脐橙(Citrus sinensis Osbeck)果实采后商业贮藏中基因表达的芯片分析及候选基因的表达验证

Genechip Analysis of Navel Orange (Citrus Sinensis Osbeck) in Commercial Storage and Expression Confirmation of Several Candidate Genes

作者: 专业:果树学 导师:邓秀新 年度:2010 学位:硕士  院校: 华中农业大学

Keywords

navel orange storage, gene expression, plant hormone, transcription factor, carbohydrate metabolism, lipid metabolism, pathogenesis-related protein

        脐橙(Citrus sinensis Osbeck)以其多汁、味美、富含维生素C等特点深受大众欢迎。由于脐橙的成熟期较为一致,因此采后长期贮藏是解决脐橙周年供应问题的重要措施。然而脐橙果实在贮藏过程中,易发生脱水、褐斑、病虫害等问题。冷藏处理可以有效减少上述症状的发生,但低温伤害亦不可避免。尤其是低温贮藏后突然恢复常温,会造成果实品质的劣变。因此,了解脐橙果实贮藏过程中的生理生化代谢特征,从而优化贮藏条件是迫切需要解决的问题。本实验以常规商业贮藏方式(低温+出库前分级升温)贮藏脐橙,采用基因芯片技术对贮藏期间的果皮果肉的基因表达谱进行分析,并采用实时定量PCR技术对部分采后贮藏相关的候选基因的表达特征进行验证,以期为柑橘贮藏技术的改进与发展奠定理论依据。获得的主要结果如下:1.从整体上看,贮藏期果实的基因差异表达主要集中在前27天,且果皮中差异基因的数目远多于果肉,这可能由于果皮与外界环境的直接接触而引起的较多的敏感反应。2.从贮藏结果中可以看出,差异基因参与众多生理生化途径,包括植物激素代谢、基因转录与调控、碳水化合物代谢、脂类物质代谢、病程相关蛋白基因等。3.贮藏过程中,编码植物激素的众多基因表达发生改变,包括乙烯合成及信号传导途径中的ACC合成酶(ACC Synthase)、ACC氧化酶(ACC oxidase)、乙烯应答因子1和2(ERF1,2)、乙烯不敏感转录调节因子4(EIN4); ABA合成代谢中的关键酶9-顺式-环氧类胡萝卜素双加氧酶3(NCED3); GA3合成代谢中的关键酶内根—贝壳杉烯酸羟化酶1(KAO1);参与JA合成中的12-氧植物二烯酸还原酶2(OPR2)以及信号转导代谢中的茉莉酸羧基甲基转移酶(JMT)和抗茉莉酮酸酯(JAR1)。上述基因的表达模式除KAO1上调外,其它均下调表达,可能意味着GA3在果实贮藏过长中合成受到促进。4.参与植物逆境响应的转录因子WRKY, MYB, NAC, BZIP, BHLH, AP2/ERF等均在表达上表现差异。在相同的代谢调控中,同一转录因子家族的不同成员会表现出不同的表达模式,说明转录调控的复杂性或冗余性。此外这些转录因子参与脐橙贮藏过程中的多种生理生化途径的调控。5.芯片数据显示贮藏期脐橙果实存在活跃的碳水化合物代谢,主要表现在糖酵解、糖异生、葡萄糖醛酸途径、淀粉及蔗糖向单糖的转化过程、碳水化合物的转运等过程。其说明贮藏中果实过程为了维持细胞的正常代谢过程,细胞内的碳水化合物之间不断进行相互转化。6.贮藏期间参与脂类代谢中的磷脂酶、脂肪酶、酯酶、脂肪酸脱饱和酶、脂肪转移酶的基因表达在脐橙的果皮中变化明显,而在果肉中,这些变化相对较弱。脂类物质代谢特征体现了贮藏过程中细胞内能量代谢及细胞膜结构及透性的改变,以更好的适应贮藏环境。7.病程相关蛋白基因的表达在脐橙的贮藏过程中发生改变,尽管果实并没有明显的病斑出现,但贮藏过程本身的逆境也可能使果实启动了相关基因的表达,其中几丁质酶及伤害响应蛋白的编码基因上调表达,而其他如β-1,3--葡聚糖酶等其他病程相关蛋白的编码基因下调表达。8.实时定量PCR的验证实验表明,本实验中贮藏27天(低温贮藏+后期分级升温)时大量基因下调表达,而这些基因在单纯低温贮藏27天过程中是上调表达的,因此可以推测芯片分析中脐橙基因表达下调是来自出库前的分级升温过程,这从基因表达的水平初步解释了分级升温可以缓解低温对果实造成的冷害,尤其是低温贮藏果实突然出库时造成的劣变。
    Navel orange (Citrus sinensis Osbeck) is one of main commercial citrus varieties in China, favored with its very juicy, tasty and richen in Vitamin C. However, due to its overlapping maturation times, it’s hard to fully satisfy the market demands on navel oranges in the whole year. And in this case, navel oranges are more depended on a proper storage environment to keep postharvest quality and prolong shelf life of fruit. Under postharvest storage, the orange fruits are prone to losing water, rind staining, and getting other pathological and physiological disorders especially traditional low temperature which may result in numerous cellular and metabolic dysfunctions, such as altered respiration rates, impaired photosynthetic activity, and changes in membrane permeability then subsequently cause serious commercial losses. Thus, Knowledge of fruit morphology, physiology and biochemistry is a prerequisite for thorough understanding of postharvest science. In present study, various navel oranges are stored in the commercial cold rooms at 5℃and put them into 12℃for four days and 20℃for two days before getting them out. After RNA extraction, the expression patterns of differently expressed genes were measured by Genechip technology. After that, we use navel oranges grown in Gannan, China as samplings and store them at the same storage environment then confirm expression of several genes related with postharvest storage by Real-time PCR. The main results are as follows:1. In general, the significantly differentially expressed gene occurred on the first 27 days after storage and the gene numbers in peels are many over than numbers in vesicles. It is probably that peels are exposure on the storage environment directly and would be more sensitive to the change of environment.2. According to the Genechip analysis, the differentially expressed genes are distributed into various plant metabolic pathways, such as plant hormone, transcriptional regulate, carbohydrate metabolism, lipid metabolism, pathogenesis-related protein and other transport or synthesis pathyways.3. During the process of storage, plant hormone genes are changed a lot, including ACC synthase, ACC oxidase, ethylene response factor 1,2 (ERF 1,2), ethylene insensitive 4 participated in the ethylene biosynthesis or catabolism; 9-cis-epoxycarotenoid dioxygenase3 (NCED3) of the ABA biosynthesis; a key enzyme ent-kaurenoic acid hydroxylase 1(KAO1) in GA3 biosynthesis, and 12-oxophytodienoate reductase 2 (OPR2), jasmonic acid carboxyl methyltransferase (JMT), jasmonate resistant 1 (JAR1) in the biosynthesis and signal transduction of JA. Most of these hormones encoded probe sets are down regulated during the storge, only ent-kaurenoic acid hydroxylase 1(KAO1) up regulated which may suggest that the GA3 are increased during the storage.4. Global transcripts profiling analysis revealed that exposure to storage environment markedly affected the expression patterns of many transcription factor genes, including Heat shock protein, WRKY, MYB, NAC, BZIP, BHLH, AP2/ERF et al. These transcription factors play various roles on different pathways. Even if they are from the same family, their expression pattens changed varied with different family members which may related to the self-regulation model or they just take part in different pathways.5. Under stress storage environment, genes encoded enzymes involved in carbohydrate metabolism regulate the balance between biosynthesis and breakdown and transverse among starch, sucrose, glucose and fructose. Simultaneously, genes involved in the process of glycolysis and fermentation, gluconeogenesis, and sugar transport are also differentially expressed, which maintain the cellular metabolism.6. Altered genes encoded esterase, fatty acid dehydrogenase, lipase, lipid transfer protein, phospholipids and etc. are mainly in the peels, while lipid related genes changes little in vesicles and differed with ones in peels, which indicates that lipid metabolism participate the cellular energetic metabolism and membrane lipid modification, subsequently change the membrane permeability, in order to help orange fruits are adaptive to storage environment.7. Although there is no obvious rind staining on the peel, transcripts encoded pathogenesis-related protein also change a lot under orange storage environment, which indicates that the storage itself activate pathogenesis-related gene express, including increased transcripts encoded chitinases and wound responsive protein, while suppressed the expression ofβ-1,3-glucanase and other pathogenesis-relate transcripts.8. The confirmed results by Real-time PCR show that pure low temperature storage increase many gene expression while these genes are down regulated by the Genechip analysis, which partially indicate that down regulated of these genes are resulted from the graded increased temperature before these samples are exposed onto the room temperature. From the results, we could deduce that alternate-temperature storage could alleviate the chilling injury, especially the fruit quality decrease because of the temperature increase suddenly.
        

