發(fā)布時間：2018-01-03 11:45

  本文關鍵詞：采前光照誘導抗氧化物積累抑制‘Anjou’梨果實虎皮病發(fā)生的生化及分子機制　出處：《山東農(nóng)業(yè)大學》2016年博士論文　論文類型：學位論文

  更多相關文章： ‘Anjou’梨 虎皮病 抗氧化物質(zhì) α-法尼烯 MYB

【摘要】：虎皮病(Superficial scald)是影響‘Anjou’梨采后貯藏壽命的主要生理性病害,嚴重影響果實的外觀和商品價值�；⑵げ〉陌l(fā)生是某些梨和蘋果品種在貯藏之后出現(xiàn)的因表皮細胞氧化造成的棕褐色病變。一般認為α-法尼烯的氧化產(chǎn)物共軛三烯是造成表皮褐化,產(chǎn)生虎皮病的關鍵因素�；⑵げ〉陌l(fā)生受采前溫度、果實成熟度、采后貯藏溫度、氣調(diào)貯藏氣體比例、乙烯合成等多個因素的影響。在本研究中,前期試驗發(fā)現(xiàn)‘Anjou’梨果實著色面在八個月的長期貯藏中也沒有虎皮病的發(fā)生,說明虎皮病的發(fā)生與采前光照有密切聯(lián)系。因此,闡明光照誘導果實著色和抑制虎皮病發(fā)生的分子機理對于梨和蘋果的采后貯藏意義重大。本研究以綠‘Anjou’(Pyrus communis L.cv.d’Anjou’)為試材,利用套袋處理,去除采前光照的影響。首先比較了套袋處理對綠‘Anjou’梨乙烯、呼吸、果實硬度、α-法尼烯、共軛三烯和虎皮病發(fā)生的影響。研究結果表明遮光處理對果實貯藏期間的乙烯、呼吸和后熟性狀沒有影響,顯著降低了可溶性固形物的含量;但是遮光處理明顯增加了α-法尼烯和共軛三烯的含量,促進了虎皮病的發(fā)生。進一步分析表明,α-法尼烯和共軛三烯僅在低溫貯藏過程中積累,在后熟過程中都呈下降的趨勢,由此說明,虎皮病的發(fā)生與低溫下α-法尼烯尤其是共軛三烯的積累量有關。研究結果表明α-法尼烯在果實陽面和果實陰面中的含量相近,但共軛三烯的含量在果實陽面中明顯低于果實陰面。果實陽面中積累了豐富的抗氧化物質(zhì)(如花青素、多酚、類黃酮、黃烷醇),從而導致總抗氧化性的提高,抑制了α-法尼烯向共軛三烯的氧化過程。另外,果實陽面中的SOD、MDAR、APX、DHAR、GR等抗氧化酶也明顯高于果實陰面,這些抗氧化酶使果實陽面具有更高的去除超氧陰離子自由基(O2-)、羥基自由基(-OH)、過氧化氫(H2O2)等自由基的能力。減輕了共軛三烯對果皮細胞的氧化傷害,從而減輕了虎皮病的發(fā)生。為了進一步闡明虎皮病發(fā)生的分子機理,本文以綠‘Anjou’和紅‘Anjou’果實為試材,克隆了兩個品種的α-法尼烯合成酶基因(AFS),命名為PcAFS1。cDNA全長1952 bp,編碼577個氨基酸,兩者同源性為99.3%。進化樹分析發(fā)現(xiàn),PcAFS1與蘋果α-法尼烯合成基因同源性最高。qRT-PCR分析發(fā)現(xiàn),PcAFS1在‘Anjou’果實、幼葉、老葉、花中都有表達,但在成熟度高的果皮中表達量最高;并且受乙烯的誘導。在采后低溫貯藏過程中表達上升,1-MCP處理可以抑制該基因的表達�？寺”容^了兩個品種PcAFS1基因的啟動子,啟動子序列全長2067 bp,同源性99.6%,兩者相差9個堿基。PLANTCARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)在線分析了啟動子上的順式元件,發(fā)現(xiàn)PcAFS1的啟動子含有HSE(熱激響應原件)和MBS(MYB結合位點),說明該基因有可能受溫度和MYB轉錄因子的調(diào)控。為了進一步確定PcAFS1基因的功能,我們構建了pBI121-PcAFS1過量表達載體,利用農(nóng)桿菌侵染法浸染擬南芥花序。分析了轉基因擬南芥的α-法尼烯積累,表明PcAFS1可以促進α-法尼烯合成。用乙烯(0.2μl l-1)處理轉基因擬南芥,α-法尼烯積累上升,并發(fā)現(xiàn)蓮座葉有向下彎曲的現(xiàn)象�；ㄇ嗨卦诠庹蘸吞状幚碇胁町愖畲�,說明花青素可能在虎皮病的發(fā)生過程中發(fā)揮了關鍵作用。為了進一步確定花青素和虎皮病發(fā)生的分子機理,以綠‘Anjou’和紅‘Anjou’果皮為試材,利用差異篩選的方法,得到一個表達差異極為顯著地花青素調(diào)控MYB轉錄因子,命名為PcMYB1。該基因只在紅‘Anjou’果皮中表達,在綠‘Anjou’沒有表達。在綠‘Anjou’和紅‘Anjou’果皮克隆了PcMYB1的啟動子序列,比對發(fā)現(xiàn)兩者同源性達99.3%,只有12個堿基的差異;克隆了兩者的基因組序列,發(fā)現(xiàn)紅‘Anjou’與綠‘Anjou’相比,在第一個內(nèi)含子上缺失了244個堿基。推測是因為內(nèi)含子的缺失造成的基因轉錄水平的差異和造成綠‘Anjou’果實變紅。在果實貯藏前期,紅‘Anjou’中α-法尼烯的積累比綠‘Anjou’要高。1-MCP處理的紅‘Anjou’PcAFS1的基因表達在果實貯藏后期可以部分恢復,在綠‘Anjou’中完全被抑制。為了確定PcMYB1是否調(diào)控PcAFS1的基因表達,用35S::PcMYB1、35S::PcbHLH3、35S::PcWD40與PPcAFS1::LUC共轉化煙草葉片,發(fā)現(xiàn)PcMYB1促進PcAFS1的基因表達,但是這種促進作用不受花青素調(diào)控因子bHLH或WD40的影響。說明PcMYB1對α-法尼烯的調(diào)控機制與花青素不同。根據(jù)本研究的結果,我們對光照抑制‘Anjou’梨虎皮病的發(fā)生做了以下總結和推測:在生化層面,光照促進了花青素、多酚、類黃酮、黃烷醇等抗氧化物質(zhì)的積累,提高了SOD、MDAR、APX、DHAR、GR等抗氧化酶的活性,從而導致總抗氧化性的提高,抑制了α-法尼烯向共軛三烯的氧化過程,構成了果實陽面對虎皮病的天然抗性。在分子機理層面,PcMYB1在紅‘Anjou’中過量表達,一方面通過結合PcAFS1基因的啟動子促進了α-法尼烯的積累,另一方面促進了花青素的合成,花青素作為抗氧化物質(zhì)通過抑制α-法尼烯向共軛三烯的氧化,因此減輕了虎皮病的發(fā)生。
[Abstract]:Skin disease (Superficial scald) is the main physiological disease Anjou 'pear postharvest life, seriously affect the appearance of the fruit and the value of the goods. There is some skin disease of pear and apple varieties during storage after oxidation caused by epidermal cell disease. Generally the brown oxidation products of conjugated alpha Pennsylvania ene three ene is caused by skin browning, key factors skin disease. Skin disease by preharvest temperature, fruit maturity, postharvest