【作者】 王贵章；

【导师】 王贵禧；

【作者基本信息】 中国林业科学研究院 ， 森林培育， 2014， 博士

【摘要】 桃（Prunus persica L. Batsch）属于呼吸跃变型果实，常温贮藏3-5d果实即发生腐败、变质，低温贮藏是目前延长桃果实贮藏时间的主要方法。但是桃属于冷敏果实，低温下容易果实发生冷害，出现果肉褐变、不能正常软化而发绵、出汁率低等现象，其中香味变淡甚至丧失是一个重要冷害特征，严重影响了果实品质。实验室前期研究发现，低温条件下，桃果实果香型香气物质含量较常温显著降低，而清香型香气物质的含量较常温有所增加；施加外源乙烯处理，在一定程度上促进了低温贮藏桃清香型香气向果香型香气的转变，部分清香型香气含量降低，而果香型香气含量上升。低温下施加外源乙烯处理，显著提高了内源乙烯的释放，乙烯合成相关基因ACO的活性降低。以上结果表明，乙烯与果实香气物质合成存在必然联系，但是关于乙烯对香气合成恢复的分子机理尚不清楚。通常代谢产物与代谢底物成正相关，因此很有必要从基因的转录水平，结合香气挥发代谢谱结果，对乙烯参与桃果实香气物质合成的调控作用进行研究。研究的主要结果如下：1、数字基因表达谱测序发现，低温贮藏条件（0℃贮藏30d的桃果实CK2，乙烯处理的桃果实T）对基因的转录表达有较大影响，低温下桃果实中能量代谢和抗病相关基因如ATP酶（ATPase）基因、防御素（cryptdin）基因、病程相关索马甜（thaumatin）基因和几丁质酶（Chitin）基因的表达水平大幅上调，这可能是对适应低温胁迫的一种应激反应。乙烯处理后，这些抗逆基因的表达显著下调，但表达水平仍高于常温贮藏桃样本CK1中的表达量，表明这些基因的转录表达可能受低温和乙烯的共同调控。低温下桃果实发生冷害，可能与能量和抗性相关代谢的失调有关。2、低温贮藏桃样本中，参与硬脂酸合成的硬脂酰CoA硫酯酶基因的表达水平较常温贮藏桃样本显著下调，低温条件下的两个样本（CK2和T）中，该基因的表达水平为0，低温下施加外源乙烯，没有改变硬脂酰CoA硫酯酶基因的转录水平。低温下硬脂酸合成关键基因的转录抑制，使不饱和脂肪酸主要通过亚油/麻酸途径进行。低温下亚油/麻酰CoA硫酯酶基因的表达上调，促进亚/麻油酸的合成，为香气物质合成保证底物的供应。低温导致某些香气物质合成代谢失调的原因，可能由于硬脂酰CoA硫酯酶基因的转录受抑引起硬脂酸代谢失调造成的。3、脂氧合酶LOX基因通过α-亚麻酸途径，参与醛类香气物质的合成。通过对LOX途径中LOX、ALDH、EH等基因家族成员的表达模式分析发现，基因家族不同成员可能参与不同的生物学过程，且它们对乙烯的敏感性不同。乙烯响应因子结合蛋白ERF12为转录抑制因子，该基因的转录受到乙烯正调控，在CK1中的表达水平最高，低温下下调表达，施加乙烯处理表达上调。乙烯响应因子结合蛋白ERF1、ERF2、ERF5为转录激活因子，三基因的转录受到乙烯负调控，在CK2中的表达水平最低。低温下，ERF12下调表达，解除了对LOX基因转录抑制作用，而促进LOX基因的上调表达，乙烯处理后ERF12表达上调，抑制LOX家族基因的转录。乙烯通过ERF1、ERF2、ERF5和ERF12等，转录激活或抑制LOX、ALDH等家族基因的转录表达，从而调控己醛、反-2-己烯醛、反-2-己烯醇等青草型香气物质的合成。4、利用同源克隆和RACE技术，从桃果实中克隆到1个LOX基因，该基因全长2909bp，ORF长2640bp，3’ UTR长269bp，编码879个氨基酸，属于LIPOXYGENASE超家族，含有植物LOX家族的两个保守结构域：PLAT/LH2和Lipoxygenase。通过实时荧光定量PCR检测，该基因在CK2样本中较CK1表达大幅下调，T样本中表达较CK2下调，与DGE测序所得的表达变化趋势一致。从‘大久保’果实中共检索到14条LOX同源基因，系统进化分析聚为两簇：13-LOX和9-LOX；有5条基因在DGE中没有检测到表达，另外9条基因中有4条基因的表达受到乙烯负调控，分别是PpLOX4、PpLOX10、PpLOX11和PpLOX12，施加乙烯处理后基本表达下调。5、利用同源克隆和RACE技术，从桃果实中克隆得到1个EH基因，该基因全长1274bp，5′UTR和3′UTR分别长161bp和143bp，最大ORF长972bp，编码323个氨基酸，属于αβ水解酶/折叠酶构型。蛋白的三维结构分析表明，该蛋白具有三位一体的活化中心Asp-His-Asp，以及Y-Y组成的帽子结构；亚细胞定位结果表明，基因定位于叶绿体；实时荧光定量PCR分析结果显示，该基因的转录表达受到低温和乙烯的双重调控，表明该基因可能同时参与多个不同的生理生化过程。6、烷烃类物质是桃果实中含量相对较高的发挥性成分，烷烃类通过CYP716A1的作用氧化生成1-醇，然后在ADH的作用下实现醛和醇的可逆互转。推测烷烃类通过为1-醇的合成提供底物，从而参与桃果实醛类和醇类物质的积累和释放，且乙烯对烷烃类物质的合成起到正向调控的作用。醇类物质的积累可能分别与烷烃-1-单加氧酶CYP716A和ALDH的联动作用及CYP716A和EH的联动作用通过脂肪酸降解途径参与；GDSL基因表达随脂肪酸水平的变化而变化，以维持机体内脂肪酸的动态平衡。更多还原

【Abstract】 Peach belongs to climacteric fruit, with fruit spoilage after3-5d storage at roomtemperature. Cold storage is the main method to extend the market supply of peach fruit. Whilepeaches are coldsensitive fruit and prone to chilling injury during cold storage. Fruits Chillinginjury syptoms show up as flesh browning, innormal soften and cotton, and low juice yield etc..Low temperature resulted in the loss of flavor peach fruit, which seriously affected the edibilityof the fruits. Early research of our laboratory showed that the content of Floral/Fruity aromavolatiles were significantly lower than which stored at the ambient temperature, while thecontent