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Cloning and identification of Mam1 in Brassica napus

【摘要】:正Glucosinolates are a group of sulfur-containing secondary metabolites derived from glucose and various amino acids,which occur almost exclusively in members of the Brassicales.The majority of glucosinolates in Arabidopsis thaliana and many other species of the Brassicaceae are formed from methionine,involving a three-step chain-elongation cycle.The first reaction of the cycle is catalyzed by a methylthioalkylmalate synthase encoded by a gene MAM1. Glucosinolates itself,located in cytoplasm,is a stable compound,but it can degraded by myrosinases released from vacuole and generate a variety of degradation products.When plant organs was damaged; they get in touch with each other and lead to the hydrolysis of glucosinolates.The degradation of glucosinolates produces a lot of metabolites,such as isothiocyanate.The smell of isothiocyanate has a very strong attraction to many insects,and so we can use this biological specialty in biological control of the crop insect and pests. In this study,the cDNA and DNA sequences of MAM1 gene in Brassica napus were cloned with Genome Walking method and named BnMAM1.Sequence analysis indicated that BnMAM1 shared high homology to MAM1 from Arabidopsis thaliana(84%identity in nucleotides sequence and 80.3%identity in amino acids sequence).We constructed a BnMAM1 overexpression vector(with a herbicide resistant gene bar),then transformed it into A.thaliana by a Agrobactierum-medicated floral dip method.To identify MAM1 activity in transformed lines and investigate whether transgenic plants are more attractive to the insects,we will detect glucosinolates and isothiocyanate content in transgenic plants and do behavioral assays with different insects.

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