storage temperature, gas storage gas ratio, influence factors such as ethylene synthesis. In this study, the test found that' Anjou 'pear fruit coloring do not skin disease in the long-term storage of eight months, that skin disease and pre harvest light are closely linked. Therefore, to clarify the light induced inhibition of fruit coloring and the molecular mechanism of the occurrence of scald in Pear And apple postharvest storage of great significance. In this study, the green "Anjou" (Pyrus communis L.cv.d 'Anjou') as test materials, the use of bagging, the removal effects of Preharvest light. The first comparison of Bagging on green 'Anjou' pear fruit firmness, respiration, ethylene, alpha farnesene three, the effects of conjugated alkenes and skin disease. The results showed that ethylene shading on Fruit during storage and ripening, did not affect the respiratory traits, decreased the content of soluble solids; however, shading treatment significantly increased the content of alpha farnesene and conjugated diene three, promote skin disease further analysis showed that alpha farnesene and conjugated three ene accumulated only during storage at low temperature, in the ripening process decreased, which indicated that the occurrence of low temperature and scald under alpha farnesene especially the accumulation of conjugated three ene related research results. That was close to that of alpha farnesene in adret fruit and fruit in the nightside, but the content of conjugated three ene in the fruit sample was significantly lower than that of fruit. The fruit has accumulated adret Yinmian rich in antioxidants (such as anthocyanins, polyphenols, flavonoids, Huang Wanchun), which leads to the increase of total antioxidant, inhibiting alpha farnesene oxidation to conjugated three ene. In addition, the fruits of adret in SOD, MDAR, APX, DHAR, GR and other antioxidant enzymes were significantly higher than those in the fruits of the nightside, these antioxidant enzymes make fruit adret has higher removal of superoxide anion radical (O2-), hydroxyl radical (-OH), hydrogen peroxide (H2O2) the ability of free radicals. Reduce the oxidative damage of the conjugate three ene pericarp cells, thereby reducing skin disease. In order to further elucidate the molecular mechanism of skin disease, the green 'Anjou' and 'Anjou' red fruit as materials, cloning Two varieties of alpha farnesene synthase gene (AFS), named PcAFS1.cDNA was 1952 BP, encoding 577 amino acids of 99.3%., phylogenetic analysis showed that the two homologous PcAFS1 and alpha farnesene synthesis gene homology analysis showed that.QRT-PCR, PcAFS1 in the "Anjou" fruits, young leaves, old leaves. The flowers are expressed, but in high maturity in the pericarp of the highest expression level; and induced by ethylene. After harvest during low temperature storage was increased, 1-MCP treatment can inhibit the expression of the gene cloning. A comparison of two varieties of PcAFS1 gene promoter, promoter sequence was 2067 BP homology 99.6%.PLANTCARE, a difference of 9 nucleotides (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) online analysis of cis elements