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

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

东北林业大学林学院，黑龙江哈尔滨 150040

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
计量
- 文章访问数: 98
- HTML全文浏览量: 63
- PDF下载量: 12
- 被引次数: 0
出版历程
- 收稿日期: 2020-12-09
- 修回日期: 2021-04-07
- 网络出版日期: 2021-06-22

Function of pine worm P glycoprotein in drug metabolism

School of Forestry, Northeast Forestry University, Harbin 150040, Heilongjiang, China

摘要

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。
- 松材线虫 /
- P糖蛋白 /
- 生物信息学 /
- 基因沉默
Abstract: Objective In order to reveal the molecular mechanism of drug sensitivity of Bursaphelenchus xylophilus to provide a theoretical basis for the control of Bursaphelenchus 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 the Bx-PGP23 were analyzed and predicted by bioinformatics. The effect of the Bx-pgp23 silencing on drug sensitivity of Bursaphelenchus xylophilus was analyzed by RNAi technology. Result Bioinformatics prediction showed 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 (NBD) and transmembrane domain (TMD). 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 showed that the mortality rate of Bursaphelenchus xylophilus in the RNAi group increased by 7.2% and 6.4% compared with the control group after 24 h of treatment with 1.5 and 2.5 g/L of matrine solution. After tremeant for 48 h, the mortality rate of Bursaphelenchus 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 Bursaphelenchus xylophilus to matrine solution and the mortality rate of Bursaphelenchus xylophilus in the RNAi group was significantly higher than the control group under the same mass concentration of matrine solution. The results indicated that the Bx-pgp23 gene plays a positive regulatory role in the regulating of drug metabolism in Bursaphelenchus xylophilus.
- Bursaphelenchus xylophilus /
- P-Glycoprotein /
- bioinformatics /
- RNAi

HTML全文

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

下载: 全尺寸图片幻灯片

图 2 Bx-PGP23保守结构域

Figure 2. Conserved domain of Bx-PGP23

下载: 全尺寸图片幻灯片

图 3 Bx-PGP23蛋白及其同源序列比对

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

下载: 全尺寸图片幻灯片

图 4 Bx-PGP23蛋白进化树分析

Figure 4. Protein evolution tree analysis of Bx-PGP23

下载: 全尺寸图片幻灯片

图 5 Bx-PGP23蛋白疏水性预测

Figure 5. The ProtScale output of Bx-PGP23

下载: 全尺寸图片幻灯片

图 6 Bx-PGP23蛋白跨膜螺旋预测

Figure 6. TMHMM posterior probabilities for Bx-PGP23.

下载: 全尺寸图片幻灯片

图 7 Bx-PGP23蛋白磷酸化位点预测

Figure 7. Phosphorylation site prediction of Bx-PGP23

下载: 全尺寸图片幻灯片

图 8 Bx-PGP23蛋白二级结构预测

Figure 8. Secondary structure prediction of Bx-PGP23

下载: 全尺寸图片幻灯片

图 9 Bx-PGP23蛋白三维结构预测图

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

下载: 全尺寸图片幻灯片

图 10 拉氏构象图

Figure 10. Laplacian conformational map

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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 Name	NCBI号 NCBI No.	比对得分 Comparison point	E	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

下载: 导出CSV

参考文献(26)

