螺虫乙酯抑制西花蓟马卵孵化的蛋白质组学分析

doi:10.3864/j.issn.0578-1752.2022.15.006

摘要/Abstract

摘要：

【目的】前期研究发现,螺虫乙酯处理西花蓟马（Frankliniella occidentalis）0 h卵24 h后,造成卵壳破裂从而抑制卵孵化出若虫。本研究旨在探究螺虫乙酯处理西花蓟马0 h卵24 h后引起卵形态结构发生变化的原因,寻找螺虫乙酯抑制西花蓟马0 h卵孵化的关键蛋白,揭示螺虫乙酯抑制西花蓟马卵孵化的分子机制。【方法】以西花蓟马0 h卵为研究对象,使用浓度为14.42 mg·L^-1的螺虫乙酯处理24 h后,采用无标记定量（Label-free）蛋白质组学技术分析螺虫乙酯处理组与对照组之间的差异表达蛋白。利用Gene Ontology（GO）富集分析和KEGG通路分析对筛选出的差异表达蛋白进行功能注释和通路分析等生物信息学分析,并通过平行反应监测（PRM）技术验证差异蛋白的表达量。【结果】螺虫乙酯处理后共有204个蛋白差异表达,其中124个蛋白表达量上调,80个蛋白表达量下调。生物信息学分析表明,上调的差异表达蛋白主要参与物质合成和代谢通路,下调的差异表达蛋白主要参与对外界刺激的防御反应通路。此外,利用PRM技术对差异表达蛋白进行鉴定,结果表明螺虫乙酯处理西花蓟马0 h卵24 h后,三磷酸鸟苷环水解酶1-1、三磷酸鸟苷环水解酶1-2、类谷胱甘肽硫-转移酶、脂酰辅酶A还原酶蛋白表达量显著上调,西花蓟马卵的类内表皮结构糖蛋白SgAbd-2、类铜/锌-超氧化物歧化酶、O-乙酰基转移酶蛋白表达量显著下调,且类内表皮结构糖蛋白SgAbd-2表达量下调最为明显。PRM技术验证结果与蛋白质组学的结果一致。【结论】上述蛋白可能在西花蓟马卵孵化过程中发挥多种功能,其中类内表皮结构糖蛋白SgAbd-2与昆虫表皮分化等生命活动相关,推测螺虫乙酯抑制了类内表皮结构糖蛋白SgAbd-2的表达,从而造成西花蓟马0 h卵出现卵壳破裂的现象,类内表皮结构糖蛋白SgAbd-2可能在西花蓟马0 h卵孵化的过程中发挥重要作用。

关键词: 西花蓟马, 螺虫乙酯, 卵, 蛋白质组学, 孵化

Abstract:

【Objective】Previous studies showed that the 0 h old eggs of Frankliniella occidentalis after 24 h treated with spirotetramat resulted in the cracking of eggs shell and inhibited the hatching of eggs. The objective of this study is to investigate the causes of morphological and structural changes of 0 h old eggs of F. occidentalis after 24 h treated with spirotetramat, and to find the key proteins and reveal the molecular mechanism of spirotetramat inhibiting the hatching of 0 h old eggs of F. occidentalis. 【Method】The 0 h old eggs of F. occidentalis were treated with 14.42 mg·L^-1 spirotetramat for 24 h, and the Label-free quantitative proteomics technique was used to analyze differentially expressed proteins between the spirotetramat-treated group and the control group. Gene Ontology (GO) enrichment analysis and KEGG pathway analysis were used for functional annotation and pathway analysis of the differentially expressed proteins, and the expression of differential expressed proteins was verified by parallel reaction monitoring (PRM).【Result】A total of 204 proteins were differentially expressed after treatment with spirotetramat, of which 124 proteins were up-regulated and 80 proteins were down-regulated. Bioinformatics analysis showed that the up-regulated differentially expressed proteins were mainly involved in substance synthesis and metabolic pathways, while the down-regulated differentially expressed proteins were mainly participated in the defense response pathway to external stimuli. In addition, the results of PRM were consistent with the proteomics results. The results showed that the protein expression levels of GTP cyclohydrolase 1-1, GTP cyclohydrolase 1-2, glutathione S-transferase-like and fatty acyl-CoA reductase were significantly up-regulated, while the expression levels of endocuticle structural glycoprotein SgAbd-2-like, superoxide dismutase [Cu-Zn]-like and O-acetyl transferase in 0 h old eggs of F. occidentalis after 24 h treated with spirotetramat were significantly down-regulated, especially endocuticle structure glycoprotein SgAbd-2-like.【Conclusion】The above proteins may play multiple functions in the hatching process of F. occidentalis eggs, the endocuticle structure glycoprotein SgAbd-2-like is related to insect epidermal differentiation and other life activities, it was speculated that the expression of this protein was inhibited by spirotetramat, which resulted in egg shell rupture of F. occidentalis 0 h old eggs, the endocuticle structure glycoprotein SgAbd-2-like may play an important role in the hatching process of F. occidentalis 0 h old eggs.

