西方蜜蜂m6A甲基转移酶的基因克隆、多克隆抗体制备及表达模式

doi:10.16380/j.kcxb.2023.07.003

昆虫学报 ›› 2023, Vol. 66 ›› Issue (7): 885-895.doi: 10.16380/j.kcxb.2023.07.003

西方蜜蜂m⁶A甲基转移酶的基因克隆、多克隆抗体制备及表达模式

吴鹰^1,#, 刘治滩^1,#, 赵浩东¹, 郭思佳¹, 刘小玉¹, 张艺琼¹, 冯佩林¹, 赵红霞³, 徐细建⁴, 陈大福^1,2, 付中民^1,2,*, 郭睿^1,2,*

(1. 福建农林大学动物科学学院(蜂学学院), 福州 350002; 2. 福建省蜂疗研究所, 福州 350002; 3. 广东省科学院动物研究所, 广州 510260; 4. 江西省养蜂研究所, 南昌 330000)

出版日期:2023-07-20 发布日期:2023-08-17

Gene cloning, polyclonal antibody preparation, and expression pattern of m⁶A methyltransferase in Apis mellifera (Hymenoptera: Apidae)

WU Ying^1,#, LIU Zhi-Tan^1,#, ZHAO Hao-Dong¹, GUO Si-Jia¹, LIU Xiao-Yu¹, ZHANG Yi-Qiong¹, FENG Pei-Lin¹, ZHAO Hong-Xia³, XU Xi-Jian⁴, CHEN Da-Fu^1,2, FU Zhong-Min^1,2,*, GUO Rui ^1,2,*

(1. College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2. Apitherapy Research Institute of Fujian Province, Fuzhou 350002, China; 3. Institute of Zoology, Guangdong Academy of Sciences, Guangzhou 510260, China; 4. Apicultural Research Institute of Jiangxi Province, Nanchang 330000, China)

Online:2023-07-20 Published:2023-08-17

摘要/Abstract

摘要： 【目的】本研究旨在克隆西方蜜蜂Apis mellifera RNA m6A甲基转移酶14 (methyltransferase 14, METTL14)基因AmMETTL14，分析AmMETTL14的理化性质和分子特性以及AmMETTL14在工蜂不同发育阶段和不同组织中的表达模式，并制备AmMETTL14多克隆抗体，为持续深入开展AmMETTL14的功能研究提供参考和基础。【方法】通过PCR扩增西方蜜蜂AmMETTL14的CDS并进行Sanger测序验证。使用相关生物信息学软件对AmMETTL14进行生物信息学分析，并构建系统进化树。通过原核表达系统诱导表达AmMETTL14融合蛋白，免疫新西兰白兔以制备多克隆抗体，利用ELISA和Western blot分别检测抗体的效价及特异性。采用RT-qPCR检测AmMETTL14在卵、幼虫、预蛹、蛹及工蜂成虫中以及刚出房工蜂成虫的触角、毒腺、脑、中肠、咽下腺、脂肪体和表皮7种组织中的相对表达量。【结果】成功克隆到西方蜜蜂AmMETTL14的CDS；AmMETTL14的分子式为C₁₉₅₇H₃₁₁₃N_567O589S₁₅，分子量约为44.49 kD，脂溶系数为79.97，理论等电点为8.58，平均亲水系数为-0.59，不含典型的跨膜结构域和信号肽，可同时定位于细胞核、线粒体和细胞质；西方蜜蜂、中华蜜蜂A. cerana cerana、黑大蜜蜂A. laboriosa、东方蜜蜂A. cerana、大蜜蜂A. dorsata、小蜜蜂A. florea、欧洲熊蜂Bombus terrestris、金环胡蜂Vespa mandarinia、美洲东部熊蜂B. impatiens和黑腹果蝇Drosophila melanogaster的METTL14均含有MT-A70结构域和3个相同的保守基序；AmMETTL14与黑大蜜蜂的METTL14的氨基酸序列一致性最高(65.60%)且亲缘关系最近。制备的AmMETTL14多克隆抗体效价较高(大于512 K)且特异性较强。AmMETTL14在7和8日龄预蛹与卵中的表达量相近且均显著高于在3日龄幼虫中的表达量，在12日龄蛹中的表达量显著高于卵、幼虫和预蛹中的表达量，在6, 12和15日龄工蜂成虫体内的表达量显著高于1日龄工蜂成虫体内的表达量；AmMETTL14在刚出房工蜂成虫脑和咽下腺中的表达量与触角中的表达量相近且均显著高于在毒腺、中肠、脂肪体和表皮中的表达量。【结论】研究结果表明，AmMETTL14可能为亲水性、非跨膜和胞内蛋白，AmMETTL14在幼虫生长发育中发挥潜在的调控作用，制备得到的AmMETTL14的多克隆抗体效价高、灵敏度高和特异性强，为进一步探究AmMETTL14的功能及其参与的表观调控机制打下了基础。

