拟南芥醛还原酶编码基因At3g04000表达模式分析及过量表达转基因植株获得

Expression pattern analysis of Arabidopsis aldehyde reductase encoding gene At3g04000 and generation of overexpression plants

关键词:  拟南芥, α,β-不饱和醛, 醛还原酶, At3g04000, 表达模式, 过量表达

Key words:  Arabidopsis, α,β-unsaturated aldehyde, aldehyde reductase, At3g04000, expression pattern, overexpression

摘要:

植物体内的α,β-不饱和活性醛类化合物对植物细胞具有毒害作用,清除这些α,β-不饱和活性醛类化合物对于植物细胞维持正常的生命活动至关重要。前人研究报道通过体外酶活测定和异源瞬时表达鉴定拟南芥At3g04000基因编码的蛋白为NADPH依赖的叶绿体醛还原酶(Arabidopsis NADPH-dependent chloroplastic aldehyde reductases, AtChlADRs),推测其在清除叶绿体中长链(≥5)α,β-不饱和醛类物质中具有重要的功能。该研究主要构建了拟南芥At3g04000基因的表达模式分析载体ProAt3g04000:GUS、亚细胞定位分析载体At3g04000-EGFP和过量表达载体At3g04000-OE,并获得了转基因拟南芥,并通过实时定量PCR分析了At3g04000基因在拟南芥不同组织中的转录水平。结果表明:拟南芥At3g04000基因在幼苗中的转录水平最高,在莲座叶、茎生叶、花序和角果中均有较高的转录水平; 而在根部和茎秆中的转录水平较低。通过对ProAt3g04000:GUS转基因植株的GUS染色分析可知,At3g04000基因在子叶、莲座叶和萼片的维管组织和保卫细胞中均有较强的表达,在根的维管组织中有较弱的表达。通过共聚焦显微镜对At3g04000-EGFP转基因植株的观察和分析发现,At3g04000不是定位于叶绿体中,而是定位在细胞质和细胞核中。该研究结果为深入研究拟南芥醛还原酶编码基因At3g04000的功能奠定了基础。

Abstract:

Reactive aldehyde, especially α,β-unsaturated aldehyde compounds are toxic to plant cells. Therefore, elimination of α,β-unsaturated aldehyde compounds is vital for plant cells to maintain normal life activities. In previous reports, the Arabidopsis At3g04000 gene was identified as encoding a NADPH-dependent chloroplastic aldehyde reductases(AtChlADRs)by enzyme activity assay in vitro and subcellular localization analyse in spiderwort cells. And it was proposed to play important role in scavenging α,β-unsaturated aldehydes with more than 5 carbons(C≥5)in chloroplasts. To further analysis the roles of At3g04000 gene, we generated transgenic Arabidopsis plants expressing a ProAt3g04000:GUS for analysis of its expression pattern, a At3g04000-EGFP translational fusion for subcellular localization analysis, and 35S:At3g04000 for overexpression of At3g04000 gene. We also used quantitative real time PCR to investigate the transcription of At3g04000 gene during the developmental process of Arabidopsis. The results showed that the transcription of At3g04000 gene was detected in all examined tissues. The highest transcription level of At3g04000 gene was detected in Arabidopsis seedlings, relative high level of At3g04000 gene transcripts were also detected in rosette leaf, cauline leaf, inflorescence and silique, while in root and stem the transcription level of At3g04000 gene was very weak. The results of GUS staining in ProAt3g04000: GUS transgenic Arabidopsis plants were in consistency with the results of the quantitative real time PCR analysis. Strong GUS staining was found in vascular tissues and guard cells of cotyledon, rosette leaf and sepal, and weak GUS staining was found in vascular tissues of root. To analyze the subcellular localization of the At3g04000 gene encoded protein, the At3g04000-EGFP transgenic Arabidopsis seedling was observed by confocal microscopy. The results showed that At3g04000 gene encoded protein was not localized in chloroplast, but localized in cytoplasm and nucleus. This study provides tools for further study of the roles of At3g04000 gene in Arabidopsis.