Abstract：
The most crucial yield constraint of pigeon pea is susceptibility to the pod borer Helicoverpa armigera, which causes extensive damage and severe economic losses every year. The Agrobacterium-mediated plumular meristem transformation technique was applied for the development of cry1Ac transgenic pigeon pea. Bioactivity of the cry1Ac gene was compared based on integration and expression driven by two promoters, the constitutive CaMV35S promoter and the green-tissue-specific ats1A promoter, in those transgenic events. The transgenic events also contained the selectable marker gene nptII flanked by loxP sites. Independent transgenic events expressing the Cre recombinase gene along with a linked bar selection marker were also developed. Integration and expression patterns of both cry1Ac and cre were confirmed through Southern and western blot analysis of T1 events. The constitutive expression of the Cry1Ac protein was found to be more effective for conferring resistant activity against H. armigera larvae in comparison to green-tissue-specific expression. Constitutively expressing Cry1Ac T1 events were crossed with Cre recombinase expressing T1 events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T2 events. These events were subsequently analyzed in the T3 generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T3 transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T3 events was found to be 80-100%. The development of such nptII selectable marker-free Cry1Ac-expressing pigeon pea transgenics for the first time would greatly support the sustainable biotechnological breeding program for pod borer resistance in pigeon pea. KEY POINTS: • Constitutive expression of Cry1Ac conferred complete resistance against Helicoverpa armigera • Green-tissue-specific expression of Cry1Ac conferred partial pest resistance • Cre/lox-mediated nptII elimination was successful in constitutively expressing Cry1Ac transgenic pigeon pea events.
摘要：
木豆最关键的产量限制是对豆荚虫棉铃虫的易感性，每年造成广泛的破坏和严重的经济损失。农杆菌介导的植物分生组织转化技术用于cry1Ac转基因木豆的开发。基于两个启动子驱动的整合和表达，比较了cry1Ac基因的生物活性，组成型CaMV35S启动子和绿色组织特异性ats1A启动子，在那些转基因事件中。转基因事件还包含侧翼为loxP位点的选择标记基因nptII。还开发了表达Cre重组酶基因以及连锁条选择标记的独立转基因事件。通过T1事件的Southern和Western印迹分析证实了cry1Ac和cre两者的整合和表达模式。与绿色组织特异性表达相比，发现Cry1Ac蛋白的组成型表达更有效地赋予针对棉铃虫幼虫的抗性活性。将组成型表达Cry1Ac的T1事件与表达Cre重组酶的T1事件杂交。基于杂交的Cre/lox介导的标记基因消除策略被证明产生无nptII的Cry1Ac表达T2事件。随后在T3代中分析这些事件的cre和bar基因的分离。五个表达Cry1Ac的T3转基因木豆事件缺乏nptII标记以及cre-bar基因。发现那些无标记T3事件中的棉铃虫幼虫死亡率为80-100%。首次开发这种无nptII可选择标记的Cry1Ac表达的木豆转基因技术将极大地支持木豆中豆荚虫抗性的可持续生物技术育种计划。关键点：•Cry1Ac的组成型表达赋予了对棉铃虫的完全抗性•Cry1Ac的绿色组织特异性表达赋予了部分害虫抗性•Cre/lox介导的nptII消除成功地组成型表达了Cry1Ac转基因鸽子豌豆事件。
