Cloningofsoybeanferritingene,constructionofitsexpression Vector And Transformation With Arabidopsis

Iron deficiency is a serious worldwide human nutrition problem. Expression of ferritin gene in cereal crops, intake of iron can be increased. This is an ideal way to overcome the obstacle of the iron deficiency. This experiment cloned the soybean's ferritin gene by RT-PCR method from Yudou 8 and the primers were designed by Prime5.0 software using the reference sequence in NCBI database. Sequencing confirmed the PCR result. Full-length of the cDNA is 752bp, encodes 252 amino acids. Sequence alignment by DNAMAN revealed that the cloned fragment shows above 98% homolog with soybean (M64337) ferritin gene. Functional analysis by bioinformatics methods showed that the gene contains one FER-RITIN-LIKE function domain, two signal sequence region existing in iron-binding protein gene family, six phosphorylation sites and other important featured functional sites.Gateway approach was exploited to construct the chimeric expression vector. By two-step LR reaction, target gene was transferred firstly into pGWc (Entry Clone) and than cloned into the expression vector. By resistance selections and PCR tests, a plant expression vector of ferritin gene, PLEELA, was confirmed. The expression vector was transformed into Agrobacterium pMP90RK by electric shock method.Using Agrobacterium-mediated pollen tube pathway, soybean ferritin gene was transferred into Arabidopsis successfully. 4 transgenic plants were obtained by herbicide resistance selection and PCR test.In this paper, clone of soybean ferritin gene, construction of its expression vector and transformation are a fundamental work to further exploit the soybean ferritin gene in cereal crops expression by genetic engineering.