家蚕Wnt信号通路下游关键基因Pangolin转录剪接体X3的克隆和分析

doi:10.16380/j.kcxb.2022.12.004

昆虫学报 ›› 2022, Vol. 65 ›› Issue (12): 1592-1597.doi: 10.16380/j.kcxb.2022.12.004

家蚕Wnt信号通路下游关键基因Pangolin转录剪接体X3的克隆和分析

王叶菁¹, 付秋杰¹, 殷子晴², 何华伟^1,*

(1. 西南大学, 家蚕基因组生物学国家重点实验室, 生物学研究中心, 西部(重庆)科学城种质创制大科学中心, 重庆400715; 2. 西南大学西塔学院, 重庆400715)

出版日期:2022-12-20 发布日期:2023-01-19

Cloning and analysis of Pangolin X3, a key gene transcript variant downstream of the Wnt signaling pathway in the silkworm, Bombyx mori

WANG Ye-Jing¹, FU Qiu-Jie¹, YIN Zi-Qing², HE Hua-Wei^1,*

(1. State Key Laboratory of Silkworm Genome Biology, Biological Science Research Center, Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Southwest University, Chongqing 400715, China; 2. Westa College, Southwest University, Chongqing 400715, China)

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

摘要/Abstract

摘要： 【目的】克隆家蚕Bombyx mori Wnt信号通路下游关键基因Pangolin isoforms A/H/I/S转录剪接体X3 (Pangolin X3)，分析其序列和表达特征。【方法】从NCBI数据库检索家蚕Pangolin X3，根据其编码序列(coding sequence, CDS)设计引物，利用PCR从家蚕幼虫中肠和血淋巴中进行克隆并测序验证。利用SilkDB 3.0, SMART, 多序列比对和系统发育树分析Pangolin X3的序列特征。利用qRT-PCR分析Pangolin X3在家蚕5龄第3 天幼虫不同组织(头、血淋巴、体壁、性腺、中肠、前部丝腺、中部丝腺、后部丝腺、脂肪体和马氏管)中的相对表达水平。【结果】从家蚕幼虫中肠和血淋巴克隆了Pangolin X3(GenBank登录号: XM_038020921)的CDS，其开放阅读框长1 560 bp，编码519个氨基酸残基，预测分子量为55.86 kD，预测等电点为7.53。Pangolin X3蛋白含有保守的βcatenin结合位点和HMG结构域，其氨基酸序列在不同的昆虫中比较保守，特别是与DNA结合的HMG结构域，而与β-catenin结合的N末端CTNNB1结构域部分氨基酸残基发生变异。组织表达谱显示，Pangolin X3在家蚕5龄第3 天幼虫中肠、血淋巴和性腺中的相对表达水平较高，在头、体壁、前部丝腺、中部丝腺、后部丝腺、脂肪体和马氏管中的相对表达水平较低。【结论】本研究克隆了家蚕Pangolin X3，分析了其序列和表达特征，为深入研究家蚕Pangolin的生物学功能提供了基础。

关键词: 家蚕, Wnt; Pangolin X3, 基因克隆；序列分析；表达特征

Abstract: 【Aim】 This study aims to clone the transcript variant X3 of Pangolin isoforms A/H/I/S (Pangolin X3), a key gene downstream of the Wnt signaling pathway in the silkworm, Bombyx mori, and analyze its sequence and expression characteristics. 【Methods】 Pangolin X3 of B. mori was retrieved from the NCBI database, and the primers were designed according to its coding sequence (CDS). Pangolin X3 was cloned from the midgut and hemolymph of B. mori larvae using PCR, and then verified by sequencing. The sequence characteristics of Pangolin X3 were analyzed using SilkDB 3.0, SMART, multiple sequence alignment and phylogenetic tree. The relative expression levels of Pangolin X3 in different tissues (head, hemolymph, integument, gonads, midgut, anterior silk gland, middle silk gland, posterior silk gland, fat body and Malpighian tubules) of the day-3 5th instar larvae of B. mori were analyzed using qRT-PCR. 【Results】 The CDS of Pangolin X3 (GenBank accession no.: XM_038020921) was cloned from the midgut and hemolymph of B. mori larvae. It has an open reading frame of 1 560 bp in length, encoding 519 amino acid residues with the predicted molecular weight of 55.86 kD and the isoelectric point of 7.53. Pangolin X3 contains a conserved β-catenin binding site and an HMG domain, its amino acid sequence is relatively conserved among different insects, especially the HMG domain that binds to DNA, but some amino acid residues in the N-terminal CTNNB1 domain that binds to β-catenin are variable. Tissue expression profile revealed that the relative expression levels of Pangolin X3 in the midgut, hemolymph and gonads of the day-3 5th instar larvae of B. mori were high, while those in the head, integument, anterior silk gland, middle silk gland, posterior silk gland, fat body and Malpighian tubules were low. 【Conclusion】 In this study, we cloned Pangolin X3 of B. mori and analyzed its sequence and expression characteristics, providing a basis for an in-depth study of the biological function of Pangolin in B. mori.

Key words: Bombyx mori, Wnt; Pangolin X3, gene cloning, sequence analysis, expression characteristics

王叶菁, 付秋杰, 殷子晴, 何华伟. 家蚕Wnt信号通路下游关键基因Pangolin转录剪接体X3的克隆和分析[J]. 昆虫学报, 2022, 65(12): 1592-1597.

WANG Ye-Jing, FU Qiu-Jie, YIN Zi-Qing, HE Hua-Wei. Cloning and analysis of Pangolin X3, a key gene transcript variant downstream of the Wnt signaling pathway in the silkworm, Bombyx mori[J]. Acta Entomologica Sinica, 2022, 65(12): 1592-1597.

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家蚕Wnt信号通路下游关键基因Pangolin转录剪接体X3的克隆和分析

Cloning and analysis of Pangolin X3, a key gene transcript variant downstream of the Wnt signaling pathway in the silkworm, Bombyx mori

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