转mgfp-5基因烟草的愈伤诱导及培养  

Callus Induction and Culture of Transgenic Tobacco with mgfp-5 Gene

作　　者：王英娟[1] 王睿劼 吕亚维 张雨靖 杨泽熵[1] 

机构地区：[1]西北大学生命科学学院/陕西省生物技术重点实验室/西部资源与现代生物技术省部共建教育部重点实验室,西安710069

出　　处：《基因组学与应用生物学》2017年第5期2061-2067,共7页Genomics and Applied Biology

基　　金：国家自然科学基金(31270411;31572665);陕西省科技厅社会发展攻关计划(2012K12-01-02);陕西省教育厅专项资金(12JK0827);陕西省教育厅重点实验室项目(12JS105)共同资助

摘　　要：海洋软体动物贻贝(Mytilidae)足丝腺分泌的贻贝粘蛋白(mussel adhesive protein,MAP)即贻贝足丝蛋白(mussel foot protein,Mfp),不仅有水中高粘性及耐腐蚀性,而且无毒、无免疫原性,生物相容性好,其中Mfp-5粘性最强。现用的贻贝粘蛋白主要是天然获取,但是其含量低易固化难获得,基因工程重组贻贝粘蛋白的研究刚刚开始。前期我们在烟草中表达了地中海贻贝蛋白(mytilus galloprovincialis foot protein type 5,Mgfp-5)基因,得到转mgfp-5基因烟草种子。本实验将转基因T1代种子无菌苗进行愈伤组织诱导,在附加不同浓度的2,4-D、6-BA、NAA的MS培养基上,诱导率≥90.0%,其中2,4-D 1.0 mg/L、6-BA 0.5 mg/L、NAA 0.1 mg/L时诱导率为100%;愈伤组织固/液培养过程中均以"S"型曲线生长,固体培养时12~27 d鲜质量增长率为3.73%,液体培养时间缩短6 d,在6~15 d时间内鲜质量会增加8.56~8.63倍;固/液培养的继代愈伤组织,PCR、RT-PCR及Western blotting分别检测到预期大小的电泳条带,显示了外源mgfp-5基因及Mgfp-5蛋白稳定表达。稳定表达Mgfp-5蛋白的愈伤组织,可以为获得重组蛋白Mgfp-5的原材料提供新途径,为研究植物表达贻贝粘蛋白提供参考。Mussel adhesive protein （MAP）, which is also named Mussel foot protein （Mfp）, is secreted by Mytilidae＇s byssus gland. It is of high adhesiveness in water and is corrosion-resistant. At the same time, it is non-toxic, non-immunogenic and of excellent biocompatibility. Protein Mfp-5 has the strongest adhesive in many kinds of MAPs. Now MAP is mainly extracted from nature. However, because of its low content and quick-curing, MAPs is difficult to obtain. The study on genetic-engineering of recombing MAPs has just begun. In previous study, we had successfully expressed Mytilus galloprovincicdis foot protein type 5 （Mgfp-5） gene in tobacco and obtained the transgenic tobacco seeds. In this experiment, we induced the callus of transgenic T1 generation seedings. The induction rate was more than 90.0% in medium containing different concentrations of 2,4-D, 6-BA and NAA on MS. However, the induction rate was 100% when 2,4-D was 1.0 mg/L, 6-BA was 0.5 mg/L and NAA was 0.1 mg/L. The growth curves of the callus in the solid and liquid culture were all in ＂S＂ shapes. During 12-27 days of solid culture, the fresh weight growth rate was 3.73%, liquid culture time was shortened by 6 days, and the quality of fresh increased 8.56-8.63 times in the first 6-15 days. In the callus of solid and liquid culture,we detected the expected bands by PCR, RT-PCR and Western blotting. This showed the stable expression of exogenous gene mgfp-5 and protein Mgfp-5. The established callus, which could express the Mgfp-5 protein stably, might provide a new way for obtaining raw materials of recombinant protein Mgfp-5, and it could provide a reference for the study of plant expression mussel adhesive proteins.

关 键 词：贻贝粘蛋白 mgfp-5基因 转基因烟草 愈伤培养 

分 类 号：Q943.1[生物学—植物学]