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向日葵谷胱甘肽

花匠小妙招
2024-11-19 23:09

摘要

为探讨谷胱甘肽-S-转移酶（GST）基因在向日葵抗病机制中的作用，在核盘菌诱导向日葵转录组文库的基础上从向日葵耐病品种龙食葵2号中克隆了1个GST的cDNA序列，并进行了表达和功能分析。结果表明：该基因开放阅读框为675bp，编码224个氨基酸残基，分子量为55.45kD，等电点为5.16，命名为HaGSTU1(GenBank登录号为KR071872)；HaGSTU1蛋白属于GST的Tau家族，推测是一种无信号肽的细胞质蛋白，其N端结构域包含结合GSH的G位点，C端结构域包含特异结合底物的H位点；HaGSTU1与一个橡胶树GST蛋白的亲缘关系最近，氨基酸序列的相似性为72%。实时荧光定量PCR结果显示，该基因在叶中表达量最高，其次是花、根、种和茎，在盘中的表达量最低；干旱、盐、草酸、核盘菌及其代谢物均能诱导向日葵HaGSTU1基因的表达量出现显著变化。利用农杆菌介导法将该基因导入烟草，提高了转基因烟草叶片对核盘菌的抗性。对抗性株系烟草GST和谷胱甘肽过氧化物酶(GPX)活性测定发现，转基因烟草叶片中GST和GPX酶活性较野生型提高约2倍，并随着接菌时间的延长，两种酶活性显著升高。初步推断HaGSTU1具有抗核盘菌的功能。

Abstract

To explore the disease resistance role of a glutathione S-transferases (GST) gene in sunflower, a full-length cDNA of GST (LSK-2) was cloned based on the transcriptome of Helianthus annuus induced by Sclerotinia sclerotiorum. Results showed that the cDNA contained a 675bp open reading frame (ORF) encoding 224 amino acids. The predicted protein was 55.45 kDa with isoelectric point of 5.16. This gene was named as HaGSTU1 (GenBank accession No. KR071872). HaGSTU1 protein contained Tau GST-specific N-terminal domain (G site) and C-terminal domain (H site). HaGSTU1 was predicted to be a cytoplasmic protein which shared the highest amino acid identity (72%) with a GST from Hevea brasiliensis. qRT-PCR results showed that HaGSTU1 was highly expressed in leaf, and less in sunflower head. Its expression was induced by drought, salt, oxalic acid, S. sclerotiorum and its metabolites. Then the HaGSTU1 gene was transformed into tobacco by Agrobacterium tumefaciens to further verifying its function. The results showed that the expression of HaGSTU1 improved the resistance of transgenic lines to S. sclerotiorum, significantly increased GST and GPX enzyme activity which were about 2 fold of those under S. sclerotiorum infection. It suggested that HaGSTU1 gene conferred a resistance to S. sclerotiorum.

关键词

向日葵 /谷胱甘肽-S-转移酶基因 /基因克隆 /功能分析{{custom_keyword}} /

Key words

/Glutathione S-transferase /Gene clone /Function analysis

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马立功，孟庆林，张匀华*，刘志华，王志英* .向日葵谷胱甘肽-S-转移酶基因的克隆及抗病功能研究[J]. 中国油料作物学报, 2015, 37(5): 635 https://doi.org/10.7505/j.issn.1007-9084.2015.05.007

MA Li-gong，MENG Qing-lin，ZHANG Yun-hua*，LIU Zhi-hua，WANG Zhi-ying* .Clone and function of a glutathione-S-transferase gene from sunflower (Helianthus annuus)[J]. CHINESE JOURNAL OF OIL CROP SCIENCES, 2015, 37(5): 635 https://doi.org/10.7505/j.issn.1007-9084.2015.05.007

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