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松材线虫P糖蛋白在药物代谢过程中的功能研究

李洋 郝昕 李明锐 刁健 马玲

李洋, 郝昕, 李明锐, 刁健, 马玲. 松材线虫P糖蛋白在药物代谢过程中的功能研究[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20200387
引用本文: 李洋, 郝昕, 李明锐, 刁健, 马玲. 松材线虫P糖蛋白在药物代谢过程中的功能研究[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20200387
Li Yang, Hao Xin, Li Mingrui, Diao Jian, Ma Ling. Function of pine worm P glycoprotein in drug metabolism[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20200387
Citation: Li Yang, Hao Xin, Li Mingrui, Diao Jian, Ma Ling. Function of pine worm P glycoprotein in drug metabolism[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20200387

松材线虫P糖蛋白在药物代谢过程中的功能研究

doi: 10.12171/j.1000-1522.20200387
基金项目: 中央高校基本科研业务费专项资金项目(2572018AA37;2572019CP01)
详细信息
    作者简介:

    李洋。主要研究方向:森林病理学。Email:523875000@qq.com 地址:150040黑龙江省哈尔滨市和兴路26号东北林业大学林学院

    责任作者:

    马玲,教授,博士生导师。主要研究方向:森林有害生物综合治理。Email:maling63@163.com 地址:150040东北林业大学林学院黑龙江省林木保护技术创新中心

  • 中图分类号: S763.1

Function of pine worm P glycoprotein in drug metabolism

  • 摘要:   目的  探究松材线虫(Bursaphelenchus xylophilus)对药物敏感能力产生的分子机制,为防治松材线虫提供理论基础。  方法  本研究从松材线虫基因组中获得秀丽线虫(Caenorhabditis elegans)同源解毒基因Bx-pgp23,并对该基因蛋白编码区进行PCR扩增,随后通过生物信息学对蛋白质Bx-PGP23理化性质、亲疏水性、跨膜区分布、磷酸化位点、二级结构和三级结构进行了分析和预测。并通过RNAi技术对Bx-pgp23进行沉默处理,分析Bx-pgp23沉默与否对松材线虫药物敏感程度的影响。  结果  生物信息预测结果显示PGP蛋白稳定系数为38.31,亲水系数为−0.018,三级结构预测PGP蛋白具有多个氨基酸参与构成α螺旋和β折叠并且具有多个核苷酸结合域(NBD)和跨膜结构域(TMD)。应用RNAi技术对Bx-pgp23基因进行基因沉默,沉默后的Bx-pgp23基因表达量变为原来的42.65%。生测实验结果显示,在1.5和2.5 g/L苦参碱溶液处理24 h后,RNAi组松材线虫死亡率与对照组相比升高了7.2%和6.4%,在1.5和2.5 g/L的苦参碱溶液处理48 h后,RNAi组松材线虫死亡率与对照组相比升高9.0%和7.2%。  结论  蛋白质Bx-PGP23是一种稳定的亲水蛋白,具有跨膜外排功能。成功克隆Bx-pgp23基因并合成了该基因dsRNA。Bx-pgp23基因沉默影响了松材线虫对苦参碱的敏感性,在相同质量浓度的苦参碱胁迫下,RNAi组线虫死亡率明显高于对照组,说明Bx-pgp23基因在松材线虫药物代谢调控中发挥着正向调控作用。

     

  • 图  1  松材线虫RNA和Bx-pgp23基因CDS区胶图

    A 松材线虫总RNA Total RNA of Bursaphelenchus xylophilus B Bx-pgp23基因CDS区 CDS region of Bx-pgp23

    Figure  1.  Glulam map of RNA and Bx-pgp23 of B. xylophilus

    图  2  Bx-PGP23保守结构域

    Figure  2.  Conserved domain of Bx-PGP23

    图  3  Bx-PGP23蛋白及其同源序列比对

    Figure  3.  Bx-PGP23 protein and its homologous sequence alignment

    图  4  Bx-PGP23蛋白进化树分析

    Figure  4.  Protein evolution tree analysis of Bx-PGP23

    图  5  Bx-PGP23蛋白疏水性预测

    Figure  5.  The ProtScale output of Bx-PGP23

    图  6  Bx-PGP23蛋白跨膜螺旋预测

    Figure  6.  TMHMM posterior probabilities for Bx-PGP23.

    图  7  Bx-PGP23蛋白磷酸化位点预测

    Figure  7.  Phosphorylation site prediction of Bx-PGP23

    图  8  Bx-PGP23蛋白二级结构预测

    Figure  8.  Secondary structure prediction of Bx-PGP23

    图  9  Bx-PGP23蛋白三维结构预测图

    Figure  9.  Three-dimensional structure prediction diagram of Bx-PGP23

    图  10  拉氏构象图

    Figure  10.  Laplacian conformational map

    图  11  松材线虫Bx-pgp23基因在不同时间的表达量

    Figure  11.  Expression of Bx-pgp23 in B. xylophilus at different time

    图  12  松材线虫Bx-pgp23-dsRNA浸泡后对苦参碱胁迫的敏感性

    A苦参碱胁迫处理24 h死亡率 Mortality rate of B. xylophilus exposed with matrine 24 h B 苦参碱胁迫处理48 h死亡率Mortality rate of B. xylophilus exposed with matrine 48 h

    Figure  12.  Sensitivity of B. xylophilus soaked in Bx-pgp23-dsRNA to matrine stress

    表  1  Bx-pgp23与8种线虫同源序列比对

    Table  1.   Alignment of Bx-pgp23 with 8 nematodes homologous sequences

    种名 Species NameNCBI号 NCBI No.比对得分 Comparison pointE同源性 Homology/%
    捻转血矛线虫(Haemonchus contortus AFX93750.1 1 402 0 54.50
    长形杯环线虫(Cylicocyclus elongatus AJM87336.1 1 398 0 53.39
    环纹背带线虫(Teladorsagia circumcincta SJL35509.1 1 380 0 54.72
    简单异尖线虫(Anisakis simplex AXS78254.1 1 375 0 54.04
    秀丽线虫(Caenorhabditis elegans NP507487.1 1 329 0 51.14
    鼠圆线虫(Strongyloides ratti XP024500556.1 1 328 0 51.24
    胎生网尾线虫(Dictyocaulus viviparus KJH44478.1 1 256 0 48.65
    锡兰钩虫(Ancylostoma ceylanicum EPB71825.1 1 240 0 51.06
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  • 收稿日期:  2020-12-09
  • 修回日期:  2021-04-07
  • 网络出版日期:  2021-06-22

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