采前光照诱导抗氧化物积累抑制‘Anjou’梨果实虎皮病发生的生化及分子机制

发布时间：2018-01-03 11:45

本文关键词：采前光照诱导抗氧化物积累抑制‘Anjou’梨果实虎皮病发生的生化及分子机制　出处：《山东农业大学》2016年博士论文　论文类型：学位论文

【摘要】：虎皮病(Superficial scald)是影响‘Anjou’梨采后贮藏寿命的主要生理性病害,严重影响果实的外观和商品价值。虎皮病的发生是某些梨和苹果品种在贮藏之后出现的因表皮细胞氧化造成的棕褐色病变。一般认为α-法尼烯的氧化产物共轭三烯是造成表皮褐化,产生虎皮病的关键因素。虎皮病的发生受采前温度、果实成熟度、采后贮藏温度、气调贮藏气体比例、乙烯合成等多个因素的影响。在本研究中,前期试验发现‘Anjou’梨果实着色面在八个月的长期贮藏中也没有虎皮病的发生,说明虎皮病的发生与采前光照有密切联系。因此,阐明光照诱导果实着色和抑制虎皮病发生的分子机理对于梨和苹果的采后贮藏意义重大。本研究以绿‘Anjou’(Pyrus communis L.cv.d’Anjou’)为试材,利用套袋处理,去除采前光照的影响。首先比较了套袋处理对绿‘Anjou’梨乙烯、呼吸、果实硬度、α-法尼烯、共轭三烯和虎皮病发生的影响。研究结果表明遮光处理对果实贮藏期间的乙烯、呼吸和后熟性状没有影响,显著降低了可溶性固形物的含量;但是遮光处理明显增加了α-法尼烯和共轭三烯的含量,促进了虎皮病的发生。进一步分析表明,α-法尼烯和共轭三烯仅在低温贮藏过程中积累,在后熟过程中都呈下降的趋势,由此说明,虎皮病的发生与低温下α-法尼烯尤其是共轭三烯的积累量有关。研究结果表明α-法尼烯在果实阳面和果实阴面中的含量相近,但共轭三烯的含量在果实阳面中明显低于果实阴面。果实阳面中积累了丰富的抗氧化物质(如花青素、多酚、类黄酮、黄烷醇),从而导致总抗氧化性的提高,抑制了α-法尼烯向共轭三烯的氧化过程。另外,果实阳面中的SOD、MDAR、APX、DHAR、GR等抗氧化酶也明显高于果实阴面,这些抗氧化酶使果实阳面具有更高的去除超氧阴离子自由基(O2-)、羟基自由基(-OH)、过氧化氢(H2O2)等自由基的能力。减轻了共轭三烯对果皮细胞的氧化伤害,从而减轻了虎皮病的发生。为了进一步阐明虎皮病发生的分子机理,本文以绿‘Anjou’和红‘Anjou’果实为试材,克隆了两个品种的α-法尼烯合成酶基因(AFS),命名为PcAFS1。cDNA全长1952 bp,编码577个氨基酸,两者同源性为99.3%。进化树分析发现,PcAFS1与苹果α-法尼烯合成基因同源性最高。qRT-PCR分析发现,PcAFS1在‘Anjou’果实、幼叶、老叶、花中都有表达,但在成熟度高的果皮中表达量最高;并且受乙烯的诱导。在采后低温贮藏过程中表达上升,1-MCP处理可以抑制该基因的表达。克隆比较了两个品种PcAFS1基因的启动子,启动子序列全长2067 bp,同源性99.6%,两者相差9个碱基。PLANTCARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)在线分析了启动子上的顺式元件,发现PcAFS1的启动子含有HSE(热激响应原件)和MBS(MYB结合位点),说明该基因有可能受温度和MYB转录因子的调控。为了进一步确定PcAFS1基因的功能,我们构建了pBI121-PcAFS1过量表达载体,利用农杆菌侵染法浸染拟南芥花序。分析了转基因拟南芥的α-法尼烯积累,表明PcAFS1可以促进α-法尼烯合成。用乙烯(0.2μl l-1)处理转基因拟南芥,α-法尼烯积累上升,并发现莲座叶有向下弯曲的现象。花青素在光照和套袋处理中差异最大,说明花青素可能在虎皮病的发生过程中发挥了关键作用。为了进一步确定花青素和虎皮病发生的分子机理,以绿‘Anjou’和红‘Anjou’果皮为试材,利用差异筛选的方法,得到一个表达差异极为显著地花青素调控MYB转录因子,命名为PcMYB1。该基因只在红‘Anjou’果皮中表达,在绿‘Anjou’没有表达。在绿‘Anjou’和红‘Anjou’果皮克隆了PcMYB1的启动子序列,比对发现两者同源性达99.3%,只有12个碱基的差异;克隆了两者的基因组序列,发现红‘Anjou’与绿‘Anjou’相比,在第一个内含子上缺失了244个碱基。推测是因为内含子的缺失造成的基因转录水平的差异和造成绿‘Anjou’果实变红。在果实贮藏前期,红‘Anjou’中α-法尼烯的积累比绿‘Anjou’要高。1-MCP处理的红‘Anjou’PcAFS1的基因表达在果实贮藏后期可以部分恢复,在绿‘Anjou’中完全被抑制。为了确定PcMYB1是否调控PcAFS1的基因表达,用35S::PcMYB1、35S::PcbHLH3、35S::PcWD40与PPcAFS1::LUC共转化烟草叶片,发现PcMYB1促进PcAFS1的基因表达,但是这种促进作用不受花青素调控因子bHLH或WD40的影响。说明PcMYB1对α-法尼烯的调控机制与花青素不同。根据本研究的结果,我们对光照抑制‘Anjou’梨虎皮病的发生做了以下总结和推测:在生化层面,光照促进了花青素、多酚、类黄酮、黄烷醇等抗氧化物质的积累,提高了SOD、MDAR、APX、DHAR、GR等抗氧化酶的活性,从而导致总抗氧化性的提高,抑制了α-法尼烯向共轭三烯的氧化过程,构成了果实阳面对虎皮病的天然抗性。在分子机理层面,PcMYB1在红‘Anjou’中过量表达,一方面通过结合PcAFS1基因的启动子促进了α-法尼烯的积累,另一方面促进了花青素的合成,花青素作为抗氧化物质通过抑制α-法尼烯向共轭三烯的氧化,因此减轻了虎皮病的发生。
[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.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.612

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