贻贝粘蛋白Mgfp-5基因在烟草中的转化

Transformation of Mytilus galloprovincialis Foot Protein Type 5(Mgfp-5) Gene in Tobacco

贻贝(Mytilus galloprovincialis)足腺分泌的粘蛋白Mgfp-5,是一种粘合强度高、生物相容性好的生物粘合剂,可以广泛应用于医学、化学表面以及海洋工程等领域。天然获取Mgfp-5时易固化且含量低,难获得、成本高制约了其应用,所以更多学者尝试基因工程方法获得粘蛋白Mgfp-5,其中鲜见借助植物基因工程表达Mgfp-5的报道。本研究构建携带Mgfp-5基因的植物表达载体p RI101-Mgfp,以秦烟95烟草叶片为受体,通过根癌农杆菌LBA4404介导方法,遗传转化外植体,历经转染、抗性筛选及分子生物学鉴定,获得转基因烟草植株。研究表明正确构建的植物表达载体p RI101-Mgfp,可以在外植体预培养3 d,农杆菌菌液OD600=0.6,侵染10 min,共培养3 d,20 mg/L卡那霉素抗性筛选培养的条件下,携带外源Mgfp-5基因,顺利转化进入烟草小苗,经过PCR、RT-PCR检测目标基因Mgfp-5在转化小苗基因组中可以整合及表达,得到转Mgfp-5基因烟草植株,生根后移栽成活。本研究为粘合蛋白Mgfp-5的表达、提取纯化及功能等深入研究提供参考,也是优质粘合剂粘合蛋白材料获得的一条新途径。

The Mytilus galloprovincialis foot protein type 5 gallqprovincialis was a kind of biological adhesive that had （Mgfp-5） which was produced by byssus of Mytilus strong adhesive ability and good biocompatibility. It can be widely used in medical, chemical surface and ocean engineering or other fields. But the natural extraction of Mgfp-5 was easy to solidify and contained very little purified protein, meanwhile, the natural Mgfp-5 was difficult to obtain and had high costing, which restricted its application. Now more and more scholars tried to obtain mucin Mgfp-5 by genetic engineering methods, but expression of Mgfp-5 of plant gene engineering was rarely reported. In this study, the plant expression vector pRI101-Mgfp with Mgfp-5 gene was constructed, we used the leaves of Qinyan 95 tobacco as receptor, the explant was genetic transformed by Agrobacterium-mediated （LBA4404） method, and the transgenic tobacco plant was obtained by transfection, resistance selection, and molecular biological identification. The results showed that the plant expression vector pRI 101-Mgfp with Mgfp-5 that was constructed correctly can be transformed into tobacco seedlings in the condition that leaves explants were immersed in the Agrobaeterium at OD600 of 0.6 for 10 min, and then co-cultivated for 3 days and by resistance selection cultivated in 20 mg/L Kanamycin （Kan）. The PCR analysis and RT-PCR analysis preliminarily provedthe Mgfp-5 gene had been integrated and expressed in the tobacco genome. It showed that the tobacco plant with Mgfp-5 was obtained and root transplanting was successful. The results provided some good research materials and foundation to the expression, purification and function of Mgfp-5 in future studies. It can also provide a new way to obtain quality glue adhesive proteins.