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| 东方蜜蜂死亡捕手蛋白的分子特征及基因表达模式 |
| Molecular characterization and gene expression pattern of CRQ in Apis cerana cerana |
| 投稿时间：2024-08-16 修订日期：2024-10-18 |
| DOI： |
| 中文关键词: 东方蜜蜂 时空表达谱 分子特性 CRQ 理化性质 系统进化 |
| 英文关键词:Apis cerana cerana spatiotemporal expression pattern molecular characteristics CRQ physicochemical property phyletic evolution |
| 基金项目: |
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| 摘要点击次数: 8 |
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| 中文摘要: |
| 【目的】本研究旨在通过检测东方蜜蜂Apis cerana cerana的croquemort基因AcCrq的时空表达谱并解析AcCRQ蛋白理化性质和分子特性,为AcCrq的功能研究提供参考和基础。【方法】RT-PCR扩增AcCrq的编码序列coding sequence, CDS。利用相关软件预测AcCRQ蛋白的理化性质和分子特性,鉴定东方蜜蜂和其他物种AcCrq的保守基序和结构域,并比对氨基酸序列和构建系统进化树。RT-qPCR检测AcCrq在东方蜜蜂工蜂的触角、中肠、脑、咽下腺、脂肪体、毒腺和表皮组织,以及卵、幼虫、预蛹、蛹和不同日龄工蜂成虫中的相对表达量。【结果】成功验证到AcCrq,CDS含有1719个核苷酸,可编码572个氨基酸。AcCRQ蛋白的分子量约为65.44 kD,分子式为C2969H45540N764O864S21,等电点为5.96,平均亲水系数为-0.30,含23个丝氨酸磷酸化位点,9个酪氨酸磷酸化位点和17个苏氨酸磷酸化位点,但不存在典型的信号肽。AcCrq在黑大蜜蜂Apis laboriosa、小蜜蜂Apis florea、西方蜜蜂Apis mellifera、兰花蜂Eufriesea mexicana、东南蓝莓蜂Habropoda laboriosa、红壁蜂Osmia bicornis bicornis和蓝果园壁蜂Osmia lignaria中具有较高的氨基酸序列一致性；东方蜜蜂与小蜜蜂和西方蜜蜂的Crq进化距离最近。AcCrq在工蜂成虫触角、中肠、脑、咽下腺、脂肪体、毒腺和表皮中均有表达,但表达量存在差异,触角中AcCrq的表达量显著高于其他组织的表达；AcCrq在东方蜜蜂工蜂卵、幼虫、预蛹和蛹中均有表达,但表达量存在差异,相较于在卵中的表达量,3日龄幼虫和8日龄预蛹中AcCrq的表达量显著降低,而7日龄预蛹和12日龄蛹中AcCrq的表达量下调但不显著；AcCrq在1, 2, 6, 12, 15和17日龄工蜂体内均有表达,但表达量也存在差异,1日龄成虫体内AcCrq的表达量均显著高于其他日龄成虫体内的表达量,2和17日龄成虫体内的表达量均低于其他日龄成虫体内的表达量。【结论】AcCRQ是潜在的亲水性蛋白但不具分泌功能；东方蜜蜂和其他8个物种的CRQ具有较高的保守性；东方蜜蜂与小蜜蜂和西方蜜蜂的CRQ进化距离最近。AcCrq在东方蜜蜂工蜂的不同组织和发育阶段动态差异表达。 |
| 英文摘要: |
| 【Objective】The aim of this study was to provide a reference and basis for the functional study of AcCrq by examining the spatiotemporal expression profile of the croquemort gene, AcCrq, in Apis cerana cerana, and analyzing the physicochemical property and molecular characteristics of the AcCRQ protein.【Methods】The coding sequence (CDS) of AcCrq was amplified by RT-PCR. The physicochemical and molecular properties of the AcCRQ protein were predicted using the relevant software, and the conserved motifs and structural domains of AcCrq from Apis cerana cerana and other species were identified, and the amino acid sequences were compared and a phylogenetic tree was constructed. RT-qPCR was performed to detect the relative expression of AcCrq in the antennae, midgut, brain, hypopharyngeal glands, fat bodies, venom glands and epidermal tissues, as well as in eggs, larvae, pre-pupae, pupae and adults of different day-old worker bees of Apis cerana cerana. 【Results】Successfully validated to AcCrq CDS contained 1,719 nucleotides, encoding a total of 572 amino acids. AcCRQ protein has a molecular weight of about 65.44 kD, a molecular formula of C2969H45540N764O864S21, an isoelectric point of 5.96, an average hydrophilicity coefficient of -0.30, and contains 23 serine phosphorylation sites, 9 tyrosine phosphorylation sites, and 17 threonine phosphorylation sites, but does not have a typical signal peptide. AcCrq has high amino acid sequence in Apis laboriosa, Apis florea, Apis mellifera, Eufriesea mexicana, Habropoda laboriosa, Osmia bicornis bicornis and Osmia lignaria consistency; Apis cerana cerana has the closest Crq evolutionary distance from Apis florea and Apis mellifera. AcCrq was expressed in the antennae, midgut, brain, hypopharyngeal glands, fat body, venom glands and epidermis of adult worker bees, but there were differences in the amount of expression, and the amount of expression in the antennae was significantly higher than that in other tissues; AcCrq was expressed in the eggs, larvae, prepupae and pupae of Apis cerana cerana workers, but there were differences in the amount of expression. Compared with the expression in eggs, the expression of AcCrq in 3-day-old larvae and 8-day-old prepupae was significantly lower, while the expression of AcCrq in 7-day-old prepupae and 12-day-old pupae was down-regulated, but not significantly; AcCrq was expressed in 1-, 2-, 6-, 12-, 15-, and 17-day-old workers but there were differences in the amount of expression. The expression of AcCrq in 1-day-old adult was significantly higher than that in other day-old adults, and the expression level of AcCrq in 2 and 17-day-old adults was lower than that in other day-old adults. 【Conclusion】AcCRQ is a putative hydrophylic but not secretory, AcCRQ among Apis cerana cerana and 8 other bees are highly conservative. Apis cerana cerana, Apis florea and Apis mellifera have the closet phyletic evolution of CRQ. AcCrq was dynamically and differentially expressed in different tissues and developmental stages of Apis cerana cerana. |
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