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| 西方蜜蜂CKI1蛋白的分子特征和基因时空表达谱 |
| Molecular feature and gene spatio-temporal expression profile of Apis mellifera CKI1 protein |
| 投稿时间：2024-03-25 修订日期：2024-06-01 |
| DOI： |
| 中文关键词: 西方蜜蜂 细胞周期蛋白依赖性激酶抑制因子1 分子特征 系统进化 表达谱 |
| 英文关键词:Apis mellifera cyclin-dependent kinase inhibitor 1 molecular characteristics phylogenetic evolution expression profile |
| 基金项目:国家自然科学基金面上项目(32372943,32172792)；国家现代农业产业技术体系建设专项资金项目(CARS-44-KXJ7)；福建省自然科学基金面上项目(2022J01131334); 福建农林大学硕士生导师团队项目(郭睿)；福建农林大学科技创新专项基金(KFb22060XA) |
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| 中文摘要: |
| 【目的】本研究对西方蜜蜂Apis mellifera细胞周期蛋白依赖性激酶抑制因子1（cyclin dependent kinase inhibitor1，CKI1）基因AmCKI1进行表达验证，并解析AmCKI1蛋白的理化性质和分子特征，进而测定工蜂不同组织和发育阶段AmCKI1的表达谱，旨在为进一步的功能研究提供参考和基础。【方法】通过PCR和Sanger测序验证AmCKI1的表达。使用相关软件预测AmCKI1的理化性质和分子特征，并进行系统进化分析。采用RT-qPCR检测AmCKI1在工蜂的触角、脑、咽下腺、中肠、毒腺、表皮和脂肪体等7个不同组织，卵、幼虫、预蛹、蛹和1, 2, 6, 12, 15和17日龄成虫中的相对表达量。【结果】AmCKI1在工蜂中真实表达。AmCKI1的分子式为C1104H1793N333O331S7，分子量约为25.26 kD，脂溶系数为71.88，等电点为10.18，平均亲水系数为?0.674，可同时定位于细胞核、线粒体和细胞质；AmCKI1含32个磷酸化位点，但不含典型的跨膜结构域和信号肽；西方蜜蜂和其他6种蜂的CKI1蛋白均包含1个CDI结构域和3个相同的保守基序；西方蜜蜂与小蜜蜂Apis florea的CKI同源性最高。AmCKI1在西方蜜蜂工蜂的脑、咽下腺、中肠、触角、毒腺、表皮和脂肪体中均有表达但表达量无显著差异（P>0.05）；AmCKI1在卵、3日龄幼虫、7和8日龄预蛹及12日龄蛹中差异表达，在卵中的表达量最高且显著高于3日龄幼虫和12日龄蛹中的表达量（P<0.05）；AmCKI1在工蜂的1, 2, 6, 12, 15和17日龄成虫体内差异表达，在17日龄成虫中的表达量最高且显著高于1, 2, 6, 12和15日龄成虫体内的表达量。【结论】AmCKI1是潜在的胞内蛋白和亲水性蛋白，CKI1在西方蜜蜂、小蜜蜂、欧洲熊峰Bombus terrestris、美洲东部熊蜂Bombus impatiens、黑大蜜蜂Apis laboriosa、东方蜜蜂Apis ceranae和大蜜蜂Apis dorsata中高度保守，西方蜜蜂与小蜜蜂的CKI1同源性最高，AmCKI1在工蜂卵和17日龄成虫中特异性高表达。 |
| 英文摘要: |
| [Objective] In this study, the expression of Apis mellifera cyclin-dependent kinase inhibitor 1 (CKI1) gene AmCKI1 was validated, and the physicochemical property and molecular feature of the AmCKI1 protein were investigated, followed by determination of the expression profile of AmCKI1 in various tissues and developmental stages of worker bees, aiming to provide reference and foundation for further functional research. [Methods] PCR and Sanger sequencing were performed to verify the expression of AmCKI1. Relative software were employed to predict the physicochemical property and molecular feature of AmCKI1 and conduct phylogenetic analysis. RT-qPCR was utilized to detect the relative expression level of AmCKI1 in seven different tissues of the workers (tentacle, brain, hypopharyngeal gland, midgut, posion gland, cuticle and fat body), in various developmental stages (egg, larva, pre-pupa, pupa and adult), and in adult workers of 1, 2, 6, 12, 15 and 17 day-old . [Results] The AmCKI1 was truly expressed in the midgut of worker. The molecular formula of AmCKI1 was C1104H1793N333O331S7, and the molecular weight was about 25.26 kD, and the lipophilicity coefficient , isoelectric point and the average hydrophilicity index were 71.88, 10.18 and -0.674, respectively. AmCKI1 were potentially localized in the nucleus, mitochondria and cytoplasm. AmCKI1 included 32 phosphorylation sites but lacked typical transmembrane domain and signal peptide. One CDI structuraldomain and three same conserved motifs were included in CKI1 proteins from A. mellifera and six other bee species.The highest homology was found in CKI1 between A. mellifera and Apis florea. AmCKI1 was expressed in the tentacle, brain, hypopharyngeal gland, midgut, posion gland, cuticle and fat body of workers, without significant difference in expression levels (P>0.05). Differential expression of AmCKI1 was observed in egg, 3-day-old larva, 7 and 8-day-old pre-pupae and 12-day-old pupa, the expression level in egg was the highest and significant higher than that in 3-day-old larva and 12-day-old pupa (P<0.05). AmCKI1 was also differentially expressed in 1, 2, 6, 12, 15 and 17-day-old adults, the expression level was the highest in 17-day-old adult and significantly higher than that in 1, 2, 6, 12 and 15-day-old adults. [Conclusion] AmCKI1 is a putative intracellular and hydrophilic protein. CKI1 was highly conserved among A. mellifera, A. florea, Bombus terrestris, Bombus impatiens, Apis laboriosa, Apis ceranae, and Apis dorsata. The homology of CKI1 between A. mellifera and A. florea was the highest. AmCKI1 was specifically and highly expressed in the egg and 17-day-old adult. |
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