因以提高抗应激适应性的意义和潜力。