on the promoter, PcAFS1 promoter containing HSE (heat shock response of the original) and MBS (MYB binding site) And that regulation of this gene may be affected by the temperature and the MYB transcription factor. In order to further determine the function of PcAFS1 gene, we constructed pBI121-PcAFS1 expression vector, using Agrobacterium mediated method. The inflorescence dip analysis of transgenic Arabidopsis alpha farnesene accumulation showed that PcAFS1 can promote alpha farnesene synthesis by ethylene (. 0.2 L L-1) treatment of transgenic Arabidopsis, alpha farnesene accumulation increased, and found the rosette leaves had a downward bending phenomenon. The biggest difference in anthocyanin light and bagging, that anthocyanins may play a key role in the skin disease process. In order to further determine the molecular mechanism of anthocyanin and skin disease the green "Anjou" and "Anjou" red peel as material, using the method of differential screening, an expression is significant to anthocyanin regulation of the MYB transcription factor, named Pc The MYB1. gene is expressed only in red pericarp, 'Anjou' in the green 'Anjou'. No expression in the green 'Anjou' and 'Anjou' red pericarp was cloned PcMYB1 promoter sequences, the two comparison found that the homology of 99.3%, only 12 base difference; the cloned genomic sequence, found red and green "Anjou" "Anjou" than in the first intron deletion of 244 nucleotides. That is because of differences in the level of gene transcription intron deletion caused by the green "Anjou" red fruit. The fruit in the early storage, red 'Anjou' alpha farnesene accumulation ratio green 'Anjou' to high.1-MCP red 'Anjou' PcAFS1 gene expression in fruit storage period can be partially restored, in the green 'Anjou' was completely suppressed. In order to determine whether PcMYB1 expression regulation of PcAFS1 gene by 35S:: PcMYB1,35S:: PcbHLH3,35S:: PcWD40 PPcAFS1:: LUC were transformed into tobacco leaves, found that PcMYB1 promotes the expression of PcAFS1 gene, but the effect is not affected by the promotion of anthocyanin regulatory factor bHLH or WD40. PcMYB1 of alpha farnesene regulation and anthocyanin. According to the results of this study, we light inhibited the 'Anjou' pear skin disease do the following summary and speculation: in the biochemical level, light promoted anthocyanins, polyphenols, flavonoids, flavanols and other antioxidants accumulation, increased SOD, MDAR, APX, DHAR, GR and the activity of antioxidant enzymes, which leads to the increase of total anti oxidation, inhibition of alpha farnesene to oxidation the process of conjugate three ene, a fruit of sun scald. The molecular mechanism of natural resistance level, overexpression of PcMYB1 in the red "Anjou", on the one hand by the combination of PcAFS1 gene promoter promotes alpha farnesene accumulation, on the other hand Promote the synthesis of anthocyanins and anthocyanin as antioxidant by inhibiting the oxidation of alpha farnesene to conjugate three ene, thus reducing skin disease.

【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S436.612

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