Acta Entomologica Sinica ›› 2021, Vol. 64 ›› Issue (1): 41-50.doi: 10.16380/j.kcxb.2021.01.005

• RESEARCH PAPERS • Previous Articles     Next Articles

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

LI Qiong-Yan1, CHEN An-Li2, XUN Li-Jie1, LIU Zeng-Hu1, LIAO Peng-Fei1, YANG Wei-Ke1DONG Zhan-Peng1,*   

  1. (1. Institute of Sericulture and Apiculture, Yunnan Academy of Agricultural Sciences, Mengzi, Yunnan 661101, China; 2. Ankang University, Ankang, Shaanxi 725000, China)
  • Online:2021-01-20 Published:2021-02-18

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