一氧化氮对采后肥城桃果实能量代谢及CBF抗冷途径的调控机理

发布时间：2018-01-11 21:34

本文关键词：一氧化氮对采后肥城桃果实能量代谢及CBF抗冷途径的调控机理　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

更多相关文章：桃 一氧化氮 能量代谢 抗冷机制 CBF

【摘要】：桃是广受消费者喜爱的水果之一,桃果实采后不易贮藏,冷藏是目前应用最广的桃果实保鲜方式之一,但桃属于冷敏性果蔬,低温贮藏容易使其发生冷害,影响其经济效益。一氧化氮(NO)作为活性气体小分子,能够缓解采后桃果实的冷害,提高采后桃果实的抗冷性,但其调控机理并不明确。因此,探明NO对采后桃果实抗冷性的调控机理,从而为NO在桃果实贮藏保鲜技术的应用提供理论依据,对于保持果实品质,延长贮藏期具有重要意义。能量状态与采后水果的成熟、衰老及生理失调紧密相关,冷害的发生与能量亏缺有着密切联系。为了研究NO处理在桃果实冷藏期间对其能量代谢的调控机理,选取八成熟肥城桃果实,分别用去离子水(对照)、5、15、30μmol L-1 NO溶液和5μmol L-1 2-(4-羧苯基)-4,4,5,5-四甲基咪唑啉-1-烃氧基-3-氧化钠盐(c-PTIO,NO清除剂)溶液处理后于0°C恒温贮藏。分别在果实储藏当天、第1、2、3、4、5周测定不同处理肥城桃果实冷害指数,硬度,丙二醛(MDA)、过氧化氢,腺苷三磷酸(ATP)、腺苷二磷酸(ADP)、磷酸单腺苷(AMP)的含量和能荷水平的变化,以及琥珀酸脱氢酶(SDH)、细胞色素氧化酶(CCO)、氢离子ATP酶(H+-ATPase)、钙离子ATP酶(Ca2+-ATPase)等线粒体呼吸代谢关键酶的活性变化情况。结果表明,在冷藏过程中,桃果实内ATP、ADP含量,能荷水平及桃果实线粒体内SDH、CCO、H+-ATPase、Ca2+-ATPase酶活性逐渐降低,而AMP、过氧化氢及MDA含量升高;与对照相比,NO处理能够显著延缓桃果实内ATP、ADP、能荷含量及能量代谢酶活性的降低,抑制过氧化氢、MDA、AMP含量的上升,其中以15μmol L-1 NO处理桃果实最为显著,而NO清除剂c-PTIO处理则加剧了桃果实ATP、能荷含量及能量代谢酶活性的下降以及过氧化氢、MDA、AMP含量的上升。上述结果表明,15μmol L-1外源NO处理能有效维持桃果实在冷藏期间的能量代谢,延缓膜脂过氧化过程,提高桃果实的抗冷性,而c-PTIO处理则促进了冷藏期间桃果实能量代谢紊乱,加剧了桃果实的冷害程度。植物具有接受低温刺激信号、自我调节抗冷机制以低于低温的能力。低温刺激信号沿着“细胞膜上的蛋白受体→Ca2+等第二信使→蛋白激酶→转录因子→抗冷基因”的途径逐级在植物细胞传递。CBF(C-repeat binding transcription factor/de-hydrate responsive element binding factor,DREB)基因是植物CBF抗冷途径的枢纽。植物在抗冷过程中通过CBF转录因子调控大量下游抗冷基因的表达来提高植物抗冷能力。本研究从肥城桃桃果实提取出总RNA并克隆获得cDNA并测得了肥城桃的CBF家族基因序列。对CBF家族基因进行荧光定量分析发现,在冷藏过程中,桃果实CBF1/5/6基因表达量迅速升高,且均维持在较高的水平,相对于对照,NO处理显著提高了冷藏过程中CBF1/5/6的基因表达量,而c-PTIO通过清除NO,使CBF1/5/6的基因表达量略低于对照,证明了NO对于CBF抗冷途径的调控作用。以克隆获得的cDNA为模板,构建pET30a-CBF重组载体,导入大肠杆菌BL21(DE3)进行蛋白表达,表达出了CBF1~3蛋白。为进一步从分子生物学方面探讨NO抗冷机制打下基础。
[Abstract]:Peach is one of the popular consumer favorite fruit, peach fruits are not easy to storage, cold storage is one of the peach fruit fresh preservation of the most widely used, but the peach belongs to the cold sensitive cold storage of fruits and vegetables, easy to make the damage, affect its economic benefits. Nitric oxide (NO) as the active gas molecules, can alleviate the chilling injury of peach fruit after harvest, improve the cold resistance of peach fruit after harvest, but its mechanism is not clear. Therefore, the NO of Postharvest Peach cold resistance mechanism, so as to the application of NO in storage technology of peach fruits provide a theoretical basis for maintaining fruit quality, has an important the significance of prolonged storage period. The ripe fruit energy state and postharvest senescence and physiological disorders are closely related, and lack of energy damage are closely related. In order to study the treatment of NO in Peach Fruits during cold storage on the energy metabolism regulation mechanism, selection Medium well Feicheng peach fruit, using deionized water (control), 5,15,30 mol L-1 NO L-1 2- solution and 5 mol (4- carboxyphenyl) -4,4,5,5- four methyl imidazoline -1- alkoxy -3- oxide sodium salt (c-PTIO, NO scavenger) solution at 0 DEG C in constant temperature storage. Fruit storage on the same day, in 1,2,3,4,5 weeks of different treatment for Feicheng peach fruit chilling injury