家蚕胚胎中胚胎发育因子基因BmEDF G4结构的验证及其结合蛋白的筛选

doi:10.16380/j.kcxb.2022.12.003

昆虫学报 ›› 2022, Vol. 65 ›› Issue (12): 1582-1591.doi: 10.16380/j.kcxb.2022.12.003

家蚕胚胎中胚胎发育因子基因BmEDF G4结构的验证及其结合蛋白的筛选

黄琼^#, 彭玉玲^#, 冯启理, 牛康康^*

(华南师范大学生命科学学院, 昆虫科学与技术研究所, 广东省昆虫发育生物学与应用技术重点实验室, 广州市昆虫发育应用重点实验室, 广州 510631)

出版日期:2022-12-20 发布日期:2023-01-19

Verification of the G4 structure of the embryonic development factor (EDF) gene BmEDF from Bombyx mori embryos and screening of its binding protein

HUANG Qiong^#, PENG Yu-Ling^#, FENG Qi-Li, NIU Kang-Kang^*

(Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Guangzhou Key Laboratory of Insect Development and Application, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, China)

Online:2022-12-20 Published:2023-01-19

摘要/Abstract

摘要： 【目的】本研究旨在通过寻找家蚕Bombyx mori胚胎发育因子(embryonic development factor, EDF)基因BmEDF G-四链体(G-quadruplex, G4)结构的结合蛋白，进一步探究G4结构调控家蚕胚胎发育的可能作用和机制。【方法】通过圆二色谱(circular dichroism, CD)和凝胶迁移实验(electrophoretic mobility shift assay, EMSA)验证G4序列在体外是否形成G4结构；通过启动子活性实验验证BmEDF启动子区G4结构对BmEDF的表达调控的影响；通过qRT-PCR检测BmEDF在家蚕胚胎发育各时期的表达量变化。通过EMSA联合质谱分析可能与BmEDF的G4结构结合的蛋白，然后将与G4结构结合的2个候选蛋白BmeIF4H和BmADDH分别进行基因克隆、表达和纯化，再通过EMSA实验分别验证候选蛋白BmeIF4H和BmADDH与BmEDF的G4结构结合与否。【结果】CD和EMSA实验都证明BmEDF的G4序列在体外可以形成G4结构。启动子活性实验表明BmEDF G4结构的存在对BmEDF转录表达具有正调控的作用。qRT-PCR结果表明BmEDF在产卵后120 h时表达量显著升高。经原核表达纯化，获得BmeIF4H和BmADDH重组蛋白。EMSA实验表明重组蛋白BmeIF4H在体外与BmEDF的G4结构结合，BmADDH不与BmEDF G4结构结合。【结论】家蚕胚胎中的BmeIF4H蛋白可能与BmEDF的G4结构结合。本研究为解析家蚕胚胎发育的DNA高级结构调控机理提供了实验证据。

关键词: 家蚕, 胚胎发育, DNA高级结构, G-四链体, 结合蛋白

Abstract: 【Aim】 This study aims to further explore the possible role and mechanism of G-quadruplex (G4) structure in the regulation of the embryonic development of Bombyx mori by searching for the binding protein of the G4 structure of the embryonic development factor (EDF) gene BmEDF of B. mori. 【Methods】 Circular dichroism (CD) and electrophoretic mobility shift assay (EMSA) were performed to verify whether G4 sequence formed G4 structure in vitro. The effect of the G4 structure of the BmEDF promoter on the regulation of BmEDF expression was verified by the promoter activity experiment. qRT-PCR was performed to determine the expression levels of BmEDF in B. mori embryo at different developmental stages. The protein that could bind to the G4 structure of BmEDF was analyzed by EMSA combined with mass spectrometry. Then, the two candidate proteins BmeIF4H and BmADDH binding with G4 structure were cloned, expressed and purified, respectively. The binding of the candidate proteins BmeIF4H and BmADDH to the G4 structure of BmEDF was verified by EMSA. 【Results】 CD and EMSA experiments demonstrated that the G4 sequence of BmEDF could form G4 structure in vitro. Promoter activity experiments showed that the presence of the G4 structure of BmEDF had a positive regulatory effect on the transcriptional expression of BmEDF. The qRT-PCR results showed that the expression level of BmEDF was significantly increased at 120 h after oviposition. The recombinant BmeIF4H and BmADDH were obtained by prokaryotic expression and purification. EMSA experiments showed that the recombinant BmeIF4H bound to the G4 structure of BmEDF in vitro, while BmADDH did not bind to the G4 structure of BmEDF. 【Conclusion】 The BmeIF4H protein in B. mori embryo may bind to the G4 structure of BmEDF. This study provides the experimental evidence for analyzing the regulatory mechanism of DNA advanced structure in the embryonic development of B. mori.

Key words: Bombyx mori; embryonic development, DNA advanced structure, G-quadruplex, binding protein

黄琼, 彭玉玲, 冯启理, 牛康康. 家蚕胚胎中胚胎发育因子基因BmEDF G4结构的验证及其结合蛋白的筛选[J]. 昆虫学报, 2022, 65(12): 1582-1591.

HUANG Qiong, PENG Yu-Ling, FENG Qi-Li, NIU Kang-Kang. Verification of the G4 structure of the embryonic development factor (EDF) gene BmEDF from Bombyx mori embryos and screening of its binding protein[J]. Acta Entomologica Sinica, 2022, 65(12): 1582-1591.

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家蚕胚胎中胚胎发育因子基因BmEDF G4结构的验证及其结合蛋白的筛选

Verification of the G4 structure of the embryonic development factor (EDF) gene BmEDF from Bombyx mori embryos and screening of its binding protein

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