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【目的】研究枣树谷胱甘肽过氧化物酶基因(Zj GPX)的功能,为其在果树抗逆基因工程改良中的利用奠定基础。【方法】以‘壶瓶枣’(Ziziphus jujuba Mill.Hupingzao)结果枝构建的c DNA文库中选取一条与其他植物谷胱甘肽过氧化物酶基因有较高同源性的序列为研究对象进行序列分析,预测其功能。通过实时荧光定量技术分析目的基因在盐胁迫及干旱胁迫条件下的表达特性,并构建植物表达载体PEZR(K)-LNY-Zj GPX,运用农杆菌介导法,将Zj GPX转入拟南芥,对转基因及野生拟南芥植株作盐胁迫及干旱胁迫处理,验证其抗逆功能。【结果】Zj GPX编码序列(CDS)长510 bp,编码169个氨基酸。Blast比对发现该基因与多种植物GPX基因具有高度同源性(>80%)。实时荧光定量分析表明,在辣椒枣组培苗中,Zj GPX能够被一定浓度的干旱胁迫和盐胁迫诱导表达,且反应迅速,仅胁迫15 min,其相对表达量与对照相比即出现大幅升高,暗示该基因可能对枣树抗旱性和耐盐性具有重要的作用。结合50μg·m L-1 Kan抗性筛选、PCR验证及激光共聚焦显微镜观察,共获得10株阳性转基因拟南芥株系,干旱及盐胁迫处理结果显示,转基因拟南芥种子萌发率均高于野生型拟南芥;成株在胁迫15 d后,野生型拟南芥出现叶片发黄、萎蔫、整株干枯、死亡的迹象,而转Zj GPX拟南芥生长基本正常。表明过表达Zj GPX拟南芥株系具有良好的耐旱性和耐盐性。【结论】Zj GPX在植物的干旱和盐胁迫应答反应机制中起重要作用,过量表达Zj GPX可提高转基因拟南芥的耐旱和耐盐能力。
【Objective】 The purpose of this study was to investigate the function of ZjGPX, which laid the foundation for the utilization of the ZjGPX gene. 【Method】 The sequences of the c DNA library constructed from the branches of Ziziphus jujuba Mill. Hupingzao were compared with those of other plant glutathione peroxidase genes for sequence analysis , Predict its function. The expression characteristics of the target gene under salt stress and drought stress were analyzed by real-time fluorescence quantitative analysis. The plant expression vector PEZR (K) -LNY-Zj GPX was constructed. The Agrobacterium-mediated transformation of Zj GPX into Arabidopsis thaliana The transgenic and wild Arabidopsis plants were treated with salt stress and drought stress to verify the anti-retrogradation function. 【Result】 The results showed that the sequence of Zj GPX coding sequence (CDS) was 510 bp, encoding 169 amino acids. The Blast alignment showed that the gene has high homology (> 80%) with many plant GPX genes. Real-time fluorescence quantitative analysis showed that ZjGPX could be induced by certain concentration of drought stress and salt stress in the tissue culture of Zizyphus jujuba cv. ZJGPX, and the response was rapid, only 15min stress, the relative expression of ZjGPX was significantly increased compared with the control High, suggesting that the gene may play an important role in drought resistance and salt tolerance of jujube. Ten positive transgenic Arabidopsis lines were obtained by 50μg · m L-1 Kan resistance screening, PCR verification and laser scanning confocal microscopy. The results of drought and salt stress showed that the germination rate of transgenic Arabidopsis thaliana seeds was high In Arabidopsis thaliana, the adult plants showed signs of yellowing, wilting and whole plant wilting and death in Arabidopsis thaliana 15 d after stress, while the growth of Arabidopsis thaliana was normal. The Arabidopsis lines overexpressing Zj GPX showed good drought tolerance and salt tolerance. 【Conclusion】 Zj GPX plays an important role in the response mechanism of drought and salt stress in plants. Overexpression of Zj GPX can improve drought tolerance and salt tolerance of transgenic Arabidopsis thaliana.