利用RNAi技术抑制籽粒PPO合成改良小麦面粉白度的研究

摘要

[Objective] To develop the transgenic wheat germplasm with low flour PPO activity and high whiteness by RNA interference (RNAi) to specially down-regulate the expression of grain ppo genes.[Method] The RNAi vector pBAC47P-ppoIR,aimed at silencing wheat grain ppo genes,was transformed into the callus from Zhongyou9507 immature embryo by biolistic bombardment,and the transgenic plants were obtained after resistance selection,differentiation and regeneration,and then screened by PCR,Southern blotting,half-quantitative RT-PCR,northern blotting and enzyme activity analysis.[Result] A total of 27 transgenic T0 plants were obtained.Twelve transgenic lines of T1 generation confirmed by PCR and genomic Southern blotting showed successful integration of RNAi construct into the wheat genome.The expression of grain ppo genes of 20 T2 plants was significantly lower than that of control.Enzyme activity analysis of T2 grains at filling stage showed that the PPO activity of 6 lines reduced obviously.Dough whiteness analysis of T4 grains showed that the whiteness of 5 lines increased clearly and 2 lines improved significantly.[Conclusion] The RNAi vector pBAC47P-ppoIR could down-reglulate the expression of grain ppo genes,reduce the PPO activity,and increase the dough whiteness significantly,so this study will provide wheat breeder with breeding material for whiteness and quality improvement.%[目的]通过RNAi策略抑制小麦籽粒ppo的表达,获得籽粒PPO酶活性低、白度高的转基因小麦新种质.[方法]构建了以小麦胚乳特异表达启动子1Dx5启动子驱动的籽粒ppo的RNAi载体pBAC47P-ppoIR,利用基因枪将该载体与含bar的表达载体基因枪共转化中优9507幼胚愈伤组织,获得T0转基因植株.通过分子鉴定(PCR、Southern杂交、半定量RT-PCR、Northern杂交)、酶活筛选和白度测定等方法对转基因植株及其后代株系进行筛选.[结果]获得了27株阳性T0转基因植株.转基因植株及其后代经PCR和Southern杂交检测表明,RNAi构件已经稳定整合到T1的12个株系中.半定量RT-PCR和Northern杂交结果表明有20株T2籽粒中ppo表达水平明显低于对照.对转基因T2灌浆期籽粒进行PPO同功酶活性分析表明,有6个株系的PPO活性有所减弱.对T4籽粒的面片白度测定表明,5个株系的白度均比对照有所提高,其中2个株系效果显著.[结论]RNAi技术的表达能抑制籽粒ppo表达,降低PPO同功酶的活性,明显提高小麦面片白度,从而为小麦面粉白度的改良、品质育种提供了材料.