index, hardness, malondialdehyde (MDA), hydrogen peroxide, adenosine phosphate (ATP) three, two (ADP), adenosine phosphate mono phosphate adenosine (AMP) content and can change load level, and succinate dehydrogenase (SDH), cytochrome oxidase (CCO), hydrogen ion ATP enzyme (H+-ATPase), calcium ATP enzyme (Ca2+-ATPase) activity changes of mitochondrial respiratory metabolism key enzyme. The results showed that during cold storage of peach fruit in ATP, the content of ADP, level of energy charge and peach fruit in mitochondrial SDH, CCO, H+-ATPase, Ca2+-ATPase enzyme activity gradually reduced Low, AMP, hydrogen peroxide and MDA content increased; compared with the control, NO treatment can significantly delay the peach fruit in ATP, ADP, the enzyme activity and the content of energy metabolism load reduced, inhibition of hydrogen peroxide, MDA, content of AMP increase, the 15 mol L-1 NO treatment of peach fruit was the most significant, and NO c-PTIO scavenger is exacerbated by the peach fruit ATP, can decrease content of enzyme activity and energy metabolism of bearing and hydrogen peroxide, MDA, content of AMP increase. The results showed that 15 mol L-1 exogenous NO treatment can effectively maintain the peach during the energy metabolism, delay the lipid peroxidation process, improve the cold resistance peach fruit, while c-PTIO treatment promoted the peach fruit disorder of energy metabolism during cold, exacerbated the chilling injury of peach fruit. Plants have received low temperature stimulation signal, self regulating mechanism under low temperature cold resistance ability. Low temperature stimulation signals along "The way the cell membrane protein, Ca2+ receptor and two Messenger, protein kinase, transcription factor, cold resistant genes of the gradual transfer of.CBF in plant cells (C-repeat binding transcription factor/de-hydrate responsive element binding factor, DREB) gene is CBF plant cold resistance pathway hub. In the process of cold resistance in plants by expression of CBF transcription factor the regulation of a large number of downstream cold resistance genes to improve plant cold resistance. This study from the Feicheng peach fruit extract total RNA and cloned cDNA and CBF genes was measured in Feicheng peach. Fluorescent quantitative analysis of CBF family genes in peach fruit during cold storage, CBF1/5/6 gene expression increased rapidly. And were maintained at a higher level, compared with control, NO significantly increased CBF1/5/6 gene expression during cold storage, and c-PTIO by removing NO, CBF1/5 The expression of /6 was slightly lower than that of the control, and prove that NO for regulation of CBF cold resistance pathway. To clone cDNA as template to construct pET30a-CBF recombinant vector into E. coli BL21 (DE3) for protein expression and expression of the CBF1~3 protein. Further from molecular biology to investigate NO cold resistance mechanism the foundation.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS255.3

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