Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo.     

Detecting un-authorized genetically modified organisms (GMOs) and derived materials

Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20 + species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance.     

Metabolon formation in dhurrin biosynthesis

Synthesis of the tyrosine derived cyanogenic glucoside dhurrin in Sorghum bicolor is catalyzed by two multifunctional, membrane bound cytochromes P450, CYP79A1 and CYP71E1, and a soluble UDPG-glucosyltransferase, UGT85B1 (Tattersall, D.B., Bak, S., Jones, P.R., Olsen, C.E., Nielsen, J.K., Hansen, M.L., H?j, P.B., M?ller, B.L., 2001. Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 293, 1826-1828). All three enzymes retained enzymatic activity when expressed as fluorescent fusion proteins in planta. Transgenic Arabidopsis thaliana plants that produced dhurrin were obtained by co-expression of CYP79A1/CYP71E1-CFP/UGT85B1-YFP and of CYP79A1/CYP71E1/UGT85B1-YFP but not by co-expression of CYP79A1-YFP/CYP71E-CFP/UGT85B1. The lack of dhurrin formation upon co-expression of the two cytochromes P450 as fusion proteins indicated that tight interaction was necessary for efficient substrate channelling. Transient expression in S. bicolor epidermal cells as monitored by confocal laser scanning microscopy showed that UGT85B1-YFP accumulated in the cytoplasm in the absence of CYP79A1 or CYP71E1. In the presence of CYP79A1 and CYP71E1, the localization of UGT85B1 shifted towards the surface of the ER membrane in the periphery of biosynthetic active cells, demonstrating in planta dhurrin metabolon formation.     

棉花杜松烯合成酶基因的克隆及其表达分析

杜松烯合成酶是棉酚合成途径中的关键酶,催化(E,E)-法呢基焦磷酸(FPP)环化形成(+)-δ-杜松烯。从陆地棉Y18R中克隆分离了杜松烯合成酶基因(GhCdn),该基因的基因组序列为2 700 bp,具有6个内含子,剪切后其ORF为1 665 bp,编码554个氨基酸,该基因属于杜松烯合成酶C亚家族。应用Overlap PCR方法将其上的2个Hind Ⅲ 酶切位点钝化后,将GhCdn基因连接到表达载体pBI121上,构建出分别由组成型启动子CaMV 35S和绿色组织高效启动子Psbp驱动的2个植物表达载体pGBI-CaMV 35S-GhCdn和pGBI-Psbp-GhCdn。通过农杆菌介导法转化棉花下胚轴并进行组织培养,获得了9个35S转基因阳性愈伤系和2个P转基因阳性愈伤系。经检测,阳性愈伤组织中GhCdn基因的mRNA表达量和棉酚含量均有所增加,但35S系普遍高于P系。本研究为通过基因工程手段提高棉花组织器官中的棉酚含量提供了依据。