本文關鍵詞：一氧化氮對采后肥城桃果實能量代謝及CBF抗冷途徑的調控機理　出處：《山東農業(yè)大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 桃 一氧化氮 能量代謝 抗冷機制 CBF

【摘要】：桃是廣受消費者喜愛的水果之一,桃果實采后不易貯藏,冷藏是目前應用最廣的桃果實保鮮方式之一,但桃屬于冷敏性果蔬,低溫貯藏容易使其發(fā)生冷害,影響其經(jīng)濟效益。一氧化氮(NO)作為活性氣體小分子,能夠緩解采后桃果實的冷害,提高采后桃果實的抗冷性,但其調控機理并不明確。因此,探明NO對采后桃果實抗冷性的調控機理,從而為NO在桃果實貯藏保鮮技術的應用提供理論依據(jù),對于保持果實品質,延長貯藏期具有重要意義。能量狀態(tài)與采后水果的成熟、衰老及生理失調緊密相關,冷害的發(fā)生與能量虧缺有著密切聯(lián)系。為了研究NO處理在桃果實冷藏期間對其能量代謝的調控機理,選取八成熟肥城桃果實,分別用去離子水(對照)、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-烴氧基-3-氧化鈉鹽(c-PTIO,NO清除劑)溶液處理后于0°C恒溫貯藏。分別在果實儲藏當天、第1、2、3、4、5周測定不同處理肥城桃果實冷害指數(shù),硬度,丙二醛(MDA)、過氧化氫,腺苷三磷酸(ATP)、腺苷二磷酸(ADP)、磷酸單腺苷(AMP)的含量和能荷水平的變化,以及琥珀酸脫氫酶(SDH)、細胞色素氧化酶(CCO)、氫離子ATP酶(H+-ATPase)、鈣離子ATP酶(Ca2+-ATPase)等線粒體呼吸代謝關鍵酶的活性變化情況。結果表明,在冷藏過程中,桃果實內ATP、ADP含量,能荷水平及桃果實線粒體內SDH、CCO、H+-ATPase、Ca2+-ATPase酶活性逐漸降低,而AMP、過氧化氫及MDA含量升高;與對照相比,NO處理能夠顯著延緩桃果實內ATP、ADP、能荷含量及能量代謝酶活性的降低,抑制過氧化氫、MDA、AMP含量的上升,其中以15μmol L-1 NO處理桃果實最為顯著,而NO清除劑c-PTIO處理則加劇了桃果實ATP、能荷含量及能量代謝酶活性的下降以及過氧化氫、MDA、AMP含量的上升。上述結果表明,15μmol L-1外源NO處理能有效維持桃果實在冷藏期間的能量代謝,延緩膜脂過氧化過程,提高桃果實的抗冷性,而c-PTIO處理則促進了冷藏期間桃果實能量代謝紊亂,加劇了桃果實的冷害程度。植物具有接受低溫刺激信號、自我調節(jié)抗冷機制以低于低溫的能力。低溫刺激信號沿著“細胞膜上的蛋白受體→Ca2+等第二信使→蛋白激酶→轉錄因子→抗冷基因”的途徑逐級在植物細胞傳遞。CBF(C-repeat binding transcription factor/de-hydrate responsive element binding factor,DREB)基因是植物CBF抗冷途徑的樞紐。植物在抗冷過程中通過CBF轉錄因子調控大量下游抗冷基因的表達來提高植物抗冷能力。本研究從肥城桃桃果實提取出總RNA并克隆獲得cDNA并測得了肥城桃的CBF家族基因序列。對CBF家族基因進行熒光定量分析發(fā)現(xiàn),在冷藏過程中,桃果實CBF1/5/6基因表達量迅速升高,且均維持在較高的水平,相對于對照,NO處理顯著提高了冷藏過程中CBF1/5/6的基因表達量,而c-PTIO通過清除NO,使CBF1/5/6的基因表達量略低于對照,證明了NO對于CBF抗冷途徑的調控作用。以克隆獲得的cDNA為模板,構建pET30a-CBF重組載體,導入大腸桿菌BL21(DE3)進行蛋白表達,表達出了CBF1~3蛋白。為進一步從分子生物學方面探討NO抗冷機制打下基礎。
[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.

【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TS255.3

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