Key words: Frankliniella occidentalis, spirotetramat, egg, proteomics, hatching

周桂盈,杨晓敏,滕子文,孙丽娟,郑长英. 螺虫乙酯抑制西花蓟马卵孵化的蛋白质组学分析[J]. 中国农业科学, 2022, 55(15): 2938-2948.

ZHOU GuiYing,YANG XiaoMin,TENG ZiWen,SUN LiJuan,ZHENG ChangYing. Quantitative Proteomic Analysis of Spirotetramat Inhibiting Hatching of Frankliniella occidentalis Eggs[J]. Scientia Agricultura Sinica, 2022, 55(15): 2938-2948.

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https://www.chinaagrisci.com/CN/Y2022/V55/I15/2938

图/表 5

图1

表1

差异表达蛋白结果统计"

蛋白名称 Protein name	蛋白登录号 Protein ID	肽段数 Peptide count	特殊肽段数 Unique peptide count	分子量 Molecular weight (kD)	变化倍数Fold change	P值 P value
三磷酸鸟苷环水解酶1-1 GTP cyclohydrolase 1-1	A0A6J1TMB5	3	3	18.50	13.65	0.00009
三磷酸鸟苷环水解酶1-2 GTP cyclohydrolase 1-2	A0A6J1T9W9	3	3	17.58	11.16	0.00062
类谷胱甘肽硫-转移酶Glutathione S-transferase-like	A0A6J1SUA2	13	13	23.29	7.37	0.00005
脂酰辅酶A还原酶Fatty acyl-CoA reductase	A0A6J1TJA8	4	4	57.99	4.04	0.00067
超长链脂肪酸蛋白质的延伸 Elongation of very long chain fatty acids protein	A0A6J1SJR5; A0A6J1SRS0	2	2	39.09	2.75	0.03058
甘露聚糖酶Mannanase	A0A6J1T5W2	8	8	108.14	2.59	0.03446
谷胱甘肽过氧化物酶Glutathione peroxidase	A0A6J1TAR5; A0A6J1T4F1	11	11	18.60	2.13	0.00450
类胰脂肪酶相关蛋白3 Pancreatic lipase-related protein 3-like	A0A6J1STX0	3	3	42.11	1.99	0.01464
酪氨酸蛋白激酶Tyrosine-protein kinase	A0A6J1RYS7	3	3	95.47	1.99	0.00560
Ras相关蛋白Rab-32亚型X9 Ras-related protein Rab-32 isoform X9	A0A6J1SY76; A0A6J1SSP5; A0A6J1STV6; A0A6J1STV8; A0A6J1T0S5; A0A6J1SY71; A0A6J1SSN8; A0A6J1STV1; A0A6J1STV2	3	3	24.05	1.96	0.00719
泛素结合酶E2 G1 Ubiquitin-conjugating enzyme E2 G1	A0A6J1TK58	3	3	19.44	1.89	0.04406
脂肪酸结合蛋白,肌肉亚型X2 Fatty acid-binding protein, muscle isoform X2	A0A6J1RYX2; A0A6J1RRR7	13	13	15.01	1.84	0.02624
类谷胱甘肽S-转移酶 θ-1 Glutathione S-transferase theta-1-like	A0A6J1RZW4	5	5	25.72	1.83	0.04168
脂酰辅酶A还原酶 Fatty acyl-CoA reductase	A0A6J1SPV4; A0A6J1SJC4	6	6	57.97	1.77	0.03626
类85/88 kD钙非依赖性磷脂酶A2 85/88 kD calcium-independent phospholipase A2-like	A0A6J1RWA1	3	3	87.87	1.65	0.03396
ATP依赖型RNA解旋酶A亚型X1 ATP-dependent RNA helicase A isoform X1	A0A6J1S206; A0A6J1S943	7	7	140.50	1.64	0.01121
Ras相关蛋白rab7 Ras-related protein rab7	A0A6J1STG7	8	8	23.30	1.60	0.01576
