Das Alok, Datta Subhojit, Thakur Shallu, Shukla Alok, Ansari Jamal, Sujayanand G K, Chaturvedi Sushil K, Kumar P A, Singh N P
Division of Plant Biotechnology, ICAR-Indian Institute of Pulses ResearchKanpur, India.
Division of Crop Protection, ICAR-Indian Institute of Pulses ResearchKanpur, India.
Front Plant Sci. 2017 Aug 21;8:1423. doi: 10.3389/fpls.2017.01423. eCollection 2017.
Domain swapping and generation of chimeric insecticidal crystal protein is an emerging area of insect pest management. The lepidopteran insect pest, gram pod borer ( H.) wreaks havoc to chickpea crop affecting production. Lepidopteran insects were reported to be controlled by () genes. We designed a plant codon optimized chimeric gene () using three domains from three different genes (domains I, II, and III from , , and , respectively) and expressed it under the control of a constitutive promoter in chickpea (. DCP92-3) to assess its effect on gram pod borer. A total of six transgenic chickpea shoots were established by grafting into mature fertile plants. The regenerated (organogenetic) shoots were selected based on antibiotic kanamycin monosulfate (100 mg/L) with transformation efficiency of 0.076%. Three transgenic events were extensively studied based on gene expression pattern and insect mortality across generations. Protein expression in pod walls, immature seeds and leaves (pre- and post-flowering) were estimated and expression in pre-flowering stage was found higher than that of post-flowering. Analysis for the stable integration, expression and insect mortality (detached leaf and whole plant bioassay) led to identification of efficacious transgenic chickpea lines. The chimeric expressed in chickpea is effective against gram pod borer and generated events can be utilized in transgenic breeding program.
结构域交换和嵌合杀虫晶体蛋白的产生是害虫治理的一个新兴领域。鳞翅目害虫豆荚螟对鹰嘴豆作物造成严重破坏，影响产量。据报道，鳞翅目昆虫可由（）基因控制。我们使用来自三个不同（）基因的三个结构域（分别来自、和的结构域I、II和III）设计了一个植物密码子优化的嵌合（）基因，并在鹰嘴豆（. DCP92 - 3）的组成型启动子控制下进行表达，以评估其对豆荚螟的影响。通过嫁接到成熟的可育植株上，共建立了六个转基因鹰嘴豆芽。根据抗生素硫酸卡那霉素（100 mg/L）筛选再生（器官发生）芽，转化效率为0.076%。基于基因表达模式和多代昆虫死亡率，对三个转基因事件进行了深入研究。估计了豆荚壁、未成熟种子和叶片（开花前和开花后）中的蛋白质表达，发现开花前阶段的表达高于开花后。通过对稳定整合、表达和昆虫死亡率（离体叶片和整株生物测定）的分析，鉴定出了有效的转基因鹰嘴豆品系。在鹰嘴豆中表达的嵌合（）对豆荚螟有效，所产生的事件可用于转基因育种计划。
