Ribeiro Thuanne Pires, Arraes Fabricio Barbosa Monteiro, Lourenço-Tessutti Isabela Tristan, Silva Marilia Santos, Lisei-de-Sá Maria Eugênia, Lucena Wagner Alexandre, Macedo Leonardo Lima Pepino, Lima Janaina Nascimento, Santos Amorim Regina Maria, Artico Sinara, Alves-Ferreira Márcio, Mattar Silva Maria Cristina, Grossi-de-Sa Maria Fatima
Brasilia Federal University (UnB), Brasília, DF, Brazil.
Embrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil.
Plant Biotechnol J. 2017 Aug;15(8):997-1009. doi: 10.1111/pbi.12694. Epub 2017 Mar 2.
Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2 analyses revealed that T GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T GM cotton plants, ranging from approximately 3.0 to 14.0 μg g fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 μg g fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.
本文首次报道了能有效防治棉铃象鼻虫(CBW)的转基因(GM)棉花植株,棉铃象鼻虫是南美洲最具破坏力的棉花害虫。这项研究成功培育出了一种新型转基因棉花,它对棉铃象鼻虫具有高抗性,这种抗性由苏云金芽孢杆菌(Bt)基因编码的Cry10Aa毒素赋予。携带由棉花泛素化相关启动子uceA1.7驱动的cry10Aa基因的植物转化载体,通过基因枪转化法导入巴西棉花品种。定量PCR(qPCR)分析显示,cry10Aa在转基因棉花叶片和花芽组织中均有高转录水平。Southern杂交和基于qPCR的分析表明,转基因植株有1个或2个转基因拷贝。对Cry10Aa蛋白表达的定量和定性分析表明,转基因棉花植株的花芽和叶片组织中蛋白表达水平各不相同,鲜组织中约为3.0至14.0μg/g。分别用转基因棉花叶片和花芽喂养成虫和幼虫进行的棉铃象鼻虫敏感性生物测定表明,具有显著的昆虫毒性作用和高水平的棉铃象鼻虫死亡率(高达100%)。分子分析表明,由于Cry10Aa毒素在两个组织中的表达水平仍然很高,鲜组织中为4.05至19.57μg/g,棉铃象鼻虫死亡率仍保持在100%左右,转基因在T代中保持了稳定性以及对棉铃象鼻虫的昆虫毒性作用。总之,这些Cry10Aa转基因棉花植株在防治毁灭性棉铃象鼻虫害虫方面取得了重大进展,并可能对棉花农业综合企业产生重大影响。