摘要
【目的】探讨猕猴桃Cu/ZnSOD的序列特征及其在不同组织部位与果实贮藏中的表达特征,丰富超氧化物歧化酶与果实采后品质关系的研究。【方法】以‘米良1号’猕猴桃为材料,采用RT-PCR法克隆得到2个Cu/ZnSOD家族成员,分别命名为AdCSD1和AdCSD2,通过生物信息学软件分析序列特征、功能结构域、基因结构及系统进化关系,应用qRT-PCR技术研究它们在不同组织部位、不同贮藏温度和激素处理后的表达模式。【结果】AdCSD1和AdCSD2的开放阅读框分别为459和621 bp,分别编码152和206个氨基酸,登录号分别为MF034869和KY471357。生物信息学分析结果表明,AdCSD1和AdCSD2对密码子选择偏性不强,均编码具亲水性的稳定酸性蛋白,且包含完整的Cu/ZnSOD结构域和保守的Cu2+/Zn2+结合位点。AdCSD1和AdCSD2的外显子内含子组成不同,且它们的序列一致性较低,在系统进化树中位于2个不同的分支,可能源自不同的基因祖先。AdCSD1和AdCSD2在猕猴桃各组织部位(根、茎、叶、花、幼果和成熟果)均有表达,但表达水平不同。猕猴桃果实中AdCSD1和AdCSD2在4℃低温贮藏过程中的表达量均比25℃常温贮藏中同期的表达量低;它们在脱落酸处理后的表达均下调,而在赤霉素处理后的表达模式不同。【结论】AdCSD1和AdCSD2均参与猕猴桃各组织部位活性氧的清除,且在果实低温贮藏中的表达受到抑制,但它们对外源脱落酸和赤霉素的应答模式不同。
【Objective】Superoxide dismutases are very important in antioxidant systems.Among them,copper/zinc superoxide dismutase(Cu/ZnSOD)is mostly abundant in plants and is involved in plant growth and development,stress responses and fruit ripening and senescence.The activities of superoxide dismutases in kiwifruit during storage were reported to change significantly.However how they were encoded and expressed still remians unknown.In view of this,we analyzed the sequence characteristics of kiwifruit Cu/ZnSOD genes and their expression patterns in different tissues and under different storage conditions in order to enrich the study of superoxide dismutase and postharvest fruit qualities of kiwifruit.【Methods】The roots,stems,leaves,flowers,young fruits(16DAF,days after flowering)and ripe fruits(160 days after flowering)of Actinidia chinensis var.deliciosa‘Miliang-1’were collected respectively as materials for tissue expression analysis.For low temperature treatment,ripe fruits(160 DAF)were stored at 4℃and sampled on 0,1,3,5,7,9 and 11th d.For abscisic acid(ABA)treatment,ripefruits were soaked in 50 mg·L^-1 ABA for 2 min and then stored at 25℃for 0,1,3,5,7 and 9 d.For gibberellin(GA3)treatment,ripe fruits were soaked in 50 mg·L^-1 GA3 for 2 min and then stored at 25℃for0,1,3,5,7,9 and 11 d.The ripe fruits stored at 25℃and sampled on 0,1,3,5,7,9 and 11th d wereused as controls.The RNAprep Pure Plant Kit was used for the extraction of total RNAs from differenttissues of Actinidia chinensis var.deliciosa‘Miliang-1’.cDNAs for gene amplification and quantitativePCR were synthesized,respectively,using SMART?RACE cDNA Amplification kit and TransScript?All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit.Based on the genome data of Actinidiachinensis var.chinensis‘Hongyang’,primers were designed using DNAMAN software for the amplificationof candidate Cu/ZnSOD genes.Then,their sequence characteristics,functional domains,genestructures and systemic phylogenetic relationships were analyzed using bioinformatics softwares.Theirexpression patterns of the genes in different tissues,under different storage temperatures and after hormonaltreatments were performed using an Eppendorf Real-time PCR detection instrument and Trans-Start?Top Green qPCR SuperMix.The actin isoform B gene was used as an internal control gene.Relativeexpression levels of Cu/ZnSOD genes were calculated using the 2-ΔΔCt method.