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【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。
【Objective】 The objective of this study was to inhibit the expression of ppo in wheat grain by RNAi strategy and to obtain new transgenic wheat germplasms with low PPO activity and high whiteness. 【Method】 The RNAi vector pBAC47P-ppoIR was constructed by using the wheat endosperm-specific promoter 1Dx5 promoter and pGAC47P-ppoIR vector was co-transformed with Zhongyou 9507 embryo callus , Obtained T0 transgenic plants. The transgenic plants and their offspring lines were screened by molecular identification (PCR, Southern hybridization, semi-quantitative RT-PCR, Northern blotting), enzymatic activity screening and whiteness determination. 【Result】 Twenty-seven positive T0 transgenic plants were obtained. Transgenic plants and their offspring PCR and Southern hybridization detection showed that RNAi components have been stably integrated into T1 of 12 lines. The results of semi-quantitative RT-PCR and Northern blot showed that the expression level of ppo in 20 T2 plants was significantly lower than that of the control. Analysis of PPO isoenzyme activity in transgenic T2 grain filling stage showed that PPO activity of 6 lines weakened. The whiteness of the surface of T4 grain showed that the whiteness of the five lines were higher than the control, of which two lines were significantly effective. 【Conclusion】 The expression of RNAi can inhibit the expression of ppo in grain, reduce the activity of PPO isoenzyme and increase the whiteness of wheat noodles obviously, so as to provide the material for the improvement of whiteness and quality breeding of wheat flour.