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2025年8月29日 星期五
  2018, Vol. 26 Issue (10): 1650-1658    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
玉米γ-Zein多肽融合增强外源重组蛋白在大豆种子中的高效积累
贺红利1,牛陆2,杨静1,邢国杰1,赵倩倩2,杜茜2,程云清2,杨向东1
1. 吉林省农业科学院
2.
Maize (Zea mays) γ-Zein Polypeptide Fusion Significantly Enhanced Accumulation of the Foreign Recombinant Protein in Soybean (Glycine max) Seeds
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摘要 大豆(Glycine max)种子的高水平蛋白合成和积累能力,为外源重组蛋白的高效表达提供了理想的表达宿主。为进一步提高外源蛋白在大豆种子中的表达水平,本研究基于多肽融合策略,研究玉米(Zea mays)醇溶蛋白γ-Zein对外源蛋白在大豆种子中积累水平的影响。采用农杆菌(Agrobacterium tumefaciens)介导法,分别将大豆种子特异性启动子基因β'-伴球蛋白基因启动子(β-conglycininalpha subunit promoter, BCSP)驱动的绿色荧光蛋白(green fluorescent protein, GFP)编码基因和Zein-GFP基因导入栽培大豆品种,共获得Zein-GFP转基因植株41株,GFP转基因植株46株。反转录PCR(reverse transcription PCR, RT-PCR)和Western blot检测表明,外源基因Zein-GFP和GFP在转基因大豆种子中均能够正常转录和翻译。选取外源基因mRNA表达水平相近的T3代转基因大豆株系进行酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)分析表明,转基因大豆种子中融合蛋白Zein-GFP平均表达水平为1.51% TSP (1.37%~1.60% TSP)(可溶性蛋白总量, total soluble protein),未融合蛋白GFP平均表达水平为0.14% TSP(0.13%~0.15% TSP)。融合蛋白Zein-GFP在转基因大豆种子中的积累水平较GFP提高10.78倍。激光共聚焦扫描分析进一步表明,融合蛋白Zein-GFP在转基因大豆种子中主要以蛋白体的形式存在,而未融合GFP蛋白则主要分布在胞质中。本研究结果表明,γ-Zein多肽融合促进了重组蛋白在细胞蛋白体中的积累,并显著提高其在大豆种子中的表达水平。本研究为加快大豆种子反应器的应用提供依据。
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贺红利
牛陆
杨静
邢国杰
赵倩倩
杜茜
程云清
杨向东
关键词 大豆γ-Zein多肽融合蛋白体外源重组蛋白    
Abstract:Soybean (Glycine max) seeds provide an ideal host for the production of foreign recombinant proteins because of their robust capability for biosynthesis and accumulation of the native proteins. To further enhance the expression of foreign proteins in soybean seeds, a polypeptide fusion strategy was utilized to investigate the influence of the maize (Zea mays) γ-Zein fusion tag on accumulation of the foreign recombinant protein. The GFP (green fluorescent protein) and Zein-GFP driven by the soybean seed-specific promoter BCSP were individually introduced into the cultivated soybean genotype by Agrobacterium tumefaciens-mediated transformation, respectively. Totally 41 Zein-GFP and 46 GFP transgenic plants were obtained in this study. Reverse transcription PCR (RT-PCR) and Western blot analysis confirmed transcript and translation of the foreign genes in the transgenic soybean seeds. The transgenic lines with similar expression of Zein-GFP and GFP at mRNA levels in seeds were selected and accumulation of the recombinant proteins was quantified by enzyme linked immunosorbent assay (ELISA). Average accumulation level of the fusion protein Zein-GFP reached to 1.51% TSP (total soluble protein) ranging from 1.37% to 1.60% TSP in the transgenic seeds, increased by 10.78 fold compared with 0.14% TSP of the unfused GFP (0.13%~0.15% TSP). Subcellular targeting analysis by laser scanning confocal microscopy showed that the γ-Zein fused GFP were present in the protein bodies (PBs), while the unfused GFP was mainly distributed in the cytoplasm. Taken together, the results showed that the γ-Zein polypeptide fusion induced the formation of PBs and significantly enhanced accumulation of the foreign recombinant protein in the transgenic soybean seeds, and thus provide the basis for application of the soybean-based bio-reactor.
Key wordsSoybean    γ-Zein    Polypeptide Fusion    Protein body    Foreign recombinant protein
收稿日期: 2018-03-05      出版日期: 2018-09-26
基金资助:国家自然科学基金面上项目;国家转基因生物新品种培育科技重大专项
通讯作者: 杨向东     E-mail: xdyang020918@126.com
引用本文:   
贺红利 牛陆 杨静 邢国杰 赵倩倩 杜茜 程云清 杨向东. 玉米γ-Zein多肽融合增强外源重组蛋白在大豆种子中的高效积累[J]. , 2018, 26(10): 1650-1658.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2018/V26/I10/1650
 
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