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    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, 2021, 43(11): 71-78. 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, 2021, 43(11): 71-78. DOI: 10.12171/j.1000-1522.20200387
    Li Yang, Hao Xin, Li Mingrui, Diao Jian, Ma Ling. Functional analysis of P-glycoprotein in drug metabolism of Bursaphelenchus xylophilus[J]. Journal of Beijing Forestry University, 2021, 43(11): 71-78. DOI: 10.12171/j.1000-1522.20200387
    Citation: Li Yang, Hao Xin, Li Mingrui, Diao Jian, Ma Ling. Functional analysis of P-glycoprotein in drug metabolism of Bursaphelenchus xylophilus[J]. Journal of Beijing Forestry University, 2021, 43(11): 71-78. 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

    Functional analysis of P-glycoprotein in drug metabolism of Bursaphelenchus xylophilus

    • 摘要:
        目的  探究松材线虫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和跨膜结构域。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%。药剂敏感性检测实验结果显示,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。

       

      Abstract:
        Objective  This paper aims to reveal the molecular mechanism of drug sensitivity of Bursaphelenchus xylophilus to provide a theoretical basis for the control of B. xylophilus.
        Method  In this study, the homologous detoxification gene Bx-pgp23 of Caenorhabditis elegans was obtained from the genome of Bursaphelenchus xylophilus, and PCR amplified the protein-coding region of the gene. Subsequently, the physicochemical properties, hydrophobicity, transmembrane distribution, phosphorylation sites, secondary structure, and tertiary structure of the protein Bx-PGP23 were analyzed and predicted by bioinformatics. The effect of Bx-pgp23 silencing on drug sensitivity of B. xylophilus was analyzed by RNAi technology.
        Result  Bioinformatics prediction indicated that the stability coefficient of PGP protein was 38.31 and the hydrophilic coefficient was −0.018. The tertiary structure predicted that PGP protein had multiple amino acids involved in the formation of α-helix and β-sheet with multiple nucleotide-binding domains and transmembrane domain. The gene silencing of Bx-pgp23 gene was performed by RNAi technology, after that, the expression of Bx-pgp23 gene was changed to 42.65% of the original. The results of the bioassay experiment suggested that after 24 h treatment with 1.5 and 2.5 g/L matrine solution, the mortality rate of pine nematode increased by 7.2% and 6.4% in the RNAi group compared with the control group, respectively. After treatment for 48 h with 1.5 and 2.5 g/L matrine solution, the mortality rate of B. xylophilus in the RNAi group increased by 9.0% and 7.2% compared with the control group.
        Conclusion  Protein Bx-PGP23 was a stable hydrophilic protein with transmembrane efflux function. The Bx-pgp23 gene was successfully cloned and the dsRNA of the gene was synthesized. Bx-pgp23 gene silencing affected the sensitivity of B. xylophilus to matrine solution and the mortality rate of B. xylophilus in the RNAi group was significantly higher than the control group under the same mass concentration of matrine solution. The results demonstrate that the Bx-pgp23 gene plays a positive regulatory role in the regulation of drug metabolism in B. xylophilus.

       

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