两种非标记定量技术在筛选草莓雌蕊发育差异表达蛋白中的应用与比较  

Application and Comparison of Two Label-free Quantitative Techniques for Screening Differentially Expressed Proteins in Strawberry(Fragaria ananassa)Gynoecium Development

作　　者：裘劼人[1] 柴伟国[1] 周历萍[1] 王淑珍[1] QIU Jie-Ren;CHAI Wei-Guo;ZHOU Li-Ping;WANG Shu-Zhen(Institute of Biotechnology,Hangzhou Academy of Agricultural Sciences,Hangzhou 310024,China)

机构地区：[1]杭州市农业科学研究院生物技术研究所,杭州310024

出　　处：《农业生物技术学报》2020年第8期1331-1342,共12页Journal of Agricultural Biotechnology

基　　金：杭州市农业科研自主申报项目(20191203B53);浙江省基础公益研究计划(LGN18C130002);杭州市农科院科技创新基金(2020HNCT-21)。

摘　　要：研究雌蕊发育进程对育种具有重要意义.蛋白质组学技术是强有力的系统生物学研究工具,目前草莓(Fragaria ananassa)雌蕊发育方面的蛋白质组学研究尚未见报道.为筛选适宜草莓雌蕊大规模蛋白质组学分析的方法,本研究采用数据依赖性采集(data-dependent acquisition,DDA)和非数据依赖性采集(data-independent acquisition,DIA)技术对小蕾期、初绽期和盛开期3个发育时期的草莓雌蕊进行非标记定量蛋白质组研究.从蛋白鉴定量、肽段离子峰强变异系数(coefficient of variation,CV)和数据缺失率3个方面评估两种技术的优劣.结果表明,DIA数据的覆盖度、重现性和准确度均高于DDA.DDA共鉴定到3687个蛋白质,而DIA鉴定到5341个蛋白质;DDA的3个时期肽段离子峰强平均CV值分别为28.4%、33.3%和28.4%,而DIA仅为10.4%、10.9%和11.9%;DDA的3个时期组内数据缺失率分别为12.4%、13.0%、13.9%,而DIA为2.7%、2.2%、3.4%.对DIA筛选到的差异表达蛋白(differentially expressed proteins,DEPs)进行基因本体(Gene Ontology,GO)功能注释,小蕾期和初绽期的DEPs主要注释到有丝分裂、甲基化和次生代谢物合成等过程;而初绽期和盛开期的DEPs则注释到能量代谢、光合作用和初级代谢等过程;KEGG富集分析显示,过氧化物酶4(peroxidase 4,POD4)等多个过氧化物酶参与了木质素合成,其表达量随花的发育进程而上升.对甲基化相关蛋白进一步分析,挖掘到开花调控相关的精氨酸N-甲基转移酶5(protein arginine methyltransferase 5,PRMT5)、转座子沉默相关的DNA(胞嘧啶5)-甲基转移酶3(DNA(cytosine 5)-methyltransferase 3,CMT3)和香气物质合成相关的苔黑酚O-甲基转移酶(orcinol O-methyltransferase,OOMT)等多个具有重要生理功能的蛋白质.并且PRMT5、CMT3和POD4等重要蛋白质仅在DIA数据鉴定到,进一步证明了DIA相对DDA的优越性.本研究发现DIA为草莓雌蕊大规模蛋白质组学分析的较优方法,并初步探索了Studying the developmental process of gynoecium is very important for breeding.Proteomic technique is a powerful tool for system biology research,and proteomics in strawberry(Fragaria ananassa)gynoecium development has not been reported to date.In order to select a suitable method for large-scale proteomics analysis of strawberry gynoecium,DDA(data-dependent acquisition)and DIA(data-independent acquisition)techniques were applied for label-free quantitative proteomics analysis of strawberry gynoecium at 3 developmental stages of small bud stage,early blooming stage and full blooming stage.The two techniques were evaluated in terms of protein identification quantity,coefficient of variation(CV)of peptide ion peak intensity and the proportion of missing data.The result shows that the coverage,reproducibility,and accuracy of DIA data were higher than those of DDA.A total of 3687 proteins were identified in DDA and 5341 proteins were identified in DIA;The average CV value of peptide ion peak intensity for the 3 periods were 28.4%,33.3%and 28.4%in the DDA data,respectively,while in DIA data the CV values were only 10.4%,10.9%and 11.9%.Gene ontology(GO)function analysis was performed in the differentially expressed proteins(DEPs)screened by DIA.The DEPs between small bud stage and early blooming stage were mainly annotated to mitosis,methylation,and secondary metabolite synthesis,while the DEPs between early and full blooming stage were annotated to energy metabolism,photosynthesis,and primary metabolism.KEGG enrichment analysis showed that multiple peroxidases including peroxidase 4(POD4)were involved in lignin synthesis,and their expression increased simultaneously with the flower developmental process.Further analysis of methylation-related proteins tapped into multiple proteins with important physiological functions,including flowering-regulated protein arginine methyltransferase 5(PRMT5),transposon silencing related DNA(cytosine 5)-methyltransferase 3(CMT3),and aromatic compounds synthesis related orcinol O-methyl

关 键 词：草莓 雌蕊 蛋白质组学 数据依赖性采集(DDA) 非数据依赖性采集(DIA) 过氧化物酶 甲基转移酶 

分 类 号：S663.9[农业科学—果树学]