温州蜜柑果实贮藏寿命生物标记的筛选

Biomarker Sieving for Fruit Storage Life of Satsuma Mandarin(Citrus unshiu Marc.)

【目的】对温州蜜柑果实贮藏期所处生理状态进行分析,挖掘能反映柑橘果实采后贮藏期所处不同生理阶段的生物标记。【方法】通过对温州蜜柑采后贮藏0—50 d的基因芯片数据和初生代谢物数据分析,结合聚类、主成分分析、偏最小二乘法判别分析和支持向量机等机器学习的方法挖掘能够预测温州蜜柑果实贮藏寿命的生物标记,并采用qRT-PCR对标记的可靠性进行验证。【结果】基于多元差异比较分析筛选出了5 232个差异基因;并将采后温州蜜柑分为贮藏初期、中期和后期3个生理阶段。Mapman功能富集分析发现氨基酸代谢和细胞壁代谢与采后温州蜜柑所处的生理状态密切相关;基因本体富集分析表明核小体组装、蛋白酶解等生物过程也发生了明显变化。qRT-PCR验证结果显示,谷胱甘肽S-转移酶(Cit.15371.1.S1_at)实时定量值具有明显的阶段性特征,可作为预测温州蜜柑采后贮藏寿命的基因标记。与芯片数据对应的初生代谢物数据同样可将温州蜜柑采后贮藏期划分为3个生理阶段,且筛选出甘氨酸(Glycine)、3-氨基-6-甲氧基吡啶甲酸(3-Amino-6-methoxypicolinicacid)、丁烯二酸(2-Butenedioic acid)、Cyclohexanone,2-[(dimethylamino)methyl]4个候选代谢物标记。【结论】筛选了5个生物标记物,包括谷胱甘肽S-转移酶、甘氨酸(Glycine)、3-氨基-6-甲氧基吡啶甲酸(3-Amino-6-methoxypicolinicacid)、丁烯二酸(2-Butenedioic acid)、Cyclohexanone,2-[(dimethylamino)methyl],可以用来预测温州蜜柑果实贮藏寿命。

【Objective】Experiments were carried out to study the physiological status of harvested Satsuma mandarin and to discover representative biomarker candidates that represent the status of citrus fruit. 【Method】Based on the data of microarray and primary metabolites of Satsuma mandarin during 0-50 DAH（days after harvest）, approaches of hierarchical clustering, principal component analysis, partial least squares discriminant analysis and support vector machine model were integratively used to assess different physiological status and to discover representative biomarker candidates representing citrus fruit storage process. Additionally, the high reliability of transcriptome data was verified by qRT-PCR. 【Result】A total of 5 232 differentially expressed genes were identified. The post-harvest storage process of Satsuma mandarin could be classified into early stage, middle stage and terminal stage according to the differentially expressed genes. Functional categorization by Mapman showed that the biological process of Satsuma mandarin during storage was closely related to amino compound metabolism and cell wall metabolism. The statistical analysis of gene ontology revealed that nucleosome assembly genes and proteolysis genes were overrepresented. Combined with the verification of qRT-PCR, the expression trend of glutathione S-transferase had obviously stage characteristics, and therefore it can be used as a biomarker evaluating Satsuma mandarin post-harvest storage status. The same classification of physiological stages of Satsuma mandarin during storage was done according to the primary metabolites data. Further, four candidate metabolite biomarkers, including Glycine, 3-Amino-6-methoxypicolinic acid, 2-Butenedioic acid and Cyclohexanone,2-[（dimethylamino）methyl] were screened.【Conclusion】Five biomarkers, including glutathione S-transferase, glycine, 3-amino-6-methoxypicolinic acid, cyclohexanone,2-[（dimethylamino）methyl], 2-butenedioic acid were screened, which could be used to predict the storage life of harvested Satsuma mandarin.