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胡杨和灰杨AOX1a基因在非生物胁迫响应中的功能分化

Functional Divergence of AOX1a from Populus Euphratica and P.pruinosa in Response to Abiotic Stresses

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【Author in Chinese】 雷文丽

【Supervisor】 万东石

【Author's Information】 兰州大学, 生态学, 2018, 硕士

【Abstract in Chinese】 高等植物的呼吸途径有两条,即细胞色素C氧化(COX)途径和交替氧化(AOX)途径。当COX途径被阻断时,AOX途径会被诱导启动。交替氧化酶(AOX)是AOX途径的关键蛋白,是由线粒体内膜上的一个小的核基因家族编码,广泛参与了植物的抗逆过程。本研究以胡杨和灰杨为实验材料,通过比较两个AOX1a直系同源基因的功能分化,探讨其在杨树抗逆响应中的作用机理。具体结果如下:(1)杨树AOX基因重复及进化。通过比较胡杨AOX基因家族序列及结构特征,发现胡杨的AOX基因家族有4个成员,AOX1a、AOX1b、AOX1c和AOX1d。染色体定位分析发现,AOX1a、AOX1b和AOX1c定位于12号染色体上,而AOX1d则定位在3号染色体上;共线性分析进一步发现,12号染色体上AOX1a、AOX1b和AOX1c串联分布,其物理距离小于200kb,所在的重复单元和3号染色体上的AOX1d具有共线性。表明AOX基因家族随杨树全基因组重复而扩张,又经历了串联重复,保留了4个重复基因。(2)杨树的AOX1a提高了抗氰呼吸在总呼吸的比重。我们克隆鉴定了胡杨和灰杨的AOX1a基因,并在aox1a的基础上分别构建了回补拟南芥和超表达拟南芥转基因株系。结果发现,转基因拟南芥在总呼吸速率(Vt)、细胞色素c呼吸速率(Vcyt)和抗氰呼吸速率(Valt)方面没有显著差异,但是和aox1a株系Vt、Vcyt和Valt之间显著差异,aox1a的Vcyt/Vt值显著高于其它株系,相应的Valt/Vt值显著低于其他株系。这些结果表明胡杨和灰杨的AOX1a过表达均可以恢复拟南芥抗氰呼吸的活性。(3)杨树AOX1a过表达可以提高拟南芥对于非生物胁迫的耐受性。通过分析上述转基因拟南芥在干旱、盐等不同处理下的种子萌发率和根长,发现PeuAOX1a、PprAOX1a;1和PprAOX1a;2基因过表达以后可以增加盐胁迫和干旱胁迫下的萌发率和根长。与此同时,PeuAOX1a、PprAOX1a;1和PprAOX1a;2基因过表达以后可以降低转基因拟南芥的MDA、EL和H2O2含量,提高脯氨酸和抗氧化酶如SOD、POD的活性,表明PeuAOX1a、PprAOX1a;1和PprAOX1a;2基因在突变体拟南芥中过表达提高了植物对于盐胁迫和干旱胁迫的耐受性。综上所述,胡杨和灰杨的AOX1a基因过表达以后可以显著提高植物对于盐胁迫和干旱胁迫的耐受性,主要是通过启动抗氰呼吸、减少活性氧的积累和提高抗氧化酶的活性来提高的,我们的结果强调了抗氰呼吸途径参与两种沙漠杨树对于胁迫的响应,从而对AOX基因家族的生物学功能有了更深的认识,为后续的研究积累资料。

【Abstract】 In higher plant,there are two respiratory pathways on mitochondrial membrane,cyanide-sensitive cytochrome C oxidase(COX)pathway and cyanide-insensitive alternative oxidase(AOX)pathway.Once plants are subjected to various stresses,the AOX pathway will be activated by alternative oxidases(AOXs),which are located in the mitochondrial inner membrane and act as terminal oxidase in the mitochondrial electron transport chain.The AOX gene family is coded by a small gene family,and AOXs have been established as one of the essential defence components in the response of plant to biotic and abiotic stresses.In this study,we have isolated and identified AOX genes from P.euphratica and P.pruinosa respectively,and compared their functions divergence after diversification of the two desert poplars.The results were as follows:(1)The duplication of AOX genes in poplars.A total of four orthologus AOX genes were identified in genome of Populus euphratica and P.pruinosa respectively.Of them,AOX1a,AOX1b and AOX1c located on the chromosome 12 while AOX1d is located on chromosome 3that would attribute to one whole genome duplication event occurred in genome of Populus species.Whereas AOX1a,AOX1b and AOX1c were suggested duplicated by tandem duplications.The collinear analysis of the four genes confirmed this deduce,it was found that the duplication block of AOX1a,AOX1b and AOX1c had a collinear relationship with that of AOX1d,and the distance between AOX1a,AOX1b and AOX1c were less than 200kb on chromosome 12.(2)The over-expression of AOX1a from P.euphratica and P.pruinosa enhance proportion of cyanide-resistant respiration in total respiration.We investigated total respiration rate(Vt),cytochrome c respiration rate(Vcyt)and cyanide-resistant respiration rate(Valt)of the transgenic Arabidopsis lines,respectively.The results showed that there was no significant difference between Vt,Vcytyt and Valtlt except for that of in aox1a lines,but the ratio of Vcyt/Vt of aox1a was significantly higher than that of other lines,and the ratio of Valt/Vt was significantly lower than that of other lines.However,in terms of Vcyt/Vt and Valt/Vt,no significant difference were found between the WT and AOX over-expressed lines.These results suggested that over-expression of AOX could increase alternate respiratory activity to WT level.(3)The over-expression of AOX1a from P.euphratica and P.pruinosa improve Arabidopsis tolerance to abiotic stresses.Seed germination rate and root length of transgenic Arabidopsis lines with PeuAOX1a and PprAOX1a were tested with drought and salt treatments.The results showed that over-expression PeuAOX1a and PprAOX1a could increase the germination rate and root length after salt and drought stress.In terms of other physiological parameters,including MDA,MDA,EL,H2O2,proline and antioxidase activity.The results showed that Peu AOX1a and PprAOX1a significantly decreased the contents of MDA,EL and H2O2 in transgenic Arabidopsis.However,the content of proline and antioxidase activities,such as SOD and POD were increased,suggesting that over-expression of AOX1a reduced the damage of stresses to transgenic Arabidopsis.These results suggested that over-expression of PeuAOX1a and PprAOX1a genes in Arabidopsis could improve the tolerance of plant to salt and drought stresses.In conclusion,over-expression of AOX1a gene from P.euphratica and P.pruinosa could significantly improve plant tolerance to salt and drought stress by activating cyanide-resistant respiration,increasing antioxidase activities and reducing the accumulation of ROS(Reactive Oxygen Species).Our results highlight the alternative pathway involved in the response of two desert poplars to stresses.

  • 【Contributor】 兰州大学
  • 【Year of Internet Publish】201811
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