甘蔗乙醇脱氢酶基因的克隆与表达  

Cloning and expression of ScADH from sugarcane

作　　者：苏炜华[1] 黄宁 凌辉[1] 刘峰[1] 曾瑞金 苏亚春[1] 吴期滨[1] 高世武[1] 阙友雄[1] 

机构地区：[1]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,福州350002

出　　处：《应用与环境生物学报》2017年第3期474-481,共8页Chinese Journal of Applied and Environmental Biology

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~~

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.The alcohol dehydrogenase gene （ADH） plays an important role in plant responses to abiotic stress such as waterlogging, chilling, and drought. Based on suppression subtractive hybridization libraries constructed from sugarcane with smut infection, a full-length cDNA sequence of sugarcane ADH was cloned by in silico cloning, followed by RT-PCR amplification. This gene was named Sugarcane Alcohol Dehydrogenase, ScADH （GenBank Accession Number： KJ577593）. Bioinformatics analysis showed that ScADH is 1 644 bp in length and contains a complete open reading frame of 1 140 bp, encoding a 379-amino acid sugarcane ADH protein. The stable, acidic, and hydrophilic ScADH protein was predicted to have no signal peptide and be located in the chloroplast stroma. Its main secondary structure is random coils. It has the structure typical of ADH and contains NAD and zinc binding sites. Real-time quantitative PCR analysis revealed that ScADH was constitutively expressed at different levels in various sugarcane tissues. The highest expression levels were observed in roots, whereas the lowest levels were observed in leaves. ScADH was first upregulated and then inhibited after SA and MeJA treatment. The highest expression levels, which was 7.34 times and 11.81 times higher than the control, were observed at 6 h after SA and MeJA treatment, respectively. ScADH transcripts after were upregulated at 12 h after ABA treatment to a level 5.53 times higher than the control, representing the highest inducible expression. During smut pathogen infection, the expression of ScADH was inhibited in the susceptible cultivar ROC22, but was enhanced in the resistant germplasm YC05-179, although inhibition of ScADH expression was also found in the primarily stage of smut infection in YC05-179. Results of this study suggested that ScADH plays an important role in plant responses to biotic and abiotic stresses.

关 键 词：甘蔗 ADH基因 生物信息学 实时荧光定量PCR 非生物胁迫 生物胁迫 

分 类 号：Q943.2[生物学—植物学] S566.1[农业科学—作物学]