| 摘要: |
| 【目的】克隆山黧豆抗氧化酶基因，并对其进行生物信息学及表达模式分析，为研究抗氧化酶基因在山黧豆中的抗旱机制奠定基础。 【方法】以萌发7 d的山黧豆叶片为材料，通过RT PCR克隆山黧豆抗氧化酶基因的编码序列(CDS);采用荧光定量PCR分析抗氧化酶基因的组织表达模式；利用在线软件ExPASy ProtParam、SignalP 4.1、TMHMM V. 2.0、TargetP 1.1分别分析抗氧化酶基因编码的蛋白质理化性质、信号肽、跨膜区域以及亚细胞定位；利用在线工具Conserved Domain、SOPMA预测蛋白质保守结构域和二级结构，利用MEGA 6.0软件构建蛋白系统进化树；并利用20% PEG 6000溶液模拟干旱处理山黧豆，采用荧光定量PCR分析干旱胁迫不同时间抗氧化酶基因在叶片中的相对表达量。 【结果】RT-PCR克隆获得山黧豆抗氧化酶基因APX、CAT、MnSOD、FeSOD和Cu/ZnSOD的编码序列，其长度分别为864，1 485，723，1 005和942 bp。生物信息学分析表明，APX、CAT、MnSOD、FeSOD和Cu/ZnSOD抗氧化酶均为酸性不稳定蛋白质，且均由α-螺旋、延伸链、β-转角和无规则卷曲共4种二级结构组成；山黧豆抗氧化酶均包含高度保守的结构域。聚类分析结果表明，山黧豆的APX、CAT、MnSOD、FeSOD、Cu/ZnSOD依次与蒺藜苜蓿、蚕豆、豌豆、豌豆和锦鸡儿具有较高的相似性、较近的亲缘关系和遗传距离。表达分析结果表明，APX、CAT、MnSOD、FeSOD和Cu/ZnSOD基因在山黧豆根、茎、叶组织中均有表达。APX、CAT和MnSOD基因均响应了干旱胁迫，其中APX、CAT基因的表达量在干旱胁迫后迅速升高，3 h后达到最高，分别是0 h的5和4.3倍。【结论】成功克隆了山黧豆抗氧化酶基因APX、CAT、MnSOD、FeSOD和Cu/ZnSOD，推测其可协同清除干旱胁迫下产生的活性氧。 |
| 关键词: 山黧豆 抗氧化酶基因 基因克隆 生物信息学分析 |
| DOI： |
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| 基金项目:国家自然科学基金项目（31401910）；河南省科技厅科技攻关计划项目（172102310208）；河南省教育厅自然科学计划项目（16A180046） |
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| Cloning and expression analysis of antioxidant enzymes in Lathyrus sativus L. |
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WANG Hui,LIU Xiaoning,XU Quanle,等
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| Abstract: |
| 【Objective】The antioxidant enzyme genes of Lathyrus sativus L.were cloned and the bioinformatics and gene expression patterns were analyzed to provide basis for exploring the drought resistance mechanism of antioxidant enzymes.【Method】Seven days after germination,the leaves,roots and stems of Lathyrus sativus L.were selected.The coding sequences of antioxidant enzyme genes were cloned by RT-PCR,and tissue expression patterns were investigated by qPCR.ExPASy ProtParam,SignalP 4.1,TMHMM V. 2.0,and TargetP 1.1 were used for analyzing protein physicochemical properties,protein signal peptide,protein transmembrane region and subcellular localization.Conserved Domain and SOPMA were used for predicting the conserved domain and secondary structure.The phylogenetic trees were constructed by MEGA 6.0,and qRT-PCR was used for analyzing relative gene expression at different points in Lathyrus sativus treated by 20% PEG 6000.【Result】The coding sequences of the antioxidant enzyme genes APX,CAT,MnSOD,FeSOD and Cu/ZnSOD with sizes of 864,1 485,723,1 005,and 942 bp were amplified.Bioinformatics analysis indicated that APX,CAT,MnSOD,FeSOD and Cu/ZnSOD were acidic and unstable protein consisted of α-helix,extended strand,β-turn and random coil.Cluster analysis showed that APX,CAT,MnSOD,FeSOD and Cu/ZnSOD with highly conserved domain had high similarity as well as close genetic relationship and genetic distance with Medicaga truncatula,Vicia faba,Pisum sativum,Pisum sativum and Caragana jubata.Gene expression profiles revealed that APX,CAT,MnSOD,FeSOD and Cu/ZnSOD were expressed in roots,stems and leaves.APX,CAT,and MnSOD responded to drought stress.The expression levels of APX and CAT increased fast after drought stress,and their peaks after 3 hours were 5 and 4.3 times of that at 0 h.【Conclusion】Antioxidant enzyme genes APX,CAT,MnSOD,FeSOD and Cu/ZnSOD from Lathyrus sativus were successfully cloned,and it is speculated that they could scavenge ROS induced by drought stress. |
| Key words: Lathyrus sativus L. antioxidant enzymes genes gene cloning bioinformatics |
