甜橙多胺氧化酶基因克隆及功能鉴定

发布时间：2018-02-28 23:22

本文关键词： 甜橙非生物逆境多胺分解代谢铜胺氧化酶多胺氧化酶活性氧　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：柑橘是世界上最重要的果树之一,然而各种生物胁迫和非生物胁迫严重影响柑橘产量和质量。多胺是普遍存在于高等植物中的一类低分子量脂肪族阳离子聚合物,其分解代谢在调节胁迫应答中多胺动态平衡中起着重要作用。多胺分解代谢主要由铜胺氧化酶(CuAO)和多胺氧化酶(PAO)催化完成。本研究利用甜橙基因组数据挖掘甜橙CuAO和PAO家族成员,分析其表达模式,最后利用遗传转化及生物化学技术揭示了部分成员的功能。主要结果如下:1.通过搜索比对共得到8个甜橙CuAO候选基因(命名为CsCuAO1-CsCuAO8),分别位于3条染色体上,编码区大小分别从1389 bp到2328bp。等电点和相对分子量预测结果显示甜橙CuAO蛋白等电点范围为6.11-8.77,相对分子量为52.1-82.8 kDa。亲缘关系分析显示CsCuAO基因家族成员可以分为两类。CsCuAO拥有植物CuAO高度保守的33个氨基酸残基,并且含有C端或者N端信号肽序列。组织表达分析发现CsCuAO1-3和CsCuAO5在根、茎、叶和子叶中表达量较高,其它基因表达水平较低。外源腐胺(Put),亚精胺(Spd)和精胺(Spm)诱导CsCuAO基因表达上升。此外,ABA、低温、盐害及渗透胁迫抑制CsCuAO基因(CsCuAO1-2,CsCuAO5,CsCuAO6和CsCuAO8)表达但诱导CsCuAO3和CsCuAO7上调表达。2.比对分析共发现6个甜橙PAO候选基因CsPAO1-CsPAO6。根据亲缘关系可将CsPAO基因分为四类。CsPAO基因在根中表达水平高于茎、叶和子叶中的水平。CsPAO基因表达也受到外源多胺处理的诱导,根据其表达模式推测CsPAO4基因可能参与多胺末端分解代谢而其余CsPAO基因可能参与多胺回复反应代谢。同时在烟草中超表达CsPAO3基因可以显著提高转基因烟草体内Put含量,并且降低Spd和Spm含量,说明该基因确实参与多胺回复反应代谢。另外,CsPAO基因同时受到ABA、低温、盐害和渗透胁迫的诱导,说明其可能参与激素和逆境胁迫应答。分析CsPAO基因启动子序列发现共有203个与激素和胁迫应答相关的顺式作用元件。3.为阐明PAO基因功能,通过遗传转化和生物化学技术分析了CsPAO4在多胺分解代谢和逆境应答中的功能。CsPAO4基因位于质外体中。体外底物催化结合HPLC检测,表明CsPAO4可以催化Spd和Spm并生成二氨基丙烷(Dap),说明该蛋白能够参与多胺末端分解代谢。在烟草中超表达CsPAO4基因,发现转基因植株PAO酶活性显著高于野生型,而Spd和Spm含量显著降低,同时Dap和过氧化氢(H2O2)含量显著上升。盐胁迫下转基因烟草比野生型烟草具有更快的种子发芽速度和更高的发芽率。然而,盐胁迫下转基因植株草营养生长和根系伸长均受到明显抑制,与此同时转基因植株体内积累更多的H2O2并且具有明显的细胞程序性死亡(PCD)现象。而外源加入过氧化氢清除剂(CAT)在一定程度上恢复转基因植株根系生长和营养生长。此外,外源Spm处理抑制转基因幼苗根系生长。
[Abstract]:Citrus is one of the most important fruit trees in the world. However, a variety of biological and abiotic stresses seriously affect the yield and quality of citrus. Polyamines are a kind of low molecular weight aliphatic cationic polymers widely found in higher plants. Its catabolism plays an important role in regulating the dynamic balance of polyamines in stress response. Polyamine catabolism is mainly catalyzed by copper amine oxidase (CuAO) and polyamine oxidase (PAO). In this study, sweet orange genome data were used to mine sweet orange. Members of the CuAO and PAO families, The main results are as follows: 1. Eight CuAO candidate genes (named CsCuAO1-CsCuAO8, named CsCuAO1-CsCuAO8) were obtained by searching and comparing. The results of isoelectric point and relative molecular weight prediction showed that the isoelectric point range of sweet orange CuAO protein was 6.11-8.77 and the relative molecular weight was 52.1-82.8 kDa. the genetic relationship analysis showed that the members of CsCuAO gene family could be classified into two classes. There are 33 amino acid residues which are highly conserved in plant CuAO. The expression of CsCuAO1-3 and CsCuAO5 was found to be high in root, stem, leaf and cotyledon. The expression level of other genes was low. Exogenous putrescine, spermidine (SPD) and spermidine (SPM) increased the expression of CsCuAO gene. Salt injury and osmotic stress inhibited the expression of CsCuAO1-2CsCuAO5, CsCuAO6 and CsCuAO8, but induced up-regulated expression of CsCuAO6 and CsCuAO8 in CsCuAO3 and CsCuAO7. Six candidate genes of PAO, CsPAO1-CsPAO6, were found by comparison analysis. According to their genetic relationship, the expression level of CsPAO gene in root was higher than that in stem. CsPAO gene expression in leaves and cotyledons was also induced by exogenous polyamines. According to its expression pattern, CsPAO4 gene may be involved in polyamine terminal catabolism and other CsPAO genes may be involved in polyamine response metabolism. Meanwhile, overexpression of CsPAO3 gene in tobacco can significantly increase Put content in transgenic tobacco. In addition, CsPAO gene was induced by ABA, low temperature, salt injury and osmotic stress. Analysis of the promoter sequence of CsPAO gene found that there were 203 cis-acting elements related to hormone and stress response. In order to elucidate the function of PAO gene, it may be involved in hormone response and stress stress response. The function of CsPAO4 in polyamine catabolism and stress response was analyzed by genetic transformation and biochemistry. The results showed that CsPAO4 could catalyze Spd and Spm and produce diaminopropane diapus, which indicated that the protein could participate in the end catabolism of polyamine. Overexpression of CsPAO4 gene in tobacco showed that the PAO enzyme activity of transgenic plants was significantly higher than that of wild-type plants. However, the contents of Spd and Spm decreased significantly, while the contents of Dap and H 2O 2 increased significantly. Transgenic tobacco under salt stress had faster seed germination rate and higher germination rate than wild type tobacco. The vegetative growth and root elongation of transgenic plants were significantly inhibited under salt stress. At the same time, the transgenic plants accumulated more H _ 2O _ 2 and had obvious programmed cell death (PCD) phenomenon. The exogenous hydrogen peroxide scavenger (CAT) could restore the root growth and vegetative growth of transgenic plants to some extent. Exogenous Spm treatment inhibited root growth of transgenic seedlings.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S666.4

【相似文献】