脐橙(Citrus sinensis Osbeck)果实采后商业贮藏中基因表达的芯片分析及候选基因的表达验证

摘要6-8
Abstract8-10
缩略词表11-12
1 前言12-20
    1.1 课题提出12-13
    1.2 柑橘果实采后贮藏生物学的国内外研究进展13-16
        1.2.1 贮藏条件对柑橘果实生理变化的影响13
        1.2.2 果实贮藏过程中植物激素的研究进展13-14
        1.2.3 与采后贮藏有关的转录因子研究进展14-15
        1.2.4 果实采后贮藏碳水化合物代谢的研究进展15
        1.2.5 果实采后贮藏脂类代谢的研究进展15-16
        1.2.6 果实采后贮藏病程相关蛋白的研究进展16
    1.3 基因表达的研究方法16-18
        1.3.1 基因芯片技术16-17
        1.3.2 基因芯片分析17-18
    1.4 本研究的目的与内容18-20
2 材料和方法20-29
    2.1 实验材料20
    2.2 实验方法20-29
        2.2.1 RNA提取20-22
        2.2.2 基因芯片分析的主要方法22-24
            2.2.2.1 数据分析22-24
            2.2.2.2 基因功能注释24
        2.2.3 定量PcR基因表达的检测24-29
            2.2.3.1 荧光定量PCR引物设计24
            2.2.3.2 第一链cDNA反转录24
            2.2.3.3 实时荧光定量PCR引物筛选24-27
            2.2.3.4 实时荧光定量PCR基因表达检测27-29
3 结果与分析29-50
    3.1 基因芯片表达分析29-48
        3.1.1 脐橙不同品种间贮藏29-30
        3.1.2 贮藏过程中脐橙基因表达的变化情况30-48
            3.1.2.1 芯片基因表达变化概况30-32
            3.1.2.2 基因注释及功能分析32-35
            3.1.2.3 植物激素生物合成35-37
            3.1.2.4 转录因子的表达变化37-40
            3.1.2.5 脂类代谢40-43
            3.1.2.6 碳水化合物代谢43-46
            3.1.2.7 植物病程相关蛋白46-48
    3.2 荧光定量PCR验证结果48-50
4 讨论50-56
    4.1 植物激素与脐橙贮藏51-53
    4.2 转录因子的调控与脐橙贮藏53-54
    4.3 碳水化合物代谢,脂类代谢与脐橙贮藏关系54-55
    4.4 病程相关蛋白与脐橙贮藏55-56
参考文献56-64
致谢64
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