核糖体生物合成蛋白BMS1同源异构体 X2 Ribosome biogenesis protein BMS1 homolog isoform X2	A0A6J1S7D5; A0A6J1S5Z1	6	6	151.97	1.57	0.01277
阿法丁异构体X6 Afadin isoform X6	A0A6J1SEG9; A0A6J1S933; A0A6J1S9F8; A0A6J1SGI5; A0A6J1SEG7; A0A6J1SAN7	10	10	221.95	1.57	0.00601
脂肪细胞质膜相关蛋白 Adipocyte plasma membrane-associated protein	A0A6J1SQ17	15	15	65.63	1.56	0.03344
ATP结合盒亚家族F成员2 ATP-binding cassette sub-family F member 2	A0A6J1SA46	13	13	71.60	0.64	0.00254
类蛋白阻塞物-E Protein obstructor-E-like	A0A6J1TAJ9	5	5	40.81	0.63	0.00146
O-乙酰基转移酶O-acetyl transferase	A0A6J1S6K4; A0A6J1S111	2	2	57.82	0.63	0.02456
RNA解旋酶RNA helicase	A0A6J1S314; A0A6J1S9L2	24	20	60.17	0.62	0.00058
类鸟嘌呤核苷酸结合蛋白1 Guanine nucleotide-binding protein-like 1	A0A6J1T023	9	9	68.61	0.59	0.03401
T复合蛋白1亚基epsilon T-complex protein 1 subunit epsilon isoform X1	A0A6J1S5J0; A0A6J1S3Y9	30	30	59.55	0.59	0.02217
δ-1-吡咯啉-5-羧酸合酶Delta-1-pyrroline-5-carboxylate synthase	A0A6J1THW9	8	8	83.84	0.59	0.03537
泛素结合酶E2 N Ubiquitin-conjugating enzyme E2 N	A0A6J1RWY7	5	5	17.19	0.58	0.01814
酪蛋白激酶I同种型X2 Casein kinase I isoform X2	A0A6J1SSW6; A0A6J1T0Y4	6	6	38.35	0.58	0.00652
微管蛋白α链Tubulin alpha chain	A0A6J1TCU9	16	1	49.99	0.55	0.00865
类Ras相关蛋白Rab-18-B Ras-related protein Rab-18-B-like	A0A6J1RZP1	2	2	23.35	0.52	0.04714
40S核糖体蛋白S24 40S ribosomal protein S24	A0A6J1TJB9; A0A6J1TJZ2	5	5	15.16	0.48	0.03436
上游激活因子亚基spp27 Upstream activation factor subunit spp27	A0A6J1RYK9	10	10	28.57	0.48	0.04572
类蛋白多糖4亚型X1 Proteoglycan 4-like isoform X1	A0A6J1TNX9; A0A6J1TSU0	14	14	146.33	0.38	0.03717
丝裂原活化蛋白激酶 Mitogen-activated protein kinase	A0A6J1SZU8; A0A6J1TD29; A0A6J1S2K1	5	4	42.50	0.33	0.01435
谷氨酰胺-果糖-6-磷酸转氨酶（异构化） Glutamine-fructose-6-phosphate transaminase (isomerizing)	A0A6J1SYR9; A0A6J1T384	22	22	76.10	0.33	0.00019
RNA解旋酶RNA helicase	A0A6J1T1T7	11	11	69.78	0.33	0.02672
细胞色素P450 Cytochrome P450 6a2-like	A0A6J1SXB7; F1JYR2; F1JYR1; A0A6J1SYI1; A0A6J1T5I4; A0A6J1T3E3; A0A6J1SJ06	11	10	60.04	0.28	0.02177
类铜/锌-超氧化物歧化酶Superoxide dismutase [Cu-Zn]-like	A0A6J1T4V3	4	4	18.72	0.19	0.00577
类内表皮结构糖蛋白SgAbd-2 Endocuticle structural glycoprotein SgAbd-2-like	A0A6J1T9H5	6	1	11.86	0.06	0.00211

