Transformation Of Mytilus Galloprovincialis Foot Protein Type 5 (Mgfp-5) Gene In Tobacco

The byssus of Marine mollusk mussels can secrete a kind of sticky protein, also known as Mussel foot protein (Mfp). Several types of Mfps were identified, and each of them contains a high ratio of a special residue named DOPA (3,4-dihydroxyphenyl-L-alanine), which is derived from hydroxylation of tyrosine and plays a dominant role in byssus's strong viscosity. In all Mfps, Mfp-5 (Mussel foot protein type 5) has the highest DOPA content (-25 to 30% mol%). Mussel adhesive proteins are widely applied in chemical surface, ocean engineering, medical adhesives, and other fields. Now the mussel adhesive proteins are mainly obtained from natural mussel, which restricts its application, since the natural extraction is a labor-intensive and inefficient process, and produces very little purified protein. Scholars have already try to express these proteins in prokaryotes (Escherichia coli) and eukaryotes (yeast), but the quantity or adhesion performance of mussels adhesive proteins are low. Plants can synthesize proteins with a higher glycosylation and phosphorylation modification, and expressing target proteins in plants also has a big advantage in massive production, safety, and the cost of production.Therefore, we tried to express Mytilus galloprovincialis foot protein type 5 (Mgfp-5) in plant. In this study, the plant expression vector pRI101-Mgfp with Mgfp-5 gene was constructed, we used the leaves of Qinyan 95 tobacco as host, the explants were genetic transformed by Agrobacteriun-mediated method, and the factors of transformation were optimized. The transgenic tobacco plants were obtained by infection, resistance selection, and molecular biological identification (such as PCR?RT-PCR). Transgenic plants with Mgfp-5 expression at transcription level were further examined at protein level. The SDS-PAGE and Western Blot identification of the T1 transgenic plants showed that the Mgfp-5 protein are expressed. The obtained results were as follows:1. The plant expression vector pRI101-Mgfp with Mgfp-5 gene was constructed and was transferred to LBA4404. To contruct pRI101-Mgfp, Mgfp-5 CDS (coding sequence) with six histidine codons added in 3'end were amplified by PCR, and then the fragment were digested and inserted into the plant expression vector pRI101-AN between Nde I and Kpn I enzyme sites.2. By optimizing the combination of different phytohormones contained in medium, an efficient protocol for the regeneration of tobacco (Qinyan 95) was established. Nearly 100% of adventitious buds were induced from leaf discs of the Qinyan 95 after being cultured on MSo medium added with 1.0mg/L 6-BA and 0.2mg/L NAA for 4 weeks; Roots were induced after the regenerated shoots had been cultured on MSo medium with 0.1mg/L NAA and the induction rate can reach 100% for 10 days.3. The optimum conditions for the transformation were:the explants were pre-cultured for 3 d, infected by OD600=0.6 Agrobacterium for 10 min, co-cultured time for 3 d, ultimately being selected by 20 mg/L Kan.4. Identification of transgenic Mgfp-5 tobacco plants:the transgenic plants were analyzed by PCR and RT-PCR. An expected fragment of about 260 bp should be amplified in transgenic lines. Out of 87 independent To plants,74.7% plants were integrated Mgfp-5, and 39.1% plants showed Mgfp-5 expression at RNA level. Transgenic To plants were single harvested, and T1 seedlings were cultured, and examined at DNA, RNA, and protein level with PCR, RT-PCR, SDS-PAGE electrophoresis and Western Blotting techniques, respectively. Consistent with expected 9.8 kDa, a fragment of about 12 kDa was detected in transgenic lines by Western Blot. Hence, Mgfp-5 protein is expressed in transgenic plants.