【作者】 王莉；

【导师】 金鹏；

【作者基本信息】 南京农业大学 ， 食品科学与工程， 2020， 博士

【摘要】 桃果实因其色泽艳丽,风味独特,柔软多汁,营养丰富而广受消费者青睐。桃果实成熟于夏季,采后果实呼吸旺盛,常温下极易后熟软化,加之其皮薄质软、汁液丰富,易受到机械损伤和病原菌侵染而腐烂变质,严重影响果实品质。低温贮藏是采后果实保鲜的重要方法,然而桃又是冷敏性果实,在不适宜的低温下长时间贮藏容易引发果实的冷害。热处理作为一种操作简单、环境友好型处理方法而广受重视。甜菜碱作为一种相容性渗透物质,可以起到渗透剂的作用来稳定细胞质的pH和渗透压,同时还可以作为分子伴侣来稳定酶、蛋白质和膜脂等生物大分子的结构和功能。本文以“雨花二号”水蜜桃为试材,研究10 mmolL-1的甜菜碱(Glycine betaine,GB)和45℃的热水(Hot water,HW)处理对冷藏桃果实的品质和冷害的影响,以期揭示GB和HW处理减轻果实冷害的机理,为其在桃果实采后贮运与保鲜的应用提供理论依据。研究结果分述如下:1.研究了 GB和HW处理对冷藏桃果实品质及其冷害的影响,发现桃果实在冷藏21 d转移至货架3d后出现果实褐变等冷害症状。与对照相比,GB和HW处理均减轻了桃果实的冷害症状,维持了较高的出汁率、可溶性固形物(TSS)含量、抗坏血酸和钙铁锌离子含量,从而较好的维持了果实品质。GB和HW处理在维持果实品质上无显著差异。2.转录组(RNA-Seq)研究结果表明,GB处理抑制了磷脂酶D(PLD)、脂氧合酶(LOX)及脂酶(Lipase)基因的表达及酶的活性,诱导了不饱和脂肪酸合成相关基因的表达(如去饱和酶),提高了活性氧清除系统中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)基因的表达及酶的活性,从而促进了果实中亚油酸和亚麻酸的积累,维持较高的不饱和脂肪酸指数和不饱和度,同时抑制了超氧阴离子(O2·-)和过氧化氢含量(H2O2)的积累以及细胞膜通透性和膜脂过氧化产物丙二醛(MDA)含量的增加。GB处理诱导提高酚类合成途径中苯丙氨酸解氨酶(PAL)、肉桂酸羟化酶(C4H)和4-香豆酸辅酶A连接酶(4CL)基因的表达和酶的活性,抑制多酚氧化酶(PPO)和过氧化物酶(POD)基因的表达和活性的增加,进而增强总酚和总黄酮积累,提高果实低温贮藏期间的抗氧化能力,同时减缓酚类物质的氧化,有效减轻果肉褐变程度。此外,GB处理还可以促进糖代谢中蔗糖磷酸合酶(SPS)和蔗糖合酶(SS-s)基因的表达和酶的活性,抑制酸性转化酶(AI)和中性转化酶(NI)基因的表达和酶的活性,从而促进蔗糖含量的积累,维护细胞膜的稳定性。2-DE蛋白组学结果表明,GB处理可诱导一些抗胁迫蛋白(如热激蛋白)的表达,增加细胞结构的强度,提高细胞的抗氧化能力,从而减缓低温胁迫对蛋白、膜脂等生物大分子的损伤;同时,提高桃果实的能量水平,维持细胞的正常生长的生命周期,抑制细胞壁的降解,从而起到对细胞的保护作用。此外,结合RNA-Seq和蛋白组深入分析热激转录因子(Hsfs)和热激蛋白(Hsps)对GB处理的响应机制发现,GB处理通过诱导可能处在胁迫应激上游的PpHsfA1a/b和PpHsfA2a基因的表达,进而诱导PpsHsps,PpHsp70s和PpHsp90s基因表达,提高sHsps和Hsp70蛋白的含量,从而减轻桃果实的冷害。3.HW处理对桃果实低温贮藏期间的研究结果表明,HW处理在调控脂肪酸代谢、活性氧代谢、酚类物质代谢和糖代谢方面与GB具有类似性。HW处理促进了不饱和脂肪酸,蔗糖和酚类物质的积累,抑制了O2·-和H2O2含量的积累及细胞膜通透性和MDA含量的增加。此外,进一步分析PpHsfs和PpHsps发现,HW处理通过诱导可能处在胁迫应激上游的PpHsfA1a和PpHsfA2a基因的表达,进而调控PpsHsps的表达,发挥sHsps分子伴侣的作用,保护低温逆境下细胞功能的完整性。磷酸化蛋白组分析结果表明,HW处理的桃果实中磷酸化蛋白主要参与新陈代谢和次级代谢。HW处理可诱导钙调素结合转录因子(CAMTA)和GATA型锌指蛋白的磷酸化修饰,提高转录因子的转录活性,进而激活下游相关基因的表达;同时提高蔗糖合酶的磷酸化修饰,促进蔗糖的合成;此外还诱导热应激相关蛋白的磷酸化修饰,诱导热应激蛋白的表达,减缓低温胁迫对蛋白、膜脂等生物大分子的损伤,从而减轻果实的冷害。更多还原