of Green aroma volatiles were higher. The aroma volatiles in cold storage peach fruitswere to some extent promoted after treated by exogenous ethylene. The release quantity ofethylene significantly reduced under low temperature. On the other hand, the activity of ACOenzyme reduced. While the opposite result was received from the fruit treated by exogeneousethylene. There was necessary connection between ethylene and fruit aroma-volatile-synthesis.But till now, the molecular mechanism on synthesis of aroma volatile regulated by ethylene isunclear. Metabolites are usually positively correlated with metabolic substrates. So it isnecessary to research the regulation of ethylene on aroma synthesis of peach fruit from thegene transcription level, combining with the aroma volatile metabolite profiling. The mainresults of this research are as following:1. Cold storage has a greate impact on gene expression in peach fruit. Compared withCK1, expression level of genes related to energy metabolism and pathogenesis-related proteingenes such as ATPase, cryptdin, thaumatin and Chitin were greatly upregulated in CK2. Thismaybe is stress response to low temperature. After treated by exogenous ethylene, theexpression of these genes were significantly downregulated, but which were still higher than T.This result reveled that the transcriptional expression of these genes were co-regulated by low temperature and ethylene. Metabolic disorders of energy and resistance-related is the reason offruit chilling injury at low temperature storage.2. As far as CK2sample, the expression levels of stearoyl CoA thioesterase gene involvedin stearic acids synthesis were significantly downregulated even no expression. Exogenousethylene does not change the expression levels of stearoyl CoA thioesterase genes. In CK2, themain unsaturated fatty acids substrates for synthesis of aroma volatiles were linolenic/linoleicacid because of transcriptional inhibition of key genes involved in stearic acid synthesis. Underlow temperature, linolenic/linoleic acid CoA thioesterase genes were upregulated. Metabolicdisorder of stearic acids leaded to lack supply of substrate in cold storaged peach fruit maybethe reason that some aroma volatiles metabolic disorder.3. LOX genes involved in the synthesis of aldehydes aroma substances via α-linolenicacid pathway. Different family members of LOX, ALDH, EH may be involved in differentbiological processes and they have different sensitivity to ethylene pathway through analysis oftheir expression patterns. Ethylene response factor binding protein transcription factor ERF12is a transcription repressor, positively regulated by ethylene. The highest expression level ofERF12was in CK1sample, which downregulated under lower temperature, and upregulatedafter treated by ethylene. Ethylene response factor binding protein ERF1, ERF2, ERF5astranscriptional activators