琥珀蚕丝腺转录因子基因AaSGF-1的克隆、原核表达及组织表达分析

doi:10.16380/j.kcxb.2021.01.005

摘要/Abstract

摘要： 【目的】本研究旨在克隆琥珀蚕Antheraea assama丝腺转录因子基因AaSGF-1，分析其序列特征及表达模式并制备多克隆抗体，为探讨该基因的生理功能奠定基础。【方法】采用RT-PCR和RACE技术从琥珀蚕丝腺中克隆AaSGF-1的cDNA序列，并进行生物信息学分析；利用qPCR检测AaSGF-1在琥珀蚕5龄第4天幼虫不同组织(头、中肠、脂肪体、丝腺、血液、表皮)中的表达模式；构建原核表达质粒载体，在大肠杆菌Escherichia coli BL21中表达AaSGF-1，利用纯化的融合蛋白免疫新西兰兔子，获得高效的抗体。利用免疫荧光技术检测AaSGF-1在琥珀蚕蚁蚕丝腺和表皮及4龄幼虫丝腺中的表达情况。【结果】克隆了琥珀蚕AaSGF-1的cDNA序列(GenBank登录号： MK889510.1)，开放阅读框(ORF)序列长1 050 bp，编码349个氨基酸残基，预测蛋白分子质量为38.8 kD，理论等电点(pI)为8.74。qPCR检测结果显示AaSGF-1在琥珀蚕5龄幼虫丝腺组织尤其是后部丝腺中高量表达，而在其他组织中几乎不表达。免疫荧光结果表明AaSGF-1在蚁蚕及4龄幼虫的丝腺中表达。【结论】本研究原核表达了琥珀蚕AaSGF-1，制备了多克隆抗体，证实了AaSGF-1在琥珀蚕幼虫的丝腺中高表达，为进一步研究该基因在琥珀蚕丝腺发育及丝蛋白合成中的作用奠定了基础。

关键词: 琥珀蚕, 丝腺转录因子, 基因克隆, 原核表达, 多克隆抗体, 组织表达谱

Abstract: 【Aim】 This study aims to clone the silk gland transcription factor gene AaSGF-1 from Antheraea assama, to analyze its sequence features and expression pattern, and to obtain the polyclonal antibody, so as to lay a basis for further studying the function of this gene. 【Methods】 The cDNA sequence of AaSGF-1 was cloned from the silk gland of A. assama by RT-PCR and RACE techniques and subjected to bioinformatical analysis. qPCR was employed to analyze the expression profile of this gene in different tissues (head, midgut, fat body, silk gland, hemolymph, and cuticle) of the day-4 5th instar larvae of A. assama. Prokaryotic expression plasmid was constructed and the fusion protein AaSGF-1 was expressed in Escherichia coli BL21. The polyclonal antibody with high serum titer was prepared using the purified fusion protein to immunize New Zealand rabbit. The protein level of AaSGF-1 in the silk gland and cuticle of the newly hatched larva and the silk gland of the 4th instar larva of A. assama was detected by immunofluorescence technique. 【Results】 The cDNA sequence of AaSGF-1 (GenBank accession no.: MK889510.1) of A. assama was cloned. The ORF of AaSGF-1 is 1 050 bp in length, encoding a polypeptide of 349 amino acids with the molecular weight of 38.8 kD and the isoelectric point (pI) of 8.74. The qPCR analysis results showed that AaSGF-1 was highly expressed in the silk gland of the 5th instar larvae of A. assama, especially in the posterior silk gland, but hardly expressed in other tissues. Immunofluorescence assay showed that AaSGF-1 was expressed in silk glands of the newly hatched larva and 4th instar larva of A. assama. 【Conclusion】 In this study AaSGF-1 was expressed by prokaryotic expression system, the polyclonal antibody was prepared, and AaSGF-1 was confirmed to be highly expressed in the silk gland of A. assama larva, providing a basis for further studying its roles in silk gland development and silk protein synthesis in A. assama.

Key words: Antheraea assama, silk gland transcription factor, gene cloning, prokaryotic expression, polyclonal antibody, tissue expression profile

李琼艳, 陈安利, 荀利杰, 刘增虎, 廖鹏飞, 杨伟克, 董占鹏. 琥珀蚕丝腺转录因子基因AaSGF-1的克隆、原核表达及组织表达分析[J]. 昆虫学报, 2021, 64(1): 41-50.

LI Qiong-Yan, CHEN An-Li, XUN Li-Jie, LIU Zeng-Hu, LIAO Peng-Fei, YANG Wei-Ke, DONG Zhan-Peng. Cloning, prokaryotic expression and tissue expression analysis of the silk gland transcription factor gene AaSGF-1 of Antheraea assama (Lepidoptera: Saturniidae)[J]. Acta Entomologica Sinica, 2021, 64(1): 41-50.

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琥珀蚕丝腺转录因子基因AaSGF-1的克隆、原核表达及组织表达分析

Cloning, prokaryotic expression and tissue expression analysis of the silk gland transcription factor gene AaSGF-1 of Antheraea assama (Lepidoptera: Saturniidae)

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