[1]	郝昕. 松材线虫多效耐药基因克隆及功能研究[D]. 哈尔滨: 东北林业大学, 2019. Hao X. Cloning and functional analysis of multidrug resistance gene in Bursaphelenchus xylophilus[D]. Harbin: Northeast Forestry University, 2019.
[2]	孙红, 周艳涛, 李晓冬, 等. 2020年全国主要林业有害生物发生情况及2021年发生趋势预测[J/OL]. 中国森林病虫 [2021-03-24]. https://doi.org/10.19688/j.cnki.issn1671-0886.20210004. Sun H, Zhou Y T, Li X D, et al. Occurrence of major forestry pests in China in 2020 and prediction of occurrence trend in 2021[J/OL]. Forest Pest and Disease [2021-03-24]. https://doi.org/10.19688/j.cnki.issn1671-0886.20210004.
[3]	胡龙娇, 吴小芹. 松树抗松材线虫病机制研究进展[J]. 生命科学, 2018, 30(6):659−666. Hu L J, Wu X Q. Research progress on the mechanism of pine response to the infection of Bursaphelenchus xylophilus[J]. Chinese Bulletin of Life Sciences, 2018, 30(6): 659−666.
[4]	徐晓朋. 松材线虫病综合防治技术[J]. 绿色科技, 2019, 10(19):102−103, 107. doi: 10.3969/j.issn.1674-9944.2019.19.040 Xu X P. Comprehensive control technology of Bursaphelenchus xylophilus[J]. Journal of Green Science and Technology, 2019, 10(19): 102−103, 107. doi: 10.3969/j.issn.1674-9944.2019.19.040
[5]	覃贵勇. 我国松材线虫病化学防治研究进展[J]. 河南农业, 2016, 8(23):38−40. Qin G Y. Research progress on chemical control of Bursaphelenchus xylophilus in China[J]. Agriculture of Henan, 2016, 8(23): 38−40.
[6]	Matsuda K, Kimura M, Komai K, et al. Nematicidal activities of (-)-N-methylcytisine and (-)-anagyrine from Sophora flavescens against pine wood nematodes (organic chemistry)[J]. Agricultural & Biological Chemistry, 1989, 53(8): 2287−2288.
[7]	Matsuda K, Yamada K, Kimura M, et al. Nematicidal activity of matrine and its derivatives against pine wood nematodes[J]. Journal of Agricultural & Food Chemistry, 1991, 39(1): 189−191.
[8]	崔慕华, 孙敦恒, 蒋显龙, 等. 苦参碱灌根防治山药根结线虫病效果初报[J]. 长江蔬菜, 2005(12):35. Cui M H, Sun D H, Jiang X L, et al. Preliminary report on the effect of matrine root irrigation on controlling yam root knot nematode[J]. Journal of Changjiang Vegetables, 2005(12): 35.
[9]	Dassa E, Bouige P. The ABC of ABCs: a phylogenetic and functional classification of ABC systems in living organisms[J]. Research in Microbiology, 2001, 152(3-4): 229.
[10]	柏家林, 蔡葵蒸, 何进全. P糖蛋白介导的寄生虫抗药性及其逆转的研究进展[J]. 中国兽医科学, 2010, 40(10):1085−1092. Ba J L, Cai K Z, He J Q. Advances in P-glycoprotein-mediated anthelmintic resistance and its reversal in parasites[J]. Chinese Veterinary Science, 2010, 40(10): 1085−1092.
[11]	Stupp G S, Reuss S, Izrayelit Y, et al. Chemical detoxification of small molecules by Caenorhabditis elegans[J]. Acs Chemical Biology, 2013, 8(2): 309−313. doi: 10.1021/cb300520u
[12]	Broeks A, Janssen H W, Calafat J, et al. A P-glycoprotein protects Caenorhabditis elegans against natural toxins[J]. The EMBO Journal, 1995, 14(9): 1858−1866. doi: 10.1002/j.1460-2075.1995.tb07178.x
[13]	Ardelli B F, Prichard R K. Inhibition of P-glycoprotein enhances sensitivity of Caenorhabditis elegans to ivermectin[J]. Veterinary Parasitology, 2013, 191(3-4): 264−275. doi: 10.1016/j.vetpar.2012.09.021
[14]	Graef J D, Demeler J, Skuce P, et al. Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones[J]. Parasitology, 2013, 140(4): 1−10.
