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白菜(Brassica rapa subsp.pekinensis)在种植加工和运输过程中,易遭受各种机械伤害。本研究对白菜叶片进行伤害处理,利用气相色谱-质谱(gas chromatography-mass spectrometry,GC-MS)联用、GC和高效液相色谱法(high performance liquid chromatography,HPLC)分别测定内源信号分子茉莉酸(jasmonic acid,JA)、水杨酸(salicylic acid,SA)和乙烯(ethylene,ET)的含量或释放量变化,并利用c DNA芯片杂交分析伤害处理后基因表达总体变化,结合半定量RT-PCR分析JA、ET和SA信号途径中标志基因表达量变化。结果显示,大量基因在白菜伤害后上调表达,包括转录因子、小G蛋白、受体蛋白激酶、蛋白激酶、防御相关基因、JA/ET合成途径基因、吲哚芥子油苷降解途径基因、细胞壁修饰相关基因、色氨酸生物合成途径基因、苯丙烷和生物碱代谢途径相关基因、分子伴侣等。对伤害处理后JA含量及其信号途径标志基因表达分析发现,在伤害处理后6、12、24和48 h,叶片JA含量显著高于未进行伤害处理的空白对照叶片中JA含量(P<0.05);JA合成途径关键酶基因丙二烯氧合酶(allene oxide synthase,AOS)、丙二烯环氧化酶(allene oxide cyclase,AOC)和脂氧合酶(lipoxygenase,LOX)、JA合成途径上游的磷脂酶D(phospholipase D,PLD)和JA途径的应答标志基因黑芥子酶作用蛋白(myrosinase-associated protein,MYAP)诱导上调表达,表明JA介导的信号途径在白菜伤害应答反应中起了重要作用。对伤害处理后ET释放速度及其信号途径基因表达分析发现,ET在伤害处理后2和6 h大量合成,ET合成途径基因1-氨基环丙烷-1-羧酸合成酶6(1-aminocyclopropane-1-carboxylicacid(ACC)synthase 6,ACS6)和ACC氧化酶(ACC oxidase,ACO)在伤害早期诱导上调表达,乙烯响应转录因子4(ethylene-responsive transcription factor 4,ERF4)在伤害后0.17、0.5、2、6、12和24 h均上调表达,表明ET途径参与了早期的伤害反应。JA和ET共同调控途径的标志基因β-1,3葡聚糖酶(β-1,3 glucanase,BGL)在伤害各时间点均上调表达,表明JA和ET协同调控白菜伤害反应。对伤害处理后SA含量及其信号途径基因表达分析发现,游离态SA和总SA的含量明显下降,SA途径的标志基因病程相关蛋白(pathogenesis-related protein 1a,PR1a)和PR5在伤害后期有一定程度的诱导表达,而在伤害早期其表达受到抑制或未发生变化,表明SA信号途径在伤害早期受到抑制,在伤害后期活化,可能参与了伤害后期的防卫反应。本研究阐明了白菜在伤害处理后,基因表达总体水平产生变化,JA、ET和SA合成及其介导的信号途径标志基因表达产生变化,有助于揭示白菜伤害反应分子机制。
Chinese cabbage (Brassica rapa subsp. Pekinensis) is susceptible to various mechanical injuries during its processing and transportation. In this study, cabbage leaves were harvested and harvested. Gas chromatography-mass spectrometry (GC-MS), GC and high performance liquid chromatography (HPLC) Changes of content or release of jasmonic acid (JA), salicylic acid (SA) and ethylene (ET), and analysis of overall changes of gene expression after injury treatment by c DNA chip hybridization. Combined with semi-quantitative RT -PCR analysis of JA, ET and SA signal pathway marker gene expression changes. The results showed that a large number of genes were up-regulated after cabbage injury, including transcription factor, small G protein, receptor protein kinase, protein kinase, defense related gene, JA / ET synthesis pathway gene, indole glucosinolate degradation pathway gene, cell wall modification Related genes, tryptophan biosynthesis pathway genes, phenylpropane and alkaloids metabolic pathway related genes, chaperones and so on. Analysis of the expression of JA and its signaling pathways after injury showed that the JA content of leaves at 6, 12, 24 and 48 h after injury was significantly higher than that of the blank control leaves without injury (P <0.05 ); The key enzyme genes of JA synthesis pathway including allene oxide synthase (AOS), allene oxide cyclase (AOC) and lipoxygenase (LOX), JA synthesis pathway Upstream phosphorylase D (PLD) and JA pathway response marker gene myrosinase-associated protein (MYAP) up-regulated expression, indicating JA-mediated signaling pathway in response to injury in cabbage played Important role. Analysis of the ET release rate and its signaling pathway gene expression after injury showed that ET synthesized in large quantities 2 and 6 h after injury, and the ET synthesis pathway 1-aminocyclopropane-1-carboxylate synthase 6 (1-aminocyclopropane- ACC-synthase 6, ACS6 and ACC oxidase (ACCO-1, ACCO-1, ACO) were upregulated in the early stage of injury, and the expression of ethylene-responsive transcription factor 4 (ERF4) The expression was up-regulated at 2, 6, 12 and 24 h, indicating that the ET pathway was involved in the early injury response. The β-1,3 glucanase (BGL), a marker of JA and ET co-regulatory pathway, was up-regulated at all time points after injury, indicating that JA and ET synergistically regulate cabbage injury. The analysis of gene expression of SA and its signal pathway after injury showed that the levels of free SA and total SA were significantly decreased, and the pathogenesis-related protein 1a (PR1a) and PR5 of SA pathway were at a certain extent The expression of SA signal was inhibited or not changed in the early stage of injury, indicating that the SA signal pathway was inhibited early in the injury and activated in the latter stage of injury, which may be involved in the defense response after the injury. This study elucidated that after injury, the gene expression level of Chinese cabbage changed. The changes of JA, ET and SA synthesis and the expression of signal transduction pathway markers were helpful to reveal the molecular mechanism of injury response in Chinese cabbage.