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【目的】克隆朱砂叶螨Tetranychus cinnabarinus几丁质合成过程中的关键酶几丁质合成酶基因,并检测该基因在朱砂叶螨生长发育不同阶段的相对表达量。【方法】本研究采用逆转录聚合酶链反应(RT-PCR)以及c DNA末端快速扩增(RACE)技术首次克隆获得朱砂叶螨几丁质合成酶基因1的全长c DNA序列(命名为Tc CHS1,Gen Bank登录号为KM242062),并使用实时荧光定量PCR技术首次检测了Tc CHS1基因在朱砂叶螨生长发育不同阶段的相对表达量。【结果】朱砂叶螨Tc CHS1基因的c DNA全长为4 881 bp,包括198 bp的5’非翻译区(5’-UTR),4 425 bp的开放阅读框(ORF),258 bp的3’非翻译区(3’-UTR),开放阅读框编码1 474个氨基酸,预测其蛋白质分子质量约为168.35 ku,理论等电点为6.26。其包含EDR和QRRRW这2个几丁质合成酶基因的标签序列。氨基酸序列同源性分析结果表明:Tc CHS1与其他昆虫该基因编码蛋白的氨基酸序列相似度在50%左右,与二斑叶螨Tetranychus urticae的氨基酸相似度最高(98%),与西方盲走螨Metaseiulus occidentalis的相似度为55%。分子系统进化的结果也表明Tc CHS1与其他昆虫的CHS1聚在一起,并且和二斑叶螨具有最近的亲缘关系。荧光定量分析表明Tc CHS1基因在朱砂叶螨生长发育的不同阶段(卵、幼螨、第1若螨、第2若螨、雌成螨和雄成螨)均有表达,在卵和雌成螨中的表达量较高,在第2若螨的表达量最低。【结论】Tc CHS1基因可能在朱砂叶螨生长发育过程中具有重要作用。
【Objective】 The objective of this study was to clone the chitin synthase gene, a key enzyme involved in the chitin synthesis of Tetranychus cinnabarinus, and to detect the relative expression of this gene in different stages of growth and development of Tetranychus cinnabarinus. 【Method】 The full-length cDNA sequence of Chitin synthase gene 1 from Spodoptera frugiperda was first cloned by reverse transcription-polymerase chain reaction (RT-PCR) and rapid cDNA amplification (RACE) Tc CHS1, Gen Bank Accession No. KM242062), and the relative expression of Tc CHS1 gene in different stages of the growth and development of Tetranychus cinnabarinus was detected by real-time fluorescent quantitative PCR. 【Result】 The results showed that the full length cDNA of Tc CHS1 gene of Tetranychus cinnabarinus was 4 881 bp, including 198 bp 5 ’untranslated region (5’-UTR), 4 425 bp open reading frame (ORF), 258 bp 3 (3’-UTR). The open reading frame encodes 1 474 amino acids. The protein molecular weight is estimated to be about 168.35 ku and the theoretical isoelectric point is 6.26. It contains the tag sequences of two chitin synthase genes EDR and QRRRW. The results of amino acid sequence homology analysis showed that the amino acid sequence similarity between Tc CHS1 and other insects was about 50%, the amino acid similarity with Tetranychus urticae was the highest (98%), The similarity for Metaseiulus occidentalis is 55%. The molecular phylogenetic tree also showed that Tc CHS1 is clustered with CHS1 of other insects and has the closest genetic relationship with Tetranychus urticae. Fluorescence quantitative analysis showed that the Tc CHS1 gene was expressed in different stages of the growth and development of Tetranychus cinnabarinus (eggs, larvae, No. 1 nymphs, No. 2 nymphs, female nymphs and male nymphs) The expression level of the second nymph was the lowest. 【Conclusion】 Tc CHS1 gene may play an important role in the growth and development of Tetranychus cinnabarinus.