紫穗槐抗坏血酸过氧化物酶(APX2)基因的克隆及抗旱性功能验证

Cloning of Ascorbate Peroxidase(APX2)Gene from Amorpha fruticosa L.and Functional Validation of Drought Resistance

为了研究紫穗槐(Amorpha fruticosa L.)在干旱条件下感知胁迫信号并通过生理和生化调节途径来维持其耐性功能作用的基因,可为紫穗槐抗旱性育种提供一个候选基因。通过对紫穗槐在干旱胁迫下的转录组数据进行分析,活性氧平衡系统的抗坏血酸过氧化物酶(Ascorbate Peroxidase,APX2)基因的表达量随胁迫时间显著升高,对紫穗槐的AfAPX2基因进行克隆,采用无缝克隆技术连接入门载体(pQB-V3),通过Gateway体系LR反应将目的基因构建到表达载体,并用电击法转化根癌农杆菌EHA105,采用农杆菌介导转化烟草(Nicotiana tabacum),愈伤组织培养成T 0代苗,收获种子后种植研究其与干旱胁迫的相关性。结果表明,AfAPX2基因表达蛋白与拟南芥和水稻的APX2同源比对,同样有过氧化物酶的结构域,推测可以通过ROS途径增强植物抗逆性。AfAPX2蛋白质二级结构有13处α-螺旋,17处β-折叠,4处β-转角和多处无规则卷曲,AfAPX2酶三级结构具有铁离子的结合位点。在干旱胁迫和胁迫后恢复供水下,过表达AfAPX2基因株系的叶片以及植株的茂盛程度明显大于野生型。本研究发现转AfAPX2基因阳性植株对自然干旱胁迫的耐受性增强,提高了烟草的抗旱性,说明AfAPX2在响应干旱胁迫中可能扮演重要的调节角色。进而验证了紫穗槐干旱胁迫转录组的上调基因与其相关,可以找到提高抗旱性的相关基因。

In order to study the genes that sense stress signals under drought conditions in Amorpha fruticosa and maintain their functional roles in tolerance through physiological and biochemical regulatory pathways,which can provide a candidate gene for drought-resistant breeding of Amorpha fruticosa.The transcriptome data of Amorpha fruticosa under drought stress were analysed,the expression of Ascorbate Peroxidase(APX2)gene,which is a system of reactive oxygen balance,increased significantly with the time of drought stress,and the AfAPX2 gene of Amorpha fruticosa was cloned,and a seamless cloning technique was used to connect to the starter vector(pQB-V3),and a Gateway system LR was developed.The target gene was constructed into the expression vector by Gateway system LR reaction,and transformed into Agrobacterium rhizogenes EHA105 by electroshock method,and Agrobacterium-mediated transformation of tobacco(Nicotiana tabacum)was adopted,and the healing tissues were cultured into seedlings of T 0 generation,and seeds were harvested and planted to study the correlation between them and drought stress.The results showed that AfAPX2 gene expressed protein was homologous to APX2 from Arabidopsis and rice,and also had the structural domain of peroxidase,which was hypothesized to enhance plant stress tolerance through the ROS pathway.The secondary structure of AfAPX2 protein had 13α-helices,17β-folds,4β-turns,and multiple irregular curls,and the tertiary structure of the AfAPX2 enzyme had a ferric ion.The tertiary structure of AfAPX2 enzyme has a binding site for iron ions.Under drought stress and restoration of water supply after stress,the leaves as well as plants of overexpressing AfAPX2 gene lines were significantly more luxuriant than those of the wild type.In this study,we found that trans-AfAPX2 gene-positive plants showed enhanced tolerance to natural drought stress and improved drought tolerance in tobacco,suggesting that AfAPX2 may play an important regulatory role in response to drought stress.It was further verified that the up-regulated genes in the drought stress transcriptome of Amorpha fruticosa were related to it,and the relevant genes for improving drought tolerance could be found.Overexpression of AfAPX2 in tobacco showed that transgene-positive plants showed enhanced tolerance to natural drought stress and improved drought tolerance in tobacco,suggesting that AfAPX2 may play an important regulatory role in response to drought stress.It was further verified that the up-regulated genes in the drought stress transcriptome of Amorpha fruticosa were related to it,and the relevant genes for improving drought tolerance could be found.It can be used as a candidate gene for breeding drought resistance in Amorpha fruticosa,which lays a solid theoretical foundation for its genetic improvement and resource utilization.