棉铃虫硫氧还蛋白类似基因HaTrx-like的分子鉴定和抗逆特征
Molecular identification of a thioredoxin-like gene in Helicoverpa armigera and its expression following different kinds of stress
张松斗** 张博宇 申忠健 刘彦君 李 贞 张青文 刘小侠***
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DOI：10.7679/j.issn.2095-1353.2015.062
作者单位：中国农业大学昆虫系，北京 100193
中文关键词：棉铃虫，硫氧还蛋白类似基因，基因克隆，时空表达，逆境处理
英文关键词：Helicoverpa armigera, thioredoxin-like, molecular cloning, temporal-spatial expression, adversity treatment
中文摘要：
【目的】 克隆棉铃虫Helicoverpa armigera硫氧还蛋白类似基因（HaTrx-like）的cDNA全长并进行序列分析，研究HaTrx-like基因的时间和空间表达模式以及参与抵抗机械损伤、紫外线照射和极端温度等逆境的特征。【方法】 利用RT-PCR的方法扩增得到HaTrx-like基因的全长cDNA并通过测序进行验证，运用几种生物信息学软件对基因和氨基酸序列进行分析，利用荧光定量PCR检测HaTrx-like基因的时间和空间分布以及4种逆境条件处理后的表达模式。【结果】 棉铃虫HaTrx-like基因的cDNA全长为526 bp，其中开放阅读框长度为381 bp，编码126个氨基酸，含有一个保守的氧化还原活性位点CXXC，其氨基酸序列和帝王斑蝶Danaus plexippus以及家蚕Bombyx mori同源物的序列一致性为72%和71%。HaTrx-like基因在幼虫5龄0 h和5龄96 h，蛹期第1天和第5天，以及成虫第1天的表达量相对较高，在幼虫的中肠、马氏管和成虫的肌肉中的表达量相对较高。在机械损伤、紫外线照射、极端低温和高温处理后，该基因的表达均显著地升高。【讨论】 获得了棉铃虫HaTrx-like基因的cDNA全长，并初步鉴定该基因为硫氧还蛋白家族成员，其表达水平受到机械损伤、紫外线照射和极端温度的上调，为进一步深入研究昆虫中硫氧还蛋白家族基因的各种生理功能提供理论基础。
英文摘要：To clone the full-length cDNA of a thioredoxin-like (HaTrx-like) gene in Helicoverpa armigera, to analyze its spatio-temporal distribution in different developmental stages and different tissues, and to determine its expression patterns following mechanical injury, exposure to UV radiation and extreme temperatures. [Methods] The full-length cDNA of HaTrx-like gene was amplified using the RT-PCR method and further validated by sequencing. The gene and protein sequences of HaTrx-like were analyzed with several kinds of bioinformatics software. The spatio-temporal distribution of HaTrx-like and its expression patterns after the different stress treatments were determined by qRT-PCR. [Results] The full-length cDNA of HaTrx-like was 526 bp and contained a 381 bp ORF encoding a 126-residue amino acid sequence. Sequence analysis indicates that HaTrx-like shares high sequence identity with its counterpart in other insects and contains a highly conserved CXXC active site in the N-terminal. The results of temporal transcript profiling indicate that HaTrx-like was more highly expressed in the 0 h and 96 h of the 5th instar, on the first and fifth day of the pupal, and on the first day of the adult, life stages. Spatial expression profiling shows that the gene was mainly expressed in the midgut and malpighian tubules of larvae, and in the muscles of adults. Meanwhile, the transcription of HaTrx-like was obviously induced by mechanical injury, ultraviolet light, cold (0°C), or high (37°C), temperatures. [Conclusion] The full-length cDNA of HaTrx-like gene was successfully cloned and preliminarily identified as a member of the thioredoxin family. Its expression is significantly upregulated by different kinds of stress. These results will contribute to further studies on the features and functions of insect thioredoxin.