外源NO对伽师瓜AOX基因克隆表达及抗氰呼吸的影响
Effect of exogenous nitric oxide on cloning and expression of AOX gene and cyanide resistant respiration in Jiashi muskmelon
-
摘要: 为了探究外源一氧化氮(nitric oxide,NO)熏蒸处理对伽师瓜(Cucumis melo L.)采后贮藏过程中AOX基因表达及抗氰呼吸途径的影响,以伽师瓜为试材,采用外源60μL/L NO熏蒸3 h,以不充入NO气体作为对照,在(5±0.5)℃条件下贮藏49 d。根据甜瓜基因组数据库,通过EST设计引物,克隆获得伽师瓜AOX基因全长序列。得到的基因片段同其他物种的AOX基因具有高度的同源性,命名为Cm AOX。通过拼接获得AOX基因的c DNA全长序列,基因c DNA全长为1300 bp,开放阅读框架为1042 bp,拟编码346个氨基酸,预测分子量为39606.6 Da,等电点为9.00。该基因已在Gene Bank登录,登录号为:KM273124。通过荧光定量分析及抗氰呼吸速率的测定表明,经60μL/L NO处理果实的Cm AOX的表达量和抗氰呼吸速率明显高于对照果实。说明NO处理对伽师瓜采后贮藏过程中抗氰呼吸途径的影响可能与其调控AOX基因的表达密切相关。Abstract: The effects of exogenous nitric oxide fumigation on the expression of AOX gene and the cyanide resistant respiratory pathway in Jiashi muskmelon during the storage were investigated. Jiashi muskmelon ( Cucumis melo L.) as test materials were fumigated with 60 μL/L NO for 3 hours in sealed boxes, not filled with NO gas as control, and then stored at ( 5 ± 0.5) ℃ for49 days.According to the muskmelon genome database, primers were designed by EST, and the full length sequence of Jiashi muskmelon AOX gene was cloned.The c DNA fragment exhibited high homology to the alternative oxidase of other species and could be referred to as Cm AOX.The full-length c DNA sequence of AOX gene was obtained by splicing.The full length c DNA of Cm AOX was 1300 bp in size with a 1042 bp open reading frame ( ORF) , and 346 amino acids was encoded. The predicted molecular weight of Cm AOX was 39606.6 Da, and the isoelectric point was 9.00.The accession number of Cm AOX in Gen Bank database was KM273124. The cyanide resistant respiration rate of Jiashi muskmelon with the treatment of 60 μL/L NO was found to be significantly higher than that of the control. Real-time PCR showed that the expression of Cm AOX with the NO treatment of Jiashi muskmelon was significantly higher than that of control muskmelons during the whole storage period.The NO treatment could induce the up regulation of AOX gene in Jiashi muskmelon during the storage.It was suggested that the effect of NO treatment on the expression of AOX gene of Jiashi muskmelon may be closely related to cyanide resistant respiration regulation during the storage.