【Abstract】 Peach fruit is popular among consumers for its bright color,unique flavor,juiceful texture,and rich in nutrient.Peach fruit matures in summer and has strong respiration after harvest.Peach fruit is easy to post-ripen and soften under ambient temperature conditions.In addition,the peach fruit skin is thin and soft.Fruit is highly susceptible to mechanical damage and pathogenic infection,which seriously affects quality.Low-temperature storage is an important method to store fresh fruit after harvest.However,peach fruit is chilling sensitive and highly vulnerable to chilling injury(CI)during long-term cold storage at unsuitable temperature.Heat treatment is widely regarded as an environmentally friendly treatment method with simple operation.Glycine betaine,as a compatible osmotic substance,can act as a penetrant to stabilize the cytoplasmic pH and osmotic pressure,and can also be used as a molecule chaperone to stabilize the structure and function of biological macromolecules such as enzymes,proteins and membrane lipids.In this paper,"Yuhua No.2" peach was treated with 10 mmol L-1 GB and 45℃ HW to study their effect on the quality and chilling injury.This study aims to reveal the mechanism of GB and HW treatment on alleviating chilling injury,and to provide a theoretical basis for its application in postharvest storage and transportation of peach fruit.The research results are summarized as follows:1.The effects of GB and HW treatments on the quality and chilling injury of cold stored peach fruit were studied.The results showed that CI index appeared after the 21 days cold storage and 3 days shelf storage and the main symptom of CI was internal browning in peach fruit.GB and HW treatments alleviated the internal browning of peach fruit,maintained higher extractable juice and the contents of TSS,ascorbic acid,calcium,iron and zinc in comparison with control,thereby maintaining better fruit quality.Furthermore,there was no significant difference in maintaining fruit quality between GB and HW treatments.2.Transcriptome(RNA-Seq)research results showed that GB treatment inhibited the gene expression and enzyme activities of phospholipase D(PLD),lipoxygenase(LOX)and lipase(Lipase),enhanced the gene expression related to unsaturated fatty acid synthesis(such as desaturase),improved the gene expression and enzyme activities of superoxide dismutase(SOD),catalase(CAT)and ascorbate peroxidase(APX)in the active oxygen scavenging system,which promoted the accumulation of linoleic acid and linolenic acid,maintained a high unsaturated fatty acid index and unsaturation,while suppressed the accumulation of superoxide anion(O2·-)and hydrogen peroxide content(H2O2),inhibited the increase of cell membrane permeability and malondialdehyde(MDA)content.GB treatment induced the up-regulated gene expression and enzyme activities of phenylalanine ammonia lyase(PAL),cinnamate hydroxylase(C4H)and 4-coumaric acid coenzyme A ligase(4CL),down-regulated the gene expression and enzyme activities of polyphenol oxidase(PPO)and peroxidase(POD),contributing to the accumulation of total phenol and total flavonoid and mitigation of phenolic compounds oxidation,increasing the antioxidant capacity and effectively alleviating the internal browning of fruit during storage.In addition,GB promoted the gene expression and enzyme activities of sucrose phosphate synthase(SPS)and sucrose synthase(SS-s),suppressed the gene expression and enzyme activities of acid invertase(AI)and neutral(NI),thereby promoting the accumulation of sucrose content,which was beneficial to maintaining the stability of cell membranes.2-DE proteomics results showed that GB treatment induced some anti-stress proteins contents(such as heat shock proteins),enhanced the strength of cell structure and improved the antioxidant capacity,thereby alleviating the chilling injury on biological macromolecules such as proteins and membrane lipids.Meanwhile,GB improved the energy level,maintained the normal growth life cycle of the cells and inhibited the degradation of the cell wall,thus playing positive role in cell protection.Moreover,further mechanism analysis of heat shock transcription factor(Hsfs)and heat shock protein(Hsps)response to GB treatment was studied by combining RNA-Seq and proteome method.Results revealed that GB treatment up-regulated gene expression of PpHsfA1a/b and PpHsfA2a,which might be upstream of stress response,induced the expression of PpsHsps,PpHsp70s and PpHsp90s,contributing to enhancing the protein contents of sHsps and Hsp70s,thus mitigating the chilling injury of peach fruit.3.The effect of HW treatment was similar to GB treatment in regulating active oxygen,fatty acid,sugar and phenylpropane metabolisms.HW treatment promoted the accumulation of unsaturated fatty acids,sucrose and phenols,inhibited the accumulation of O2·-and H2O2 content,and the increase of cell membrane permeability and MDA content.In addition,further analysis of PpHsfs and PpHsfs revealed that HW treatment up-regulated the gene expression of PpHsfA1a and PpHsfA2a,then promoted the gene expression of PpsHsps,exerted the role of sHsps molecular chaperone,thereby protecting the integrity of cell function under cold stress.The results of phosphorylated proteome analysis showed that phosphorylated proteins in HW-treated peach fruit were mainly involved in metabolism and secondary metabolism.HW treatment induced the phosphorylation modification of calmodulin binding transcription factor and GATA-type zinc finger protein,improved the transcriptional activity of transcription factors,then activated the expression of downstream related genes.Meanwhile,HW induced the phosphorylation modification of sucrose synthase,which promoted sucrose synthesis.Furthermore,HW treatment induced phosphorylation modification of heat stress-related proteins,enhanced the expression of heat stress proteins,contributing to alleviating chilling injury on biological macromolecules and delaying the development of chilling injury.更多还原