Department of Biotechnology, St. Xavier's College (Autonomous), 30, Mother Teresa Sarani, Kolkata - 700016, West Bengal, India.
Department of Genetics, University of Calcutta, 35, Ballygunge Circular Road, Kolkata - 700019, West Bengal, India.
Appl Microbiol Biotechnol. 2022 Apr;106(8):3051-3067. doi: 10.1007/s00253-022-11922-1. Epub 2022 Apr 20.
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 T 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 T events were crossed with Cre recombinase expressing T events. The crossing-based Cre/lox-mediated marker gene elimination strategy was demonstrated to generate nptII-free Cry1Ac-expressing T events. These events were subsequently analyzed in the T generation for the segregation of cre and bar genes. Five Cry1Ac-expressing T transgenic pigeon pea events were devoid of the nptII marker as well as cre-bar genes. H. armigera larval mortality in those marker-free T 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.
木豆的主要产量限制因素是对豆荚螟 Helicoverpa armigera 的易感性，后者每年都会造成广泛的损害和严重的经济损失。应用根癌农杆菌介导的茎尖分生组织转化技术培育 cry1Ac 转基因木豆。基于两个启动子（组成型 CaMV35S 启动子和绿色组织特异性 ats1A 启动子）驱动的整合和表达，比较了 cry1Ac 基因的生物活性。这些转基因事件还含有由 loxP 位点侧翼的选择标记基因 nptII。还开发了表达 Cre 重组酶基因和连接的 bar 选择标记基因的独立转基因事件。通过对 T 事件的 Southern 和 Western blot 分析，证实了 cry1Ac 和 cre 的整合和表达模式。与绿色组织特异性表达相比，Cry1Ac 蛋白的组成型表达被发现对赋予对 H. armigera 幼虫的抗性活性更有效。组成型表达 Cry1Ac 的 T 事件与表达 Cre 重组酶的 T 事件杂交。证明基于杂交的 Cre/lox 介导的标记基因消除策略可产生无 nptII 的 Cry1Ac 表达 T 事件。随后在 T 代中分析这些事件以分离 cre 和 bar 基因。五个无 nptII 标记和 cre-bar 基因的 Cry1Ac 表达 T 转基因木豆事件。这些无标记 T 事件中 H. armigera 幼虫死亡率为 80-100%。首次开发这种无 nptII 选择标记的 Cry1Ac 表达木豆转基因品系，将极大地支持木豆对豆荚螟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 Helicoverpa armigera 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 消除在组成型表达 Cry1Ac 的转基因木豆事件中是成功的。
