›› 2018, Vol. 61 ›› Issue (4): 423-431.doi: 10.16380/j.kcxb.2018.04.004

• 研究论文 • 上一篇    下一篇


郗学鹏#, 魏伟#, 张卫星, 王红芳, 胥保华*   

  1. (山东农业大学动物科技学院, 山东泰安 271000)
  • 出版日期:2018-04-20 发布日期:2018-04-20

Molecular cloning and characterization of transcription factor AP-1 gene in Apis cerana cerana (Hymenoptera: Apidae)

CHI Xue-Peng#, WEI Wei#, ZHANG Wei-Xing, WANG Hong-Fang, XU Bao-Hua*   

  1. (College of Animal Science and Technology, Shandong Agricultural University, Tai′an, Shandong 271018, China)
  • Online:2018-04-20 Published:2018-04-20

摘要: 【目的】克隆获得中华蜜蜂Apis cerana cerana转录因子AP-1(transcription factor AP-1)基因AccAP-1序列,分析其在中华蜜蜂不同发育时期和组织中以及不同非生物应激条件下的表达差异,为该基因的功能研究提供参考。【方法】以中华蜜蜂全组织cDNA为模板,利用RT-PCR技术扩增克隆获得中华蜜蜂转录因子AP-1基因cDNA序列;利用生物信息学软件预测分析其编码蛋白的理化特性和结构特征;采用MEGA 5.2软件中的邻接法(neighbor-joining, NJ)构建中华蜜蜂转录因子AP-1与其他膜翅目昆虫同源AP-1蛋白系统发育树;通过实时定量PCR分析中华蜜蜂转录因子AP-1基因在不同发育时期(1-6日龄幼虫、预蛹、白眼蛹、红眼蛹、褐眼蛹、1日龄成年工蜂和15日龄成年工蜂)以及15日龄成年工蜂不同组织(头、肌肉、表皮、中肠、直肠和毒腺)中的mRNA表达情况;将15日龄成年工蜂分别置于极端温度(4℃, 16℃和44℃)下,以及饲喂含有农药(0.01 mL/L百草枯)和重金属(1 mg/mL CdCl2)的蔗糖溶液条件下,分析目的基因mRNA的表达情况。【结果】克隆获得了中华蜜蜂转录因子AP-1基因AccAP-1(GenBank登录号: MF994311)开放阅读框(ORF)的序列,其长度为813 bp,编码270个氨基酸,预测蛋白分子量为30.24 kD,理论等电点为8.31。保守结构域分析表明,在第7-137位氨基酸之间存在一个Jun超家族的保守结构域,在第195-255位氨基酸之间存在一个bZIP_Jun 超家族的保守结构域。氨基酸序列比对以及进化树结果显示,中华蜜蜂与西方蜜蜂Apis mellifera的亲缘关系最近。荧光定量PCR结果显示,AccAP-1在各发育时期表达量差异显著(P<0.05),其中,在1日龄幼虫和15日龄成蜂期表达量最高;AccAP-1在测试的各组织中均有表达,在肌肉中的表达量最高(P<0.05)。不同非生物应激条件下的表达量分析表明,4℃低温可以诱导AccAP-1表达量显著升高(P<0.05),16℃处理0.5~2 h可显著提高其表达量(P<0.05),2 h后表达量逐渐降低,而44℃处理时除了在15 min时表达量显著下降(P<0.05),其余时间点表达量差异不显著(P>0.05);在饲喂含有百草枯的蔗糖溶液后,其表达量显著降低(P<0.05),而CdCl2处理诱导其表达量显著升高(P<0.05)。【结论】结果提示AccAP-1基因可能参与中华蜜蜂机体抵抗外界环境压力的过程。

关键词: 中华蜜蜂, 转录因子AP-1, 基因克隆, 基因功能, 胁迫反应

Abstract: 【Aim】 The objective of this study is to clone the cDNA sequence of transcription factor AP-1 gene (AccAP-1) from Apis cerana cerana and to explore its expression profiles in A. cerana cerana at different developmental stages, in different tissues and under different abiotic stress, so as to provide fundamental evidence for the future study of the physiological function of AccAP-1. 【Methods】 The full-length cDNA sequence of AP-1 gene was cloned from the entire tissues of A. cerana cerana by RT-PCR. Physiochemical properties and structure characteristics of the deduced amino acid sequence were analyzed by multiple bioinformatics methods. Phylogenetic tree between AP-1 from A. cerana cerana and its homologous AP-1 proteins from other hymenopteran insects was constructed using neighbor-joining method of MEGA5.2. The expression levels of AP-1 gene in A. cerana cerana at different developmental stages (1-6 d old larva, prepupa, white-eyed pupa, pink-eyed pupa, dark-eyed pupa, 1 d-old adult worker, and 15 d-old adult worker), in different tissues (head, muscle, epidermis, midgut, rectum and venom gland) of 15 d-old adult workers, and in 15 d-old adult workers under stress of extreme temperatures (4, 16 and 44℃) and fed with sucrose solutions containing pesticide (0.01 mL/L paraquat) and heavy metal (1 mg/mL CdCl2), respectively, were detected by real-time PCR. 【Results】 We obtained the full-length open reading frame (ORF) of AccAP-1 (GenBank accession no.: MF994311) from A. cerana cerana, which is 813 bp in length, encoding 270 amino acids with an estimated molecular weight of 30.24 kD and a predicted theoretical isoelectric point (pI) of 8.31. Conserved domain analysis indicated that AccAP-1 contains two highly conserved structures, i.e., Jun superfamily (AA 7-137) and bZIP_Jun (AA 195-255). The results of homologous sequence alignment and phylogenetic tree analysis indicated that A. cerana cerana was most closely related to Apis mellifera. The results of real-time PCR indicated that the expression levels of AccAP-1 at different developmental stages were significantly different (P<0.05) and the highest in 1 d-old larva and 15 d-old adult. AccAP-1 transcripts were expressed in all tissues tested, and the expression level was higher in muscles than in other tissues (P<0.05). The expression levels of AccAP-1 under different abiotic stress indicated that the low temperature 4℃ upregulated its expression (P<0.05), while the treatment at 16℃ for 0.5-2 h upregulated its expression (P<0.05), but extended treatment time at 16℃ (over 2 h) downregulated its expression. The expression level of AccAP-1 at 44℃ for 15 min was downregulated significantly (P<0.05), while those at 44℃ for the other treatment time had no significant difference (P>0.05). The expression of AccAP-1 was downregulated when the workers were fed with the sucrose solution containing paraquat (P<0.05); the expression of AccAP-1, however, was induced by CdCl2 (P<0.05). 【Conclusion】 The results suggest that AccAP-1 might play an important role in the response to abiotic stress in A. cerana cerana.

Key words: Apis cerana cerana; transcript factor AP-1, gene cloning, gene function, stress response