negatively regulated by the ethylene, which the lowest level ofexpression of the CK2. Under low temperatures, the expression of ERF12gene downregulated,lifting the inhibition of transcription for LOX gene. In peach fruit T sample, the expression ofLOX family genes downregulated after treated by ethylene because of the inhibitiontranscription of ERF12. Ethylene regulated synthesis of green aroma volatiles such as hexanal,trans-2-hexenal, trans-2-hexenol and hexenyl acetate and fruity aroma volatiles such as hexylacrylate, o-aminobenzaminicacid-1,5-dimethyl-1-ethylene-4-hexenyl and dibutyl glutaratethrough transcriptional activition or repression of LOX, ADH, AAT, ALDH controlled by ERF1,ERF2, ERF5and ERF12. 4. Lipoxygenase gene was cloned from peach fruit by homologous cloning and RACEtechnology. The gene belong to LIPOXYGENASE superfamily with full length2640bp,3’UTR269bp and encoding879amino scids. The deduced amino acid sequence contains twoconserved domains: PLAT/LH2and Lipoxygenase. The result of real-time quantitative PCRshowed that the expression of this gene significantly downregulated in CK2than CK1andwhich was higer than in T. This result was consistent with the result of DGE sequencing. Total14LOX homologous genes were retrieved from okubo fruit with two clusters afterphylogenetic analysis:13-LOX and9-LOX. Five genes were not detected by DGE. There werefour of the nine genes negative regulated by ethylene respectively as PpLOX4, PpLOX10,PpLOX11and PpLOX12.5. Epoxide hydrolase gene was cloned by homologous cloning and RACE technologywith1274bp full length,161bp and143bp5’UTR and3’UTR respectively, and with972bpORF. This gene encoded323amino acids. The deduced amino acid sequence are αβ hydrolase/folding enzymes configuration with a trinity of Asp-His-Asp activation center and Y-Y capstructure. The protein was located in the chloroplast. The result of Real-time PCR analysisindicated that this ge ne was co-regulated by low temperature and ethylene. It maybeparticipate in a number of different physiological and biochemical processes.6. Alkanes were the high level volatile ingredients from ‘okubo’ peach fruit in relativecontent. The alkane were oxidized to1-alcohol by CYP716A1.Then the aldehydes and alcoholsconverted into each other under the activition of ADH. The substances involved in thesynthesis of alkanes were positively controlled by ethylene. Alkanes participated in theaccumulation and release aldehydes and alcohols from peach fruit by providing a substrate forsynthesis of1-ol. Accumulation of alcohols were effected by the linkage of alkane-1-monooxygenase CYP716A and ALDH and the linkage of CYP716A and epoxide hydrolaseEH respectively through fatty acid degradation pathways. The transcription expression level ofGDSL gene changed with changed in fatty acid levels and was to maintain the homeostasis of fatty acid in organ body.更多还原