甘蔗乙醇脱氢酶基因的克隆与表达

发布时间：2018-11-14 15:25

【摘要】：乙醇脱氢酶基因(Alcohol dehydrogenase,ADH)在植物抵御涝害、冷害、干旱等逆境中起着重要作用.基于前期已构建的甘蔗受黑穗病菌胁迫后基因差异表达的抑制消减杂交(Suppression subtractive hybridization,SSH)文库,利用电子克隆和RT-PCR技术,从甘蔗品种ROC22中获得一条ADH基因的c DNA全长序列,命名为Sugarcane Alcohol Dehydrogenase(Sc ADH;Gen Bank登录号为KJ577593).生物信息学分析显示,Sc ADH基因全长为1 644 bp,含有1 140bp的开放阅读框,编码一个379个氨基酸的蛋白质.Sc ADH蛋白为稳定、亲水的酸性非分泌蛋白,定位于叶绿体基质上.蛋白二级结构元件多为无规则卷曲,具有典型的ADH蛋白结构域以及NAD和锌的结合位点.实时荧光定量PCR(Real-time quantitative PCR)表达分析表明,该基因在甘蔗各组织中组成型表达,其中根中表达量最高,而叶中的表达量最低.在SA和Me JA胁迫下,Sc ADH基因的表达趋势为"先扬后抑",均在胁迫6 h达到最大值,分别为对照的7.34倍和11.81倍.在ABA胁迫下,该基因均上调表达,在胁迫12 h达到最大值,为对照的5.53倍.在黑穗病菌胁迫下,该基因在感病品种ROC22中的表达受到抑制,而在抗病品种YC05-179中的表达模式为"先抑后扬".综上推测Sc ADH基因在甘蔗应答非生物胁迫和生物胁迫中发挥重要作用.
[Abstract]:Ethanol dehydrogenase gene (Alcohol dehydrogenase,ADH) plays an important role in plant resistance to waterlogging, chilling and drought. Based on the previously constructed suppressive subtractive hybridization (Suppression subtractive hybridization,SSH) library of differentially expressed genes of sugarcane under Smut stress, a full-length sequence of ADH gene c DNA was obtained from sugarcane variety ROC22 by electronic cloning and RT-PCR techniques. Named Sugarcane Alcohol Dehydrogenase (Sc ADH; Gen Bank login number is KJ577593). Bioinformatics analysis showed that the full length of, Sc ADH gene was 1 644 bp, with an open reading frame of 1 140bp, encoding a 379-amino acid protein,. Sc ADH protein, which was stable and hydrophilic acid non-secretory protein, located on chloroplast matrix. Most of the protein secondary structural elements are irregularly curled with typical ADH protein domains and binding sites of NAD and zinc. Real-time fluorescence quantitative PCR (Real-time quantitative PCR) analysis showed that the expression of the gene was the highest in the root and the lowest in the leaf. The expression trend of, Sc ADH gene under SA and Me JA stress was "first rising and then suppressive", and reached the maximum at 6 h, which was 7.34 times and 11.81 times of that of the control, respectively. Under ABA stress, the expression of the gene was up-regulated, and reached the maximum at 12 h, 5.53 times of the control. Under the stress of smut, the expression of the gene was inhibited in the susceptible variety ROC22, but the expression pattern in the resistant variety YC05-179 was "suppresses first and then rose". It is concluded that Sc ADH gene plays an important role in the response of sugarcane to abiotic stress and biological stress.
【作者单位】：福建农林大学农业部福建甘蔗生物学与遗传育种重点实验室;
【基金】：福建省杰出青年科学基金(2015J06060) 现代农业产业技术体系建设专项资金(CARS-20) 国家公益性行业(农业)科研专项(201503119) 福建省高等学校新世纪优秀人才支持计划(JA14095)资助~~
【分类号】：Q943.2;S566.1

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