【Results】Two Cu/Zn-SOD genes were isolated from Actinidia chinensis var.deliciosa‘Miliang-1’and were named as AdCSD1and AdCSD2,respectively.Sequence analysis showed that AdCSD1 had an open reading frame(ORF)of 459 bp and encoded 152 amino acids,while the ORF of AdCSD2 was 621 bp encoding 206amino acids.Their accession numbers were MF034869 and KY471357,respectively.Bioinformaticsanalysis showed that both AdCSD1 and AdCSD2 were hydrophilic and stable acidic proteins althoughtheir molecular weight,isoelectric point,instability index and garnd average of hydropathicity weresomewhat different.Subcellular localization analysis showed that AdCSD1 might be located mainly incytoplasm while AdCSD2 might be mainly located in the cytoplasm and chloroplast.Both AdCSD1 andAdCSD2 harbored intact Cu/ZnSOD domians and conserved Cu^2+binding sites and Zn2+binding sites.The codon bias analysis revealed that the effective number of codons in AdCSD1 and AdCSD2 were47.93 and 54.46,respectively,suggesting that their condon bias levels be low.Additionally,their GC3svalues were less than 50%,indicating that they were biased toward the synonymous codons with A or Ton the 3rd site.Gene structure analysis revealed that AdCSD1 had 7 extrons and 6 introns while AdCSD2harbored 8 extrons and 7 introns.All introns of AdCSD1 and AdCSD2 were cleaved at"GT-AG",whichwas consistent with the splicing pattern of eukaryotic introns.Moreover,AdCSD1 and AdCSD2 had lowsequence similarities and were clustered in different evolutionary branching in the phylogenetic tree,which implied that AdCSD1 and AdCSD2 were originated from different ancestral genes.qRT-PCR analysisshowed that AdCSD1 was expressed strongly in leaves,moderately in stems,young fruits,and flowers,and weakly in ripe fruits and roots,while AdCSD2 showed the maximum expression in leaves,followedby young fruits,roots,and stems,and the minimum expression in flowers and ripe fruits.The expressionlevels of both AdCSD1 and AdCSD2 in ripe fruits during storage at 4℃were low,but significantlyincreased when the fruits were stored at 25℃for 5 days and peaked at 7 to 9 days.Moreover,theexpression levels of both AdCSD1 and AdCSD2 in ripe fruits stored at 4℃was lower than that of thesame time stored at 25℃.Additionally,both AdCSD1 and AdCSD2 were down-regulated after beingtreated with abscisic acids,while their expression patterns were different after being treated with gibberellins.【Conclusion】Both AdCSD1 and AdCSD2 were involved in the scavenging of reactive oxygen speciesin various tissues of kiwifruit,and their expressions in the fruits during storage at low temperaturewere inhibited,while their response patterns to exogenous abscisic acid and gillberellin were different.
作者
冯新
赖瑞联
高敏霞
陈文光
吴如健
陈义挺
FENG Xin;LAI Ruilian;GAO Minxia;CHEN Wenguang;WU Rujian;CHEN Yiting(Fruit Research Institute,Fujian Academy of Agricultural Sciences/Research Centre for Engineering Technology of Fujian Deciduous Fruits,Fuzhou 350013,Fujian,China)
出处
《果树学报》
CAS
CSCD
北大核心
2019年第8期980-991,共12页
Journal of Fruit Science
基金
福建省属公益类科研院所基本科研专项（2018R1013-2,2015R1014-3）
福建省自然科学基金（2018J05051,2017J01044）
福建省农业科学院项目（YC2017-2,STIT2017-3-6,A2017-11,AC2017-19）