关键词: 西方蜜蜂, N⁶-甲基腺苷, 甲基化转移酶14, 时空表达模式, MT-A70, 抗体

Abstract: 【Aim】 This study aims to offer reference and foundation for continuous and further study on RNA m⁶A methyltransferase 14 (METTL14) gene of Apis mellifera AmMETTL14 through cloning AmMETTL14, analyzing the physicochemical properties and molecular characteristics of AmMETTL14, detecting the expression pattern of AmMETTL14 in different developmental stages and various tissues of workers, and preparing the polyclonal antibody to AmMETTL14. 【Methods】 The CDS of AmMETTL14 in A. mellifera was amplified by PCR followed by Sanger sequencing validation. Bioinformatic analysis and construction of phylogenetic tree of AmMETTL14 were conducted using relevant bioinformatics software. Inducible expression of AmMETTL14 fusion protein was performed through prokaryotic expression system, followed by immunization of New Zealand white rabbits to prepare the polyclonal antibody, and the titer and specificity of the antibody were evaluated by ELISA and Western blot, respectively. RT-qPCR was used to detect the relative expression level of AmMETTL14 in the egg, larva, prepupa, pupa and adult worker, and seven tissues including antenna, venom gland, brain, midgut, hypopharyngeal gland, fat body and cuticle of the newly emerged adult worker.【Results】 The CDS of AmMETTL14 of A. mellifera was successfully cloned. The molecular formula of AmMETTL14 is C₁₉₅₇H₃₁₁₃N_567O589S₁₅, the molecular weight is about 44.49 kD, the liposoluble coefficient is 79.97, the theoretical isoelectric point is 8.58, and the average hydrophilic coefficient is -0.59. AmMETTL14 contains no typical transmembrane domain and signal peptide, and is simultaneously located to the nucleus, mitochondria and cytoplasm. METTL14s in A. mellifera, A. cerana cerana, A. laboriosa, A. cerana, A. dorsata, A. florae, Bombus terrestris, Vespa mandarinia, B. impatien and Drosophila melanogaster contain the structural domain MT-A70 and three same conserved motifs. AmMETTL14 and METTL14 of A. laboriosa had the highest amino acid sequence identity (65.60%) and the closest relationship. The prepared polyclonal antibody to AmMETTL14 had high titer (more than 512 K) and strong specificity. The expression levels of AmMETTL14 in the 7- and 8-day-old prepupae and eggs were similar and significantly higher than that in the 3-day-old larva, that in the 12-day-old pupa was significantly higher than those in the egg, larva and prepupa, and those in the 6-, 12- and 15-day-old adult workers were significantly higher than that in the 1-day-old adult worker. Additionally, the expression levels of AmMETTL14 in the brain and hypopharyngeal gland were similar to that in the antenna, and significantly higher than those in the venom gland, midgut, fat body and cuticle of the newly emerged adult worker. 【Conclusion】 This study result indicates that AmMETTL14 may be a hydrophilic, non-transmembrane and intracellular protein, AmMETTL14 plays potential regulatory role in larval growth and development, and the prepared polyclonal antibody to AmMETTL14 has high titer, sensitivity and specificity, laying a foundation for further exploring the function of AmMETTL14 and the involved epigenetic regulation mechanisms.

Key words: Apis mellifera, N⁶-methyladenosine, methyltransferase 14, spatiotemporal expression pattern, MT-A70, antibody

吴鹰, 刘治滩, 赵浩东, 郭思佳, 刘小玉, 张艺琼, 冯佩林, 赵红霞, 徐细建, 陈大福, 付中民, 郭睿. 西方蜜蜂m⁶A甲基转移酶的基因克隆、多克隆抗体制备及表达模式[J]. 昆虫学报, 2023, 66(7): 885-895.

WU Ying, LIU Zhi-Tan, ZHAO Hao-Dong, GUO Si-Jia, LIU Xiao-Yu, ZHANG Yi-Qiong, FENG Pei-Lin, ZHAO Hong-Xia, XU Xi-Jian, CHEN Da-Fu, FU Zhong-Min, GUO Rui , . Gene cloning, polyclonal antibody preparation, and expression pattern of m⁶A methyltransferase in Apis mellifera (Hymenoptera: Apidae)[J]. Acta Entomologica Sinica, 2023, 66(7): 885-895.

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西方蜜蜂m⁶A甲基转移酶的基因克隆、多克隆抗体制备及表达模式

Gene cloning, polyclonal antibody preparation, and expression pattern of m⁶A methyltransferase in Apis mellifera (Hymenoptera: Apidae)

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