干旱和盐胁迫条件下枣树谷胱甘肽过氧化物酶基因(ZjGPX)的差异表达及功能分析

Differential Expression and Functional Analysis of Glutathione Peroxidase Gene from jujube(Zj GPX ) Under Drought and Salt Stresses

【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】As a foundation for further studying on the application of fruit stress tolerance genetic engineering, this study analyzed the function of a c DNA encoding GPX, which was isolated from the fruit-bearing shoot of Ziziphus jujuba Mill. Hupingzao and designated as Zj GPX（Gen Bank accession No. AB608053）. 【Method】 The Zj GPX sequence was analyzed and expression functions were forecasted by using bioinformatics analysis tool first. The differential expressions of Zj GPX on drought and salt stresses were tested by real-time quantitative PCR, and a vector named PEZR（K）-LNY-Zj GPX was constructed and introduced into Arabidopsis by floral dip method. Transgenic and wild Arabidopsis plants were tested under Na Cl and drought stresses to verify Zj GPX’s function.【Result】Sequence analysis indicated that Zj GPX was 510 bp in length, encoding 169 amino acids. Blast analysis exhibited that Zj GPX was highly homologous to other GPXs（〉80%）. Real-time quantitative PCR analysis showed that Zj GPX expression could be rapidly induced by drought and salt stresses just in 15 minutes in Z. jujuba Mill. Lajiaozao test tube seedling. After agrobacterium-mediated transformation, 10 Zj GPX transgenic Arabidopsis lines were obtained. Results of the drought resistance and salt tolerance tests pointed out that in the stress treatments, the transgenic plants exhibited higher seed germination rates than the untransformed control plants. After 15 days of stress treatment, the untransformed control plants showed signs of death, but the transgenic plants maintained normal growth.【Conclusion】The results showed that Zj GPX was likely related to drought and salt resistances and over-expression of Zj GPX gene increased the drought resistance and salt tolerance of the transgenic Arabidopsis.