本文關(guān)鍵詞：一氧化氮對(duì)采后肥城桃果實(shí)能量代謝及CBF抗冷途徑的調(diào)控機(jī)理　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 桃 一氧化氮 能量代謝 抗冷機(jī)制 CBF

【摘要】：桃是廣受消費(fèi)者喜愛的水果之一,桃果實(shí)采后不易貯藏,冷藏是目前應(yīng)用最廣的桃果實(shí)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(jì)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(shí)的冷害,提高采后桃果實(shí)的抗冷性,但其調(diào)控機(jī)理并不明確。因此,探明NO對(duì)采后桃果實(shí)抗冷性的調(diào)控機(jī)理,從而為NO在桃果實(shí)貯藏保鮮技術(shù)的應(yīng)用提供理論依據(jù),對(duì)于保持果實(shí)品質(zhì),延長(zhǎng)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(diào)緊密相關(guān),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(shí)冷藏期間對(duì)其能量代謝的調(diào)控機(jī)理,選取八成熟肥城桃果實(shí),分別用去離子水(對(duì)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(shí)儲(chǔ)藏當(dāng)天、第1、2、3、4、5周測(cè)定不同處理肥城桃果實(shí)冷害指數(shù),硬度,丙二醛(MDA)、過(guò)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(xì)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(guān)鍵酶的活性變化情況。結(jié)果表明,在冷藏過(guò)程中,桃果實(shí)內(nèi)ATP、ADP含量,能荷水平及桃果實(shí)線粒體內(nèi)SDH、CCO、H+-ATPase、Ca2+-ATPase酶活性逐漸降低,而AMP、過(guò)氧化氫及MDA含量升高;與對(duì)照相比,NO處理能夠顯著延緩桃果實(shí)內(nèi)ATP、ADP、能荷含量及能量代謝酶活性的降低,抑制過(guò)氧化氫、MDA、AMP含量的上升,其中以15μmol L-1 NO處理桃果實(shí)最為顯著,而NO清除劑c-PTIO處理則加劇了桃果實(shí)ATP、能荷含量及能量代謝酶活性的下降以及過(guò)氧化氫、MDA、AMP含量的上升。上述結(jié)果表明,15μmol L-1外源NO處理能有效維持桃果實(shí)在冷藏期間的能量代謝,延緩膜脂過(guò)氧化過(guò)程,提高桃果實(shí)的抗冷性,而c-PTIO處理則促進(jìn)了冷藏期間桃果實(shí)能量代謝紊亂,加劇了桃果實(shí)的冷害程度。植物具有接受低溫刺激信號(hào)、自我調(diào)節(jié)抗冷機(jī)制以低于低溫的能力。低溫刺激信號(hào)沿著“細(xì)胞膜上的蛋白受體→Ca2+等第二信使→蛋白激酶→轉(zhuǎn)錄因子→抗冷基因”的途徑逐級(jí)在植物細(xì)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(guò)程中通過(guò)CBF轉(zhuǎn)錄因子調(diào)控大量下游抗冷基因的表達(dá)來(lái)提高植物抗冷能力。本研究從肥城桃桃果實(shí)提取出總RNA并克隆獲得cDNA并測(cè)得了肥城桃的CBF家族基因序列。對(duì)CBF家族基因進(jìn)行熒光定量分析發(fā)現(xiàn),在冷藏過(guò)程中,桃果實(shí)CBF1/5/6基因表達(dá)量迅速升高,且均維持在較高的水平,相對(duì)于對(duì)照,NO處理顯著提高了冷藏過(guò)程中CBF1/5/6的基因表達(dá)量,而c-PTIO通過(guò)清除NO,使CBF1/5/6的基因表達(dá)量略低于對(duì)照,證明了NO對(duì)于CBF抗冷途徑的調(diào)控作用。以克隆獲得的cDNA為模板,構(gòu)建pET30a-CBF重組載體,導(dǎo)入大腸桿菌BL21(DE3)進(jìn)行蛋白表達(dá),表達(dá)出了CBF1~3蛋白。為進(jìn)一步從分子生物學(xué)方面探討NO抗冷機(jī)制打下基礎(chǔ)。
[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(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS255.3

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