桃PpCuAO家族基因鉴定及在果实发育和成熟中的表达分析

Genomic identification and expression pattern of copper-amine oxidase genes during peach fruit development and ripening

【目的】明确铜胺氧化酶(CuAO)基因与桃果实发育和成熟过程中多胺积累的关系,探究CuAO介导的多胺分解在桃果实成熟中的作用。【方法】采用乙腈浸提法提取黄水蜜桃果实发育过程中的多胺,采用高效液相质谱联用方法测定多胺含量。挖掘并分析桃基因组CuAO基因家族成员在不同组织及桃果实不同发育时期的表达水平,并利用转基因技术验证关键成员在多胺分解代谢中的作用。【结果】多胺含量分析发现,伴随着桃果实发育和成熟过程,多胺含量逐渐降低。在桃基因组中鉴定了4个CuAO候选基因(PpCuAO1~4),各基因呈现明显的组织表达特异性,其中PpCuAO4在果实发育后期受到明显诱导。利用外源多胺处理及转基因技术分析,表明PpCuAO4可能参与亚精胺(spermidine,Spd)的氧化分解代谢。【结论】PpCuAO4介导的多胺分解是导致桃果实成熟过程中多胺积累减少的重要原因。

【Objective】Peach(Prunus persica L.)is one of the important fruits worldwide.However,peach fruits are highly perishable and deteriorate after ripening.Many studies have shown that polyamines(PAs)serve as endogenous anti-senescence agents.PAs are usually found at high levels during early stages of fruit development but at relatively low levels in mature fruits.Recent studies report that PA catabolism is associated with decreased PA content during fruit ripening.In addition,there is an increased interest in the function of PA catabolism during fruit ripening,but little is known about the role of PA catabolism during the ripening of peach Putrescine(Put),spermidine(Spd)and spermine(Spm)are the most common PAs in plants.PA catabolism is mediated by two kinds of amine oxidases,copperamine oxidase(CuAO)and polyamine oxidase(PAO).It has been reported that CuAOs play important functions in plant growth,fruit development and ripening,as well as abiotic and biotic stress response.However,the potential role of CuAOs in peach fruit development and ripening are still unknown.This study aimed to identify the CuAO genes in peach genome and investigate the potential function of CuAO genes in PA catabolism as well as peach fruit development and ripening.【Methods】The free PA during Huangshuimi peach fruit development and ripening was ertracted by acetonitrile and its concentration was detected with HPLC-MS method.Genes encoding potential copper-containing amine oxidases(CuAOs),which are involved in PA catabolism,were identified across the peach genome.The expression patterns of PpCuAOs were analyzed in different tissues and different development stages of peach fruit,as well as with the treatments of exogenous PAs.The relevance of PA concentration and the expression of PpCuAOs was analyzed.Transient transformation system was used to investigate the function of PpCuAO4 in PA catabolism.The expression level of PpCuAO4,NtCuAO1 and NtMPO1 were analyzed in control and tobacco transgenic lines.DAB staining and detection of PA contents were carried out for transgenic lines.【Results】The contents of free PAs including Put,Spd and Spm markedly decreased as the peach fruit ripened.Four putative CuAO genes(PpCuAO1-PpCuAO4)were identified in peach using the recently released genome database.The PpCuAO genes exhibited tissue-specific expression patterns.Specifically,PpCuAO1 and PpCuAO2 exhibited the highest expression level in old leaves and the lowest expression level in flowers.Conversely,PpCuAO3 and PpCuAO4 presented the highest transcription level in flowers and the lowest expression level in fruits.The PpCuAO genes also exhibited different expression patterns during peach fruit development and ripening.The expression level of PpCuAO1 decreased during fruit development and ripening,except a slight increase at S2/S3 stage.Interestingly,the expression level of PpCuAO2 significantly increased from S1 to S3 stage and exhibited the highest expression level at S2/S3 stage,which was nearly 20 folds of S1 level.Subsequently,the expression level of PpCuAO2 sharply decreased at S3/S4 stage and kept a low level up to S4 stage.The mRNA level of PpCuAO3 gradually increased during fruit development and ripening and exhibited two peaks at the stage of S3/S4(5 folds of S1 level)and S4(10 folds of S1 level),respectively.The transcript level of PpCuAO4 gradually increased during fruit development and ripening except a slight decrease at S2 stage.The transcription of PpCuAOs with exogenous PAs treatment was also investigated.The result showed PpCuAO3 was remarkably induced by Put treatment.The expression of PpCuAO4 was dramatically induced by Spd treatment while obviously inhibited by Put and Spm treatments.To further investigate the function of the PpCuAOs in PA catabolism,PpCuAO4 was selected and transiently expressed in tobacco.The expression level of PpCuAO4 significantly increased in transgenic lines while the transcript level of NtCuAO1 and NtMPO1 exhibited no difference between control and transgenic lines.The levels of free PAs markedly decreased in PpCuAO4 transgenic lines compared to the empty vector(EV)control line.In addition,the DAB staining of tobacco leaves showed PpCuAO4 transgenic lines produced more H2 O2 than control tobacco plants.These data indicated PpCuAO4 was probably involved in the terminal catabolism of Spd but not Spm.【Conclusion】In our study,the contents of free PAs markedly decreased as the peach fruit ripened.Four putative CuAO genes were identified in peach genome.The PpCuAO1-4 genes exhibited obviously tissue-specific expression patterns.The expression level of PpCuAO4 increased during fruit development and ripening and was significantly induced by Spd treatment but inhibited by Put and Spm treatments.In addition,the levels of free PAs markedly decreased in PpCuAO4 transgenic lines.These results suggested PpCuAO4 was probably involved in Spd terminal catabolism.Furthermore,PpCuAO4-mediated PA catabolism was associated with decreased PA content during peach fruit development and ripening.Our study provides valuable knowledge for better understanding the roles of PA catabolism in peach development and ripening.However,the specific roles of PpCuAOs,especially PpCuAO4,in peach fruit ripening needs to be further investigated.