表1

图2

螺虫乙酯处理西花蓟马0 h卵24 h后差异表达蛋白的GO富集聚类分析 A：上调差异表达蛋白的GO分析GO enrichment analyses of up-regulated differentially expressed proteins。1：谷胱甘肽衍生物代谢过程Glutathione derivative metabolic process;2：谷胱甘肽衍生物生物合成过程Glutathione derivative biosynthetic process;3：应对受伤过程Response to wounding;4：酪氨酸生物合成多巴胺的过程Dopamine biosynthetic process from tyrosine;5：谷胱甘肽代谢过程Glutathione metabolic process;6：化学稳态Chemical homeostasis;7：二氢生物蝶呤代谢过程Dihydrobiopterin metabolic process;8：细胞因子分泌Cytokine secretion;9：听觉感受器细胞静纤毛组织Auditory receptor cell stereocilium organization;10：单有机体运输过程Single-organism transport;11：细胞外区域Extracellular region;12：细胞外区域部分Extracellular region part;13：细胞外分泌体Extracellular exosome;14：细胞外囊泡Extracellular vesicle;15：细胞外细胞器Extracellular organelle;16：细胞外空间Extracellular space;17：溶解液泡Lytic vacuole;18：初级溶酶体Primary lysosome;19：嗜苯胺蓝细胞颗粒Azurophil granule;20：空泡部分Vacuolar part;21：抗氧化活性Antioxidant activity;22：氧化还原酶活性Oxidoreductase activity;23：过氧化物酶活性Peroxidase activity;24：氧化还原酶活性 Oxidoreductase activity, acting on peroxide as acceptor;25：以过氧化氢为受体的谷胱甘肽过氧化物酶活性Glutathione peroxidase activity with hydrogen peroxide as receptor;26：清道夫受体结合Scavenger receptor binding;27：钙转运ATP酶活性Calcium-transporting ATPase activity;28：胆固醇结合Cholesterol binding;29：苯乙醛合酶活性 Phenylacetaldehyde synthase activity;30：磷脂酶抑制剂活性 Phospholipase inhibitor activity B：下调差异表达蛋白的GO分析 GO enrichment analyses of down-regulated differentially expressed proteins。1：核糖体生物合成Ribosome biogenesis;2：核糖核蛋白复合物生物合成 Ribonucleoprotein complex biogenesis;3：核糖体RNA代谢过程 rRNA metabolic process;4：核糖体RNA加工过程 rRNA processing;5：翻译过程Translation process;6：ncRNA代谢过程ncRNA metabolic process;7：肽代谢过程Peptide metabolic process;8：肽生物合成过程 Peptide biosynthetic process;9：细胞质转化延伸Cytoplasmic translational elongation;10：毒素代谢过程Toxin metabolic process;11：细胞内的核糖核蛋白复合体Intracellular ribonucleoprotein complex;12：核糖核蛋白复合体Ribonucleoprotein complex;13：胞质小核糖体亚基 Cytosolic small ribosomal subunit;14：核糖体 Ribosome;15：密集染色体着丝粒Condensed chromosome kinetochore;16：胞质核糖体 Cytosolic ribosome;17：小核糖体亚基Small ribosomal subunit;18：核仁Nucleolus;19：凝聚染色体,着丝粒区Condensed chromosome, centromeric region;20：主轴Spindle;21：RNA结合RNA binding;22：mRNA结合mRNA binding;23：mRNA 5′-UTR 结合mRNA 5′-UTR binding;24：翻译起始因子结合Translation initiation factor binding;25：结构分子活性Structural molecule activity;26：核酸结合 Nucleic acid binding;27：核糖核蛋白复合物结合Ribonucleoprotein complex binding;28：核糖体的结构成分Structural constituent of ribosome;29：缬氨酸-tRNA连接酶活性Valine-tRNA ligase activity;30：去甲基化酶活性Demethylase activity"

图2

图3

图4

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