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【目的】克隆棉铃虫Helicoverpa armigera硫氧还蛋白类似基因(Ha Trx-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Ha Trx-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c DNA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。
【Objective】 The full-length cDNA of Ha Trx-like gene of Helicoverpa armigera was cloned and sequenced to investigate the temporal and spatial expression pattern of HaTrx-like gene and its roles in resistance to mechanical injury, ultraviolet radiation and extreme Temperature and other adversities. 【Method】 The full-length cDNA of Ha Trx-like gene was amplified by RT-PCR and verified by sequencing. Several bioinformatics softwares were used to analyze the gene and amino acid sequence. HaTrx-like The temporal and spatial distribution of genes and expression patterns after four stress conditions. 【Result】 The results showed that the full-length c DNA of HaTrx-like gene was 526 bp in length. The open reading frame (ORF) of this gene was 381 bp in length and encoded a protein of 126 amino acids with a conserved redox active site CXXC. Sequence identities of Danaus plexippus and Bombyx mori homologues of Bombyx mori were 72% and 71%, respectively. The expression of HaTrx-like gene in larvae at 0 h, 5 h, 96 h, 1 and 5 days of pupal stage and on the 1st day of adult larva was relatively high. The expression of HaTrx- The amount of muscle expression is relatively high. The expression of this gene was significantly increased after mechanical injury, UV irradiation, extreme low temperature and high temperature treatment. 【Discussion】 The full-length cDNA of HaTrx-like gene was obtained from Helicoverpa armigera, and the gene was initially identified as a member of thioredoxin family. Its expression level was up-regulated by mechanical damage, ultraviolet radiation and extreme temperature. Thioredoxin gene family of various physiological functions provide the theoretical basis.