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家蚕BmMet2基因的组织定位与激素诱导表达分析

童春梅 易文瑾 马琼 邓惠敏 朱子丹

童春梅, 易文瑾, 马琼, 邓惠敏, 朱子丹. 家蚕BmMet2基因的组织定位与激素诱导表达分析[J]. 华南师范大学学报(自然科学版), 2022, 54(2): 63-69. doi: 10.6054/j.jscnun.2022027
引用本文: 童春梅, 易文瑾, 马琼, 邓惠敏, 朱子丹. 家蚕BmMet2基因的组织定位与激素诱导表达分析[J]. 华南师范大学学报(自然科学版), 2022, 54(2): 63-69. doi: 10.6054/j.jscnun.2022027
TONG Chunmei, YI Wenjin, MA Qiong, DENG Huimin, ZHU Zidan. The Tissue Localization and Hormone-induced Expression of BmMet2 in Silkworm, Bombyx mori[J]. Journal of South China normal University (Natural Science Edition), 2022, 54(2): 63-69. doi: 10.6054/j.jscnun.2022027
Citation: TONG Chunmei, YI Wenjin, MA Qiong, DENG Huimin, ZHU Zidan. The Tissue Localization and Hormone-induced Expression of BmMet2 in Silkworm, Bombyx mori[J]. Journal of South China normal University (Natural Science Edition), 2022, 54(2): 63-69. doi: 10.6054/j.jscnun.2022027

家蚕BmMet2基因的组织定位与激素诱导表达分析

doi: 10.6054/j.jscnun.2022027
基金项目: 

国家自然科学基金项目 31872969

国家自然科学基金项目 32000338

详细信息
    通讯作者:

    朱子丹,Email: zhuzidanbb@163.com

  • 中图分类号: Q751

The Tissue Localization and Hormone-induced Expression of BmMet2 in Silkworm, Bombyx mori

  • 摘要: Met作为保幼激素(JH)的受体, 能编码bHLH-PAS结构域结合DNA并调控下游基因的表达。为了探究BmMet2是否作为JH的受体参与调控家蚕的生长发育,以家蚕为材料,用生物信息学方法分析了BmMet2蛋白结构,利用实时定量PCR检测其在翅原基不同时期和激素诱导下的表达情况;原核表达了BmMet2中能与DNA结合的蛋白区域BmMet2DBD,并制备了抗体;利用蛋白质印迹法(Western blotting)和免疫组化检测了BmMet2在家蚕多个时期和各组织中的定位表达情况。结果显示:在翅原基中,BmMet2在5龄第3天和5龄第6天有较高水平的表达,蛹期的表达量较低,这与家蚕体内JH的滴度一致;BmMet2在5龄第6天、吐丝期和预蛹期的胸节表皮、脂肪体及翅原基等组织中都有较高的表达;JH和20E均对BmMet2有不同程度的诱导,说明其可能同时参与了JH和20E的调控, 进而参与调控家蚕变态发育过程。
  • 图  1  BmMet2蛋白的序列分析

    Figure  1.  The sequence analysis of BmMet2 protein

    图  2  BmMet2在翅原基中的时期表达谱及激素诱导表达分析

    注:L5D3为5龄第3天;L5D6为5龄第6天;W1为游走期第1天;PP为预蛹期;P0为蛹期0天;P3为蛹期第3天;数据差异分析采用单因素方差分析,P<0.05,字母不同表示具有显著性差异;*表示P<0.05,**表示P<0.01,***表示P<0.001。

    Figure  2.  The expression profile and hormone-induced expression analysis of BmMet2 in wing discs

    图  3  BmMet2DBD的cDNA序列和PCR扩增产物

    注:有下划线的碱基为引物设计位点,红色氨基酸序列为HLH结构域,绿色氨基酸序列为PAS结构域,紫色氨基酸序列为PAC结构域;M为DNA标准物。

    Figure  3.  The cDNA sequence and the PCR product of BmMet2DBD

    图  4  重组蛋白BmMet2DBD的表达及BmMet2DBD抗体的专一性分析

    注:M为蛋白标准物; (A)图中:1为经IPTG诱导的pPROEX-HTa菌体蛋白,2为经IPTG诱导的BmMet2DBD-pPROEX-HTa菌体重组总蛋白(未破碎),3为诱导的BmMet2DBD-pPROEX-HTa菌体重组蛋白上清,4为诱导的BmMet2DBD-pPROEX-HTa菌体重组蛋白沉淀;(B)图中:1为经IPTG诱导的BmMet2DBD-pPROEX-HTa菌体重组总蛋白,2为经IPTG诱导的pPROEX-HTa菌体总蛋白。

    Figure  4.  The expression of recombinant protein BmMet2DBD and the analysis of the specificity of BmMet2DBD antibody

    图  5  BmMet2蛋白在家蚕翅原基中各个时期表达的分析

    注:M为蛋白标准物,L5D3为5龄第3天,L5D6为5龄第6天,W1为游走期第1天,PP为预蛹期,P0为蛹期0天,P3为蛹期第3天。

    Figure  5.  The analysis of the expression profile of BmMet2 protein in the wing discs during the development of Bombyx mori

    图  6  BmMet2蛋白在不同发育时期和组织中的免疫组织化学定位分析

    Figure  6.  The immunohistochemical localization of BmMet2 protein in different tissues and at developmental stages of Bombyx mori

    表  1  本研究所使用到的引物

    Table  1.   The primers used in this study

    引物 序列(5’-3’) 用途
    BmMet2-qRT-F TCTGCTGCGAACCTGGAATCTG 定量PCR
    BmMet2-qRT-R TCCAAGAAACTTTCGAGACACGC
    BmRp49-F CAGGCGGTTCAAGGGTCAATAC 定量PCR
    BmRp49-R TACGGAATCCATTTGGGAGCAT
    BmMet2DBD-F1 GAATTCATGAATAAAGCTGAGAAGCAGCG 蛋白表达
    BmMet2DBD-R1 CTCGAGTTACTCTTCTCCAATAACGGTGT
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-04-23
  • 网络出版日期:  2022-05-12
  • 刊出日期:  2022-04-25

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