转mgfp-5基因烟草的愈伤诱导及培养

发布时间：2018-02-23 17:25

  本文关键词： 贻贝粘蛋白 mgfp-基因 转基因烟草 愈伤培养　出处：《基因组学与应用生物学》2017年05期 　论文类型：期刊论文

【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。
[Abstract]:The mussel adhesive adhesive secreted by the foot silk gland of the marine mollusk Mytilida (Mytilida), which is called mussel foot protein, not only has high viscosity and corrosion resistance in water, but also has no toxicity, no immunogenicity, and good biocompatibility. The viscosity of Mfp-5 is the strongest. The mussel mucin in use is mainly obtained naturally, but its content is easy to solidify and difficult to obtain. Genetic engineering of recombinant mussel mucin has just begun. Previously, we expressed the MMP gene of mytilus galloprovincialis foot protein type 5 in tobacco. Mgfp-5 transgenic tobacco seeds were obtained. In this experiment, transgenic T1 seeds were induced by callus induction on MS medium supplemented with different concentrations of 2H4 DX 6-BANAA. The induction rate was more than 90.0. The inductive rate of callus was 100 at 1.0 mg / L 1.0 mg / L 6-BA 0.5 mg / L NAA 0.1 mg/L, and the callus grew on a "S" curve during solid / liquid culture, and the induction rate was more than 90% at 0. 1 mg 路L ~ (-1) 路L ~ (-1) 路L ~ (-1). The growth rate of fresh mass was 3.73, the time of liquid culture was shortened by 6 days, and the fresh mass increased by 8.56 ~ 8.63 times in 6 ~ 15 days after solid / liquid culture, and the electrophoresis bands of expected size were detected by RT-PCR and Western blotting in subculture callus of solid / liquid culture, respectively. The stable expression of exogenous mgfp-5 gene and Mgfp-5 protein and the stable expression of Mgfp-5 protein in callus could provide a new way to obtain the raw material of recombinant protein Mgfp-5 and provide a reference for studying the expression of mussel mucin in plants.
【作者单位】： 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;
【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
【分类号】：Q943.1
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本文编号：1526968