Functional analysis of P-glycoprotein in drug metabolism of Bursaphelenchus xylophilus
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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. -
Key words:
- Bursaphelenchus xylophilus /
- P-glycoprotein /
- bioinformatics /
- RNAi
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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 3 Bx-PGP23蛋白及其同源序列比对
Figure 3. Bx-PGP23 protein and its homologous sequence alignment
图 9 Bx-PGP23蛋白三维结构预测图
Figure 9. Three-dimensional structure prediction diagram of Bx-PGP23
图 11 松材线虫Bx-pgp23基因在不同时间的表达量
1.5、2.5 g/L为苦参碱溶液的质量浓度。下同。1.5 and 2.5 g/L are the mass concentrations of matrine. The same below.
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 for 24 h; B. 苦参碱胁迫处理48 h死亡率 Mortality rate of B. xylophilus exposed with matrine for 48 h
Figure 12. Sensitivity of B. xylophilus soaked in Bx-pgp23-dsRNA under 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 nematode homologous sequences
种名 Species name NCBI号 NCBI No. 比对得分 Comparison score E值 E value 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 注：E值表示在随机的情况下，其他序列与目标序的列相似度要大于这条显示的序列的可能性。Note: E value represents the similarity between other sequences and the target sequence which is greater than the possibility of this displayed sequence, in the case of random. 
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