枇杷果实品质形成过程中差异表达蛋白的变化

Study on differentially expressed proteins during the fruit quality formation in loquat(Eriobotrya japonica)

【目的】研究枇杷果实成熟过程中不同发育阶段(青果期、转色期和成熟期)的生理代谢变化及果实品质形成机制。【方法】以‘解放钟’枇杷品种为试验材料,采用蛋白质双向凝胶电泳及MALDI-TOF/TOF-MS技术分离并鉴定蛋白质,采用RT-qPCR技术分析基因表达水平变化,同时应用生理生化技术分析生理代谢变化。【结果】成功鉴定出55个差异表达蛋白,这些蛋白主要参与糖代谢(16%)、三羧酸循环与能量代谢(9%)、抗坏血酸-谷胱甘肽循环(14%)、类胡萝卜素合成及乙烯合成(11%)、蛋白质合成(13%)、细胞结构(8%)、防御与胁迫(9%)以及其它调控(13%)等生物学过程。随着果实成熟,参与糖代谢、三羧酸循环与能量代谢以及类胡萝卜素合成及乙烯合成过程的相关蛋白均上调表达;RT-qPCR的结果与蛋白质丰度变化趋势相一致;同时,糖类物质、类胡萝卜素以及维生素C等的含量不断积累,有机酸含量不断减少。【结论】在蛋白质丰度、生理生化水平以及mRNA水平上的结果共同表明糖代谢、三羧酸循环及能量代谢、类胡萝卜素合成以及乙烯合成等代谢途径在枇杷果实品质形成过程中起着重要作用。这些结果为枇杷果实品质改良提供重要的理论依据,并为今后枇杷新品种的培育奠定分子生物学基础。

【Objective】Loquat[Eriobotrya japonica(Thunb.)Lindl.]is a species of Eriobotrya plants belonging to Maloideae in family Rosaceae,which is an important fruit in Asia that ripens in late spring and early summer.Its fruit contains rich nutrients such as carotenoids,vitamins,minerals and amygdalin in flesh.It also has some health-beneficial effects,for example,expelling phlegm,arresting coughing,moistening lung,promoting appetite and helping digestion.Different regulation metabolisms in 3 different development periods(green fruit period,color changing period and yellow ripening period)were revealed during the fruit quality formation in loquat.【Methods】‘Jiefangzhong’loquat trees were planted in the National Germplasm Resources Nursery for Loquat in Fruit Research Institute,Fujian Academy of Agricultural Sciences,Fuzhou,China.The fruits were harvested during 3 different fruit development periods from 7-year-old loquat trees.Total 27 fruits at each period were randomly divided into three groups,with each group having 9 fruits.The fruits were peeled and the pulps were immediately frozen in liquid nitrogen,and stored at-80℃for further analysis.The proteins of loquat pulp were extracted using the Tris-buffered phenol method.The total proteins were separated by using two-dimensional gel electrophoresis(2-DE)with 24 cm,pH 4-7 linear immobilized pH gradient(IPG)strip in the first dimension,and after isoeletric focusing(IEF),with 12%polyacrylamide gels in the second dimension(namely SDS-PAGE).Differentially expressed protein spots with more than 2-fold differences in abundance at the three fruit development periods were found from the 2-DE gels by using the ImageMaster software.These differentially expressed proteins were identified using the matrix-assisted laser desorption/ionization tandem time-of-flight mass spectroscopy(MALDI-TOF/TOF-MS)and protein database search.The nutrient contents,including the total soluble sugar,reducing sugar,sucrose,carotenoid and ascorbic acid(AsA),were measured by plant biochemistry methods at three fruit development periods.Meanwhile,the total RNA was extracted from the fruit pulp using the CTAB-LiCl method.The relative mRNA expression was analyzed using the RT-qPCR method in a CFX ConnectTM(BioRad)platform.The relative m RNA expression level of each gene was quantified using the 2-ΔΔCtmethod.【Results】To identify the proteins involved in the fruit development periods,2-DE analysis was performed in 3 different development periods,in which more than 1 200 protein spots were detected for each period by using Image Master 2 D Platinum 5.0,respectively.Statistical analysis(p<0.05)revealed a total of 56 protein spots with at least 2-fold differences in abundance as differentially expressed proteins in the green fruit period,color changing period,and yellow ripening period.Among them,36 proteins were found to be up-regulated,12 were down-regulated and the rest were up-regulated or down-regulated at the color changing period and yellow ripening period.These differentially expressed protein spots were excised from the gels,digested,and then analyzed by matching MALDI-TOFTOF/MS data to determine the biochemical characteristics of proteins.Fifty-five proteins were successfully identified,which were divided into 9 classes:sugar metabolism(16%),tricarboxylic acid(TCA)cycle and energy metabolism(9%),AsA-GSH cycle(14%),carotenoid biosynthesis and ethylene formation(11%),amino acid and protein synthesis(13%),cell structure(8%),defense stress(9%),other regulatory proteins(13%)and so on,according to their physiological function.The proteins from sugar metabolism,TCA cycle energy metabolism and carotenoid biosynthesis and ethylene formation were upregulated,while the ones in the AsA-GSH cycle pathway were down-regulated.Visual and biochemical analysis in the fruit development of loquat revealed that the contents of total soluble sugar,reducing sugar,sucrose,carotenoid,and AsA increased with the fruit development,while the organic acid content decreased.To ascertain whether the protein expression levels were correlated with their mRNA contents,several genes encoding proteins related to carotenoids synthesis and ethylene formation metabolism(namely phytoene synthase(PSY),chromoplaste beta lycopene cyclase(CYCB),lycopene beta cyclase(LCYb),lycopene epsilon cyclase(LCYe),beta carotene ring hydroxylase(BCH),zeaxanthin cyclase(ZEP),1-aminocyclopropane-1-carboxylate oxidase(ACO))were examined through quantitative RTPCR analyses.The result of RT-qPCR was similar to protein data.Compared with green fruit period,the relative mRNA expression levels of PSY,BCH,ZEP,LCYb,CYCB,and ACO were remarkably up-regulated in both color changing period and yellow ripening period.【Conclusion】The proteomic analysis of the loquat fruits at different development periods was performed in the present study.Fifty-five differential expressed protein spots were identified by MALDI-TOF/TOF-MS.The consistent results in the physiological analysis and relative mRNA expression level with the protein data reinforced each other to demonstrate an important scientific basis during the quality formation of loquat fruits.The quality formation of loquat fruits was closely related to these pathways including sugar metabolism,TCA cycle energy metabolism,carotenoid biosynthesis and ethylene formation.