基于差异蛋白质组学的低温胁迫下蓝莓果蒂凹陷的机理研究

Blueberry fruit is harvested normally at high temperature, and the picking time is concentrated. Refrigeration can effectively retard the senescence of the fruit. However, in the previous research, the applicant found that the blueberry fruit was prone to pedicel pitting during shelf life after cold storage, which influences its commercial value seriously, and has proved that the cold stress was the primary cause of the pedicel pitting which happened at shelf life after the cold storage. And the relationship has also been explored between the pedicel pitting and the reactive oxygen metabolism, energy metabolism, membrane lipid metabolism and the cell ultrastructure. Up to now, the molecular mechanism of this phenomenon is not clear. So in this research, the fluorescent difference two-dimensional electrophoresis will be used to separate and screen the proteins which are related to the pedicel pitting of blueberry. And then the screened differential protein will be authenticated by the mass spectrometric detection. Functional classification and potential metabolic pathways involved will be cleared up by means of bioinformatics analysis. Also, the applicant will use real-time fluorescent quantitative PCR techniques, to select the differentially expressed protein, analyze and verify the corresponding genes on the RNA level, probing into the molecular mechanism of the blueberry pedicel pitting. By integrating the results of proteomics and physiological and biochemical metabolism data in early research, we will ascertain the metabolic pathways related to the blueberry pedicel pitting after the cold stress, which will allow us to elucidate at the molecular mechanism of the pedicel pitting of blueberry. The results of this program will lay theoretical foundation for improving the adjustment measures of blueberries storage.

蓝莓果实的采收正值高温季节，采收期集中，冷藏能够延缓果实的后熟衰老过程。但申请者前期研究发现，蓝莓果实在冷藏后常温货架期，果蒂部易发生凹陷，严重影响了果实的商品品质，并证明了冷藏期间的低温胁迫是引发常温货架期蓝莓果蒂凹陷的主要原因，同时，探索了果蒂凹陷与活性氧代谢、能量代谢、膜脂代谢和细胞超微结构的关系，其分子机制尚不清楚。本项研究拟利用荧光差异双向电泳技术，分离和筛选与蓝莓果蒂凹陷相关的蛋白质，对筛选出的差异蛋白利用质谱检测技术进行鉴定，并通过生物信息学分析明确其功能分类和可能参与的代谢途径。同时，利用实时荧光定量PCR技术，选择主要的差异表达蛋白质的相应基因进行mRNA水平的分析与验证，探索蓝莓果蒂凹陷的分子机理。综合蛋白质组学与前期生理生化代谢的研究结果，探明与低温胁迫后蓝莓果蒂凹陷相关的代谢途径，揭示冷藏蓝莓果蒂凹陷的分子机理，为改进蓝莓果实贮藏调控措施提供理论依据。

冷藏蓝莓果实转入常温货架期后果蒂极易出现凹陷现象，随着果实贮藏时间的延长，果蒂凹陷症状加重，严重影响了蓝莓果实的商品品质。本项目利用iTRAQ技术分析共鉴定蓝莓果蒂凹陷相关差异表达蛋白质163个，82个蛋白表达量上调，81个蛋白表达量下调。.采用GO分析和KEGG分析对鉴定的蛋白质进行功能分析和归类。GO分析表明，可能与冷藏后蓝莓果蒂凹陷相关的差异蛋白质主要参与到代谢进程、绑定、催化活性等进程。KEGG分析表明，鉴定的差异蛋白质主要集中到了碳水化合物代谢中的糖酵解途径和丙酮酸代谢通路，能量代谢中的生物固碳作用通路、氧化磷酸化通路以及脂质代谢中的花生四烯酸代谢通路、脂肪酸生物合成代谢通路等。.为了验证iTRAQ结果，本项目筛选得到12个差异表达显著的蛋白进行Western blot和real-time PCR，对其进行了蛋白水平以及转录组水平上的分析。通过对不同代谢途径差异蛋白表达水平研究表明，差异蛋白的表达和iTRAQ结果的表达趋势基本一致。.针对差异蛋白质比较集中的膜脂代谢通路和能量代谢通路研究发现：冷藏后货架期间，蓝莓果实细胞膜相对电导率和膜脂代谢产物丙二醛含量均呈现上升趋势。随着冷藏时间越长，货架期间磷脂酰胆碱（PC）含量减少越明显，磷脂酸（PA）含量增加越明显，膜损伤越严重，蓝莓果实膜脂过氧化不断加深。PLDα、PLDδ在冷藏过程中均呈下调表达。说明长时间冷藏造成PLD活性及基因表达量提高，加速了对膜脂的降解作用，从而导致细胞膜损伤而发生果蒂凹陷。冷藏后转入常温货架期的蓝莓果实，ATP含量和EC值都显著下降，糖酵解途径中葡糖激酶基因（GK）表达量发生显著下调；氧化磷酸化代谢途径中的NADH脱氢酶基因、细胞色素C氧化酶基因和ATP合成酶（ATPase）基因的表达量都发生下调。说明了能量代谢和膜质代谢与冷藏蓝莓果蒂凹陷是密切相关的。.间歇升温处理可以有效减轻冷藏后蓝莓果蒂凹陷的发生，使果实保持较高的硬度和脂肪酸不饱和度，同时增加脯氨酸含量和CBF转录因子的基因表达量，进而增加蓝莓果实的低温抗性，减少果蒂凹陷的发生。