三个棉蚜谷胱甘肽-S-转移酶（GST）基因克隆及在不同寄主专化型中的表达分析 *

doi:10.7679/j.issn.2095-1353.2020.084

摘要/Abstract

摘要：

【目的】明确棉蚜Aphis gossypii Glover体内3个高表达谷胱甘肽-S-转移酶（GST）基因不同发育阶段的表达谱,并比较它们在不同寄主专化型棉蚜中的表达差异。【方法】 从棉蚜的伏蚜和苗蚜转录组数据库中挑选GSTs并克隆得到全长cDNA序列,使用qRT-PCR分析这些基因在不同寄主专化型棉蚜不同龄期中的相对表达量。【结果】 克隆到3个高表达的GSTs基因,分别命名为AgoGST-s1（GenBank登录号：MN688789）、AgoGST-d1（GenBank登录号：MN688790）、AgoGST-d2（GenBank登录号：MN688791）。这3个基因在不同龄期均表达,不同发育阶段的表达谱基本一致,表达量呈现从低到高的变化趋势。AgoGST-s1基因在各个发育阶段相对表达量的变化幅度较小,而AgoGST-d1、AgoGST-d2的变化幅度较大。对比两种寄主专化型棉蚜相同龄期时的表达情况发现,黄瓜型棉蚜中GSTs基因的表达量在大部分龄期都高于棉花型棉蚜。【结论】 AgoGST-s1、AgoGST-d1、AgoGST-d2基因表达量伴随棉蚜生长发育变化趋势明显,在不同寄主专化型棉蚜体内的表达量存在差异,可能与棉蚜对寄主植物的适应性有关。

关键词: 棉蚜, 谷胱甘肽-S-转移酶, 发育阶段, 寄主专化型, 表达谱

Abstract:

[Objectives] To determine the profiles of three high expression glutathione-S-transferase (GST) genes in various developmental stages of Aphis. gossypii Glover, and compare the expression of these genes in different host-races of this pest. [Methods] Full-length cDNA sequences of GSTs genes were cloned based on transcriptome data of A. gossypii specimens collected from seedling and mid-summer growth stages of cotton. The relative expression levels in different developmental stages (nymph and adult) and host-races (cucumber and cotton host-races) were investigated with real-time quantitative PCR. [Results] Three high expression GSTs were identified and named AgoGST-s1 (GenBank accession no.: MN688789), AgoGST-d1 (GenBank accession no.: MN688790) and AgoGST-d2 (GenBank accession no.: MN688791). These 3 genes were expressed in all development stages and their temporal expression profiles all tended to increase over time. The relative expression of AgoGST-s1 gene was less in all developmental stages, whereas that of AgoGST-d1 and AgoGST-d2 was more variable. Expression of these 3 GSTs in the cucumber host-race was higher than in the cotton host-race for most age-classes. [Conclusion] Expression of the AgoGST-s1, AgoGST-d1 and AgoGST-d2 genes is significantly associated with the developmental stage of A. gossypii. The expression patterns of these 3 GST genes in two host-races differed, which may reflect adaptation to different host plants.

Key words: Aphis gossypii, glutathione-S-transferase (GST), host specialization, development stage, expression profile

王传鹏, 张帅, 高雪珂, 雒珺瑜, 朱香镇, 王丽, 张开心, 杨亦桦, 崔金杰. 三个棉蚜谷胱甘肽-S-转移酶（GST）基因克隆及在不同寄主专化型中的表达分析 ^*[J]. 应用昆虫学报, 2020, 57(4): 823-832.

Chuan-Peng WANG, Shuai ZHANG, Xue-Ke GAO, Jun-Yu LUO, Xiang-Zhen ZHU, Li WANG, Kai-Xin ZHANG, Yi-Hua YANG, Jin-Jie CUI. Clone and expression analysis of three glutathione-S-transferase genes in different host-specific types of Aphis gossypii Glover[J]. Chinese Journal of Applied Entomology, 2020, 57(4): 823-832.

图/表 5

表1 实时荧光定量PCR引物

Table 1 Primers used for real-time quantitative PCR

基因 Gene	引物序列 Primer sequence（5?-3?）	扩增效率（%） Amplification efficiency	相关系数（R²） Correlation coefficient
AgoGST-s1	F-TGATAATGGCACTTAACTACTA R-AACCACTATCCGAACAAC	90	0.994
AgoGST-d1	F-ACAGTCAGTCGTCATACAC R-TTCTGAATCGGTCGGTTC	91	0.997
AgoGST-d2	F-GTGATTGGCTTCGTTGTAAT R-ATCTGACCAGTTATCCTAATGT	94	0.995
AgoGAPDH	F-ATCATTCCAGCATCTACT R-TCCTTAACCTTATCCTTGA	90	0.999

