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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Development of a Gateway-compatible pCAMBIA binary vector for RNAi-mediated gene knockdown in plants
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Development of a Gateway-compatible pCAMBIA binary vector for RNAi-mediated gene knockdown in plants

机译：植物中RNAi介导的基因敲低的网关兼容的PCAMBIA二元载体的开发

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摘要

RNA interference (RNAi), based on hairpin RNA (hpRNA) expression, plays an important role in functional analysis of plant genes. Traditional methods for making RNAi constructs usually involve multiple time-consuming cloning steps. We have developed a Gateway-compatible binary vector for RNAi-mediated gene knockdown in plants from pCAMBIA2301 and pHANNIBAL vectors. The new plant RNAi binary vector, named pCAMBIA2301-GW-RNAi, has two inverted repeated Gateway cassettes driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter. This enables site-specific recombination at two sites by one Gateway LR reaction without restriction enzymes and ligases. The pCAMBIA2301-GW-RNAi vector's effectiveness was evaluated by Agrobacterium-mediated transient co-expression assays of overexpression and silencing constructs of HvCEBiP in Nicotiana benthamiana followed by western blot analysis. Obtained results show that the developed RNAi vector successfully knocked down 35S-driven expression of HvCEBiP, as expression levels of the encoded HvCEBiP protein were significantly reduced.
机译：基于发夹RNA（HPRNA）表达的RNA干扰（RNAi）在植物基因的功能分析中起重要作用。用于制造RNAi构建体的传统方法通常涉及多次耗时的克隆步骤。我们已经开发了一种从PCAMBIA2301和Phannibal载体的植物中的RNAi介导的基因敲低的网关兼容的二元载体。名为PCAMBIA2301-GW-RNAi的新植物RNAi二进制向量，具有由花椰菜马赛克病毒35s（CAMV 35S）启动子驱动的两条反转的重复网关盒。这使得在两个位点在没有限制酶和连接酶的情况下通过一个网关LR反应来实现特异性的重组。通过农杆菌介导的过表达和沉默的瞬态共同表达测定和尼古尼尼斯·本艾氏菌的耐过表达和沉默构建，随后进行蛋白质印迹分析，评估PCAMBIA2301-GW-RNAi载体的有效性。得到的结果表明，发育的RNAi载体成功敲击了35s驱动的HVCEBIP表达，因为编码的HVCEBIP蛋白的表达水平显着降低。

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    Yu Deshui; Liao Libing; Zhang Yi; Xu Kedong; Zhang Ju; Liu Kun; Li Xiaoli; Tan Guangxuan; Zheng Jurui; He Yong; Xu Changling; Zhao Jinjin; Fu Beibei; Xie Jiaxing; Mao Jie; Li Chengwei;

  • 作者单位

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Henan Key Lab Crop Mol Breeding &

    Bioreactor Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

    Zhoukou Normal Univ Key Lab Plant Genet &

    Mol Breeding Zhoukou 466001 Peoples R China;

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  • 中图分类 医学遗传学;
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