[15]	许佳瑶, 陈俏丽, 张瑞芝, 等. 松材线虫Bx-ubc-3基因克隆及泛素通路鉴定[J]. 森林工程, 2019, 35(5):9−15. doi: 10.3969/j.issn.1006-8023.2019.05.002 Xu J Y, Chen Q L, Zhang R Z, et al. Genetic cloning of Bx-ubc-3 and identification of ubiquitin pathway from Bursaphelenchus xylophilus[J]. Forest Engineering, 2019, 35(5): 9−15. doi: 10.3969/j.issn.1006-8023.2019.05.002
[16]	郝昕, 王峰, 马玲, 等. 松材线虫耐药基因克隆及其功能[J]. 东北林业大学学报, 2018, 46(9):89−92+97. doi: 10.3969/j.issn.1000-5382.2018.09.019 Hao X, Wang F, Ma L, et al. Cloning and function of resistance gene of Bursaphelenchus xylophilus[J]. Journal of Northeast Forestry University, 2018, 46(9): 89−92+97. doi: 10.3969/j.issn.1000-5382.2018.09.019
[17]	Kikuchi T, Cotton J A, Dalzell J J, et al. Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus[J]. Plos Pathogens, 2011, 7(9): e1002219. doi: 10.1371/journal.ppat.1002219
[18]	Seddigh S, Darabi M. Functional, structural, and phylogenetic analysis of mitochondrial cytochrome b (cytb) in insects[J]. Mitochondrial DNA Part A, 2018, 29(2): 236−249. doi: 10.1080/24701394.2016.1275596
[19]	Diao J, Hao X, Ma W, et al. Bioinformatics analysis of structure and function in the MRP gene family and its expression in response to various drugs in Bursaphelenchus xylophilus[J]. Journal of Forestry Research, 2021, 32(2): 779−787. doi: 10.1007/s11676-019-01086-6
[20]	王军伟, 吴秋云, 毛舒香, 等. 外源物质调控芥蓝幼苗CYP83A1基因表达及其生物信息学分析[J]. 分子植物育种, 2019, 17(24):7996−8004. Wang J W, Wu Q Y, Mao S X, et al. Regulation of CYP83A1 gene expression in Cabbage seedling by exogenous substances and its bioinforma analysis[J]. Molecular Plant Breeding, 2019, 17(24): 7996−8004.
[21]	念波. 松材线虫谷胱甘肽巯基转移酶基因BxGST3和BxGST1全长克隆和功能分析[D]. 南京: 南京林业大学, 2017. Nian B. Full-length cloning and functional analysis of glutathione S-transferase genes BxGST3 and BxGST1 from Bursaphelenchus xylophilus[D]. Nanjing: Nanjing forestry university, 2017.
[22]	王博文, 刘伟璐, 王峰, 等. 低温调控Bx-SCD促进松材线虫脂肪积累[J]. 东北林业大学学报, 2017, 45(7):89−93. doi: 10.3969/j.issn.1000-5382.2017.07.018 Wang B W, Liu W L, Wang F, et al. Fat accumulation in Bursaphelenchus xylophilus by positively regulating Bx-SCD under low temperature[J]. Journal of Northeast Forestry University, 2017, 45(7): 89−93. doi: 10.3969/j.issn.1000-5382.2017.07.018
[23]	Ali S, Zhang C, Wang Z, et al. Toxicological and biochemical basis of synergism between the entomopathogenic fungus Lecanicillium muscarium and the insecticide matrine against Bemisia tabaci (Gennadius)[J]. Scientific Reports, 2017, 7: 1−14. doi: 10.1038/s41598-016-0028-x
[24]	Li Y, Zheng C, Liu K, et al. Transformation of multi-antibiotic resistant stenotrophomonas maltophilia with GFP gene to enable tracking its survival on pine trees[J]. Forests, 2019, 10(3): 231. doi: 10.3390/f10030231
[25]	Kerboeuf D, Guegnard F. Anthelmintics are substrates and activators of nematode P glycoprotein[J]. Antimicrobial Agents & Chemotherapy, 2011, 55(5): 2224−2232.
[26]	Dermauw W, van Leeuwen T. The ABC gene family in arthropods: comparative genomics and role in insecticide transport and resistance[J]. Insect Biochemistry and Molecular Biology, 2014, 45(1): 89−110.