Study On Isolation And Transformation Of Soybean Ferritin Gene

Ferritin is a long-term iron binding and storage protein widely in the organism. It regulates iron absorption and release.It is a major form of iron storage in plant.Soybeans in particular have a high iron,and about 90% of Total iron in seeds stored inside ferritin.It has been demonstrated that iron from soybean ferritin is equal in bioavailability to FeSO4. In this study, soybean ferritin gene was transferred into maize by Agrobacterium-mediated transformation, in order to realize the specific expression of soybean ferritin gene in maize endosperm driven by the promoter of endosperm-specific expression gene 15 kDβ-Zein.With this study, we expect to obtain maize with higher iron content. And this would lay the foundation for the enhancement nutritional quality of maize and iron deficiency of human corn-based food. The main results were summarized as follows:1. The promoter of endosperm-specific expression gene 15 kDβ-Zein was cloned from maize inbred lines Lu 2548 genomic. Sequence analysis and promoter function prediction indicated that the cloned sequence would function as a promoter and make the endosperm-specific expression. In order to confirm its function, we construct the plant expressing vector pBI121-Zein-GUS.2. According to the reported sequence of soybean ferritin, ferritin gene cDNA sequence was cloned from soybean varieties Zhongpin 95-5883. The ferritin gene cDNA sequence is 753 bp including all encoding sequence,and 251 amino acid residues.In order to study soybean ferritin gene expression in maize, we constructed the plant expression vector pBI121-35s-Ferritin, pCUbi1390-Ferritin, pBI121-Zein- Ferritin, pCUbi1390-Ferritin-Epsps.3. In order to provide receptor material for the Agrobacterium-mediated transformation of maize, we cultivated embryogenic callus from maize inbred lines R18-599 and Qi319. Through the approach of Triparental mating, the plant expression vector pCUbi1390-Ferritin, pBI121-Zein-Ferritin were introduced into an Agrobacterium strain LBA 4404, then were transformed embryogenic callus of maize by Agrobacterium-mediated transformation. And transgenic plants had been obtained. The genome DNA was extracted from transformed maize leaves. And it was briefly proved that Ferritin gene cDNA had been integrated into the transgenic maize genome by PCR analysis.