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抗坏血酸过氧化物酶(ascorbate peroxidase,APX)是清除活性氧(reactive oxygen species,ROS)的重要酶类。本研究对针刺接种黑粉菌(Sporisorium scitamineum)后的抗、感基因型甘蔗(Saccharum officinarum)进行APX活性测定。结果表明,甘蔗接种黑穗病菌48 h内,抗病品种(崖城05-179)APX活性显著高于感病品种(柳城03-182)(P<0.05)。借助电子克隆技术,结合RT-PCR方法,从甘蔗中分离到一个APX基因,并命名为Sc APX(Gen Bank登录号:KJ7565501)。生物信息学分析显示,Sc APX基因全长1 171bp,包含一个1 038 bp的完整开放阅读框,编码345个氨基酸。Sc APX编码的蛋白不含信号肽,推测其为非分泌蛋白,定位于线粒体基质和叶绿体基质中的概率分别为91.1%和88.7%。实时荧光定量PCR检测结果显示,Sc APX在甘蔗根、芽、叶、蔗皮和蔗髓中均有表达,为组成型表达基因,表达量在蔗皮中最高,叶中最低,前者是后者的19.7倍;在外源因子处理后0~48 h,Sc APX基因的表达受水杨酸(salicylic acid,SA)、茉莉酸甲酯(methyl jasmonate,Me JA)、过氧化氢(hydrogen peroxide,H2O2)、脱落酸(abscisic acid,ABA)、氯化钠(sodium chloride,Na Cl)和聚乙二醇(polyethylene glycol,PEG)诱导,SA、Me JA和H2O2处理后的Sc APX转录本积累量峰值较外源激素(ABA)和环境(Na Cl和PEG)胁迫早。前者的Sc APX转录本变化呈现出胁迫初期积累量增加,达到峰值后又逐步下降的特点;在同样的测试时间(处理后24 h)内,ABA、Na Cl和PEG处理后Sc APX转录本在达到峰值后未见下降。虽然在外源胁迫下,Sc APX表达量变化存在明显差异,但其表达均表现出正响应上述外源因子对甘蔗的胁迫。本研究为后续深入鉴定该基因的功能与进一步应用提供基础资料。
Ascorbate peroxidase (APX) is an important enzyme that scavenges reactive oxygen species (ROS). In this study, the APX activity of resistant and susceptible sugarcane (Saccharum officinarum) after inoculation with Sporisorium scitamineum was determined. The results showed that the APX activity of resistant varieties (Yacheng 05-179) was significantly higher than that of susceptible varieties (Liucheng 03-182) within 48 h (P <0.05). An APX gene was isolated from sugarcane by electronic cloning and RT-PCR and named as Sc APX (Gen Bank accession number: KJ7565501). Bioinformatics analysis showed that Sc APX gene was 1 171 bp in length and contained a complete open reading frame of 1 038 bp encoding 345 amino acids. The protein encoded by Sc APX did not contain the signal peptide and was presumed to be a non-secreted protein with a probability of 91.1% and 88.7%, respectively, located in the mitochondrial matrix and chloroplast matrix. Real-time quantitative PCR results showed that Sc APX was expressed in sugarcane roots, buds, leaves, cane husk and pith, which were constitutively expressed genes with the highest expression in cane husk and the lowest in leaves, the former being the latter 19.7-fold higher than that of the control group. Sc APX gene expression was inhibited by salicylic acid (SA), methyl jasmonate (Me JA), hydrogen peroxide (H2O2) ), Abscisic acid (ABA), sodium chloride (Na Cl) and polyethylene glycol (PEG), the peak value of Sc APX transcripts accumulation after treatment with SA, Me JA and H2O2 Compared with ABA and NaCl and PEG, the stress was earlier. The former Sc APX transcript showed the accumulation increased in the initial stage of stress and then decreased gradually after reaching the peak value. Sc APX transcripts of ABA, NaCl and PEG treated in the same test time (24 h after treatment) After reaching the peak no decline. Although the expression level of Sc APX varied significantly under exogenous stress, the expression of Sc APX showed a positive response to stress of sugarcane under the above exogenous factors. This study provides basic information for further identification of the function and further application of this gene.