Transgenic Bt cotton driven by the green tissue-specific promoter shows strong toxicity to lepidopteran pests and lower Bt toxin accumulation in seeds
Transgenic Bt cotton driven by the green tissue-specific promoter shows strong toxicity to lepidopteran pests and lower Bt toxin accumulation in seeds
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DOI：
10.1007/s11427-015-4920-6
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发表时间：
2016-01
期刊：
影响因子：
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通讯作者：
Qing Wang;Yi Zhu;Lin Sun;Le-gong Li;Shuangxia Jin;Xianlong Zhang
中科院分区：
文献类型：
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作者：
Qing Wang;Yi Zhu;Lin Sun;Le-gong Li;Shuangxia Jin;Xianlong Zhang
从牵牛中克隆了PNZIP（Pharbitisnilleucinzipper）基因的启动子（1.459kb），该启动子与GUS（β-葡萄糖醛酸酶）基因和苏云金芽孢杆菌毒素基因（Cry 9 C）融合。通过农杆菌介导的转化获得了几个转PNZIP：：GUS和PNZIP：：Cry 9 C基因的棉花品系。在转基因PNZIP：：GUS棉花植物的绿色组织中检测到强GUS染色。与此相反，在生殖结构，如花瓣，花药，和未成熟的种子PNZIP：：GUS棉花的GUS染色是非常微弱的。选择两个转PNZIP：：Cry 9 C株系和一个转花椰菜花叶病毒（CaMV）35 S：：Cry 9 C株系进行酶联免疫吸附测定（ELISA）和昆虫生物测定。Cry 9 C蛋白在35 S：：Cry 9 C系的大部分组织中保持高水平表达，范围为24.6 - 45.5 μg g-1鲜重。在PNZIP：：Cry 9 C品系的叶片、铃皮和苞片等绿色组织中，Cry 9 C蛋白的积累量分别达到50.2、39.7和48.3 μg g-1鲜重。相比之下，PNZIP：：Cry 9 C系（PZ1.3）的种子仅积累0.26 μg g-1鲜重的Cry 9 C蛋白，这比CaMV 35 S：：Cry 9 C系的种子所记录的低100倍。转PNZIP：：Cry 9 C基因抗虫棉对棉铃虫和红铃虫均表现出较强的抗性。PNZIP启动子能有效驱动Bt毒素在棉花绿色组织中表达，降低Bt蛋白在种子中的积累水平。这些特点应该可以减轻公众对转基因食品安全性的担忧。我们提出了PNZIP作为棉花生物技术中一种经济、环境友好的促进剂的未来效用。
A promoter of thePNZIP(Pharbitis nilleucine zipper) gene (1.459 kb) was cloned fromPharbitis niland fused to theGUS(β-glucuronidase) andBacillus thuringiensisendotoxin (Cry9C) genes. Several transgenic PNZIP::GUS and PNZIP::Cry9C cotton lines were developed by Agrobacterium-mediated transformation. Strong GUS staining was detected in the green tissues of the transgenic PNZIP::GUS cotton plants. In contrast, GUS staining in the reproductive structures such as petals, anther, and immature seeds of PNZIP::GUS cotton was very faint. Two transgenic PNZIP::Cry9C lines and one transgenic cauliflower mosaic virus (CaMV) 35S::Cry9C line were selected for enzyme-linked immunosorbent assay (ELISA) and insect bioassays. Expression of the Cry9C protein in the 35S::Cry9C line maintained a high level in most tissues ranging from 24.6 to 45.5 μg g-1fresh weight. In green tissues such as the leaves, boll rinds, and bracts of the PNZIP::Cry9C line, the Cry9C protein accumulated up to 50.2, 39.7, and 48.3 μg g-1fresh weight respectively. In contrast, seeds of the PNZIP::Cry9C line (PZ1.3) accumulated only 0.26 μg g-1fresh weight of the Cry9C protein, which was 100 times lower than that recorded for the seeds of the CaMV 35S::Cry9C line. The insect bioassay showed that the transgenic PNZIP::Cry9C cotton plant exhibited strong resistance to both the cotton bollworm and the pink bollworm. The PNZIP promoter could effectively drive Bt toxin expression in green tissues of cotton and lower accumulated levels of the Bt protein in seeds. These features should allay public concerns about the safety of transgenic foods. We propose the future utility of PNZIP as an economical, environmentally friendly promoter in cotton biotechnology.