Genetic Transformation Of Tobacco With Earthworm Metallothionein Gene

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich and metal-binding proteins. They are present in the majority of organisms. In molecular evolution, MTs have conservative structure and similar function. Many studies demonstrated MTs may be involved in heavy metal resistance in both plants and animals.In order to improve heavy metal resistance, mammal MT has been introduced into plants. However, transformation of invertebrate MT gene into plants has not been reported so far. Many kinds of metal-binding proteins, such as metallothioneins, have been found in earthworm, which is capable of binding and enrich cadmium of soil. In this study, an earthworm metallothionein gene, efMet, was introduced into tobacco plants to evaluate the efficacy in heavy metal clearance in plants. First, we added suitable restriction enzyme cutting site to MT gene from Eisenia foetida by PCR amplification. Second, the MT gene was integrated into pPZP111 binary vector, generating the constitutive expression vector pPZP-efMT. Recombinant plasmid was identified by PCR and restriction digestion. Third, the expression vector pPZP-efMT was introduced into agrobacterium tumefaciens strains EHA105 and LBA4404 respectively. Result of PCR amplification and restriction digestion showed that the pPZP-efMT was transferred into agrobacterium. Finally, tobacco primary transformants were produced by leaf disc method transformation. Based on these experiments, a set of efficient and simple agrobacterium-mediated transformation system was established for our laboratory. Preculturing leaves are easier to differentiate than non-preculturing leaves. The optimal concentration of agrobacterium is OD600 =0.6. Suitable infection time is 7 min. During the course of selective culture, leaves should be placed in close contact with culture medium. Genomic DNA of regenerated plants was extracted with CTAB method, and 7 transgenic plants were screened out as positive plants after the preliminary identification with PCR. Such positive plants could lay the groundwork for research of heavy metal tolerance in transgenic tobacco.