图1 棉蚜AgoGST-s1与其他昆虫Sigma类GSTs氨基酸序列多重比较 AgoGST-s1：棉蚜Aphis gossypii,XP_027840459.1;BmGST-s2：家蚕Bombyx mori,NP_001036994.1;DvGST-s1：葡萄根瘤蚜Daktulosphaira vitifoliae,AUN35386.1;AmGST-s1：西方蜜蜂Apis mellifera,NP_001153742.1;LmGST-s4：飞蝗Locusta migratoria,AEB91976.1。黑色方框为AgoGST-s1 N端结构域;红色方框为AgoGST-s1 C端结构域;预测的GSH结合位点（G-site）和底物结合位点（H-site）分别标注为红色和绿色,Sigma家族特有的催化位点残基“Y8”用黑色箭头表示。

Fig. 1 Amino acid sequence alignment of AgoGST-s1 from Aphis gossypii with Sigma-class homologues from other insects Black box region is the N-terminal domain of AgoGST-s1; Red box region is the C-terminal domain of AgoGST-s1; Predicted GSH binding sites (G-sites) and substrate binding sites (H-sites) are shaded red and green, respectively. The typical catalytic site residue “Y8” of Sigma-class is indicated in black arrow.

图2 棉蚜AgoGST-d1、AgoGST-d2与其他昆虫Delta类GSTs氨基酸序列多重比较 AgoGST-d1：棉蚜Aphis gossypii,AFM78644.1;AgoGST-d2：棉蚜Aphis gossypii,XP_027847501.1;BmGST-d2：家蚕Bombyx mori,NP_001036974.1;DvGST-s1：葡萄根瘤蚜Daktulosphaira vitifoliae,AUN35382.1;AmGST-d1：西方蜜蜂Apis mellifera,NP_001171499.1;LmGST-d1：飞蝗Locusta migratoria,AHC08057.1。黑色方框为AgoGST-s1 N端结构域;红色方框为AgoGST-s1 C端结构域;预测的GSH结合位点（G-site）和底物结合位点（H-site）分别标注为红色和绿色,Delta家族特有的典型序列“T*AD”用黑色箭头标注。

Fig. 2 Amino acid sequence alignment of AgoGST-d1, AgoGST-d2 from Aphis gossypii with Delta-class homologues from other insects Black box region is the N-terminal domain of AgoGST-d1, AgoGST-d2; Red box region is the C-terminal domain of AgoGST-d1, AgoGST-d2; Predicted GSH binding sites (G-sites) and substrate binding sites (H-sites) are shaded red and green, respectively. The typical sequence “T*AD” of Delta-class is marked by black arrow.

图3 棉蚜与其他不同昆虫GSTs系统进化分析进化树使用邻接法构建,各分支置信度经由1 000次Bootstrap检验。棉蚜GST基因用红色字体表示。 So：米象;Bg：德国小蠊;Lm：东亚飞蝗;Cs：二化螟;Px：柑橘凤蝶;Aga：冈比亚按蚊;Dv：葡萄根瘤蚜;Dm：黑腹果蝇;Dp：黑脉金斑蝶;Tm：黄粉虫;Bm：家蚕;Pa：美洲大蠊;Zn：湿木白蚁;Api：豌豆蚜;Sl：斜纹夜蛾;Ls：烟草甲;Bt：烟粉虱;Ape：柞蚕;Ago：棉蚜。

Fig. 3 Phylogenetic analysis of GSTs of Aphis gossypii and other insects The tree is constructed by using neighbor-joining method. Bootstrap support values are based on 1 000 replicates. The GSTs of cotton aphid is marked with the red. So: Sitophilus oryzae; Bg: Blattella germanica; Lm: Locusta migratoria; Cs: Chilo suppressalis; Px: Papilio xuthus; Aga: Anopheles gambiae; Dv: Daktulosphaira vitifoliae; Dm: Drosophila melanogaster; Dp: Danaus plexippus; Tm: Tenebrio molitor; Bm: Bombyx mori; Pa: Periplaneta americana; Zn: Zootermopsis nevadensis; Api: Acyrthosiphon pisum; Sl: Spodoptera littoralis; Ls: Lasioderma serricorne; Bt: Bemisia tabaci; Ape: Antheraea pernyi: Ago: Aphis gossypii.

图4 AgoGST-s1、AgoGST-d1、AgoGST-d2在棉花型和黄瓜型棉蚜中的相对表达量 2 L：2龄若蚜;3 L：3龄若蚜;4 L：4龄若蚜;1 D：1 日龄成蚜;3 D：3 日龄成蚜;5 D：5 日龄成蚜;7 D：7 日龄成蚜。柱上标有不同小写字母代表基因表达量的差异显著（ANOVA, P<0.05）。

Fig. 4 Relative expression levels of AgoGST-s1, AgoGST-d1, AgoGST-d2 in cotton specialized and cucumber specialized aphids 2 L: 2nd instar nymph; 3 L: 3rd instar nymph; 4 L: 4th instar nymph; 1 D: 1st adult; 3 D: 3rd adult; 5 D: 5th adult; 7 D: 7th adult. Different lowercase letters above histograms indicate significant difference among various genes (ANOVA, P<0.05).

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