在长期自然选择状态下,昆虫对不同的寄主植物进化出了相应的适应能力,有些种类逐渐演化出寄主专化型（陈文胜等,1997）。我国的棉蚜主要分为黄瓜型棉蚜和棉花型棉蚜（刘向东等,2003）,相关研究发现黄瓜和棉花上的棉蚜相互转接后,棉蚜既不能正常生长也不能顺利产生后代（Wang et al.,2016）。目前蚜虫的寄主专化现象已经可利用分子生物学技术进行鉴别,如利用随机扩增多态性DNA（RAPD技术）发现寄主上的棉蚜存在分化现象（Masutti and Chavigny,1998）,微卫星标记技术可将棉花、西葫芦、黄瓜等20多个寄主植物上的棉蚜群体分为16个基因型（Masutti et al.,1999）。
蚜虫寄主专化性长期保持的结果导致了不同寄主专化型蚜在许多方面存在差异。在外部形态上,通过观察喙末端、各足跗节Ⅰ、跗节Ⅱ和爪等特征,发现蚜虫与寄主植物之间存在明显的形态适应关系（方燕等,2011）。生理生化的研究表明,瓜类上的棉蚜与茄子和马铃薯上的棉蚜存在酯酶活性上的差异（Saito,1990）。李飞等（2002）报道与取食棉花的棉蚜相比,取食南瓜的棉蚜乙酰胆碱酯酶对甲胺磷的敏感性下降,羧酸酯酶活性下降,分析得知寄主植物影响羧酸酯酶基因的转录或翻译过程。任柯昱等（2018）证实在相同发育时期,唾液蛋白C002基因在棉花型棉蚜和黄瓜型棉蚜表达水平不同,预示着两寄主专化型棉蚜在基因表达方面存在着分化。张元臣（2016）通过转录组测序发现,P450基因CYP302a1、CYP6a14、CYP4c1、CYP6a2和消化酶基因CL1593-1、CL382-1H、CL1471-2在黄瓜型棉蚜中表达量显著高于棉花型棉蚜。
谷胱甘肽-S-转移酶（GST）是生物体内的一类重要的代谢酶,参与外源和内源有毒物质的代谢（李亚红,2014）。GST通过催化还原型的谷胱甘肽（GSH）和有毒物质偶联反应,使有毒化合物的水溶性增加从而更容易排出体外,最终达到解毒的作用（Dixon and Edwards,2005）。在一些对杀虫剂有抗性的昆虫中,GST基因表达量明显上调,同时GST酶的活性也升高（刘佳等,2013;王圣印等,2013）。在植食性昆虫中谷胱甘肽-S-转移酶可以有效代谢有毒的植物次生代谢物,如α, β-不饱和羰基化合物、异硫氰酸盐、硫氰酸盐等（Wadleigh and Yu,1987,1988）。另外在一些昆虫中还发现GST具有过氧化物酶的活性,可以保护细胞免受氧化损伤从而在许多生理过程中发挥重要的作用（Vontas et al.,2001）。
本文基于棉蚜转录组数据中的相关信息,克隆获得了3个高表达GST基因的全长（AgoGST- s1、AgoGST-d1和AgoGST-d2）,对它们进行了序列和进化树分析;同时研究了这3个基因在棉花型和黄瓜型两种寄主专化型棉蚜不同发育阶段的表达特性,比较了在两种不同寄主专化型棉蚜中的表达差异,为进一步研究棉蚜寄主专化型形成的分子机理提供了重要信息。
1 材料与方法
1.1 供试植物
实验所采用的棉花品种为中棉所49,由中国农业科学院棉花种质资源中期库提供;黄瓜品种为新津优一号,购买于宁阳县金鑫种子销售中心。植物在人工气候室内种植,设定条件：温度（26±1）℃,相对湿度70%-80%,光周期L:D= 16:8。
1.2 供试棉蚜
供试所用的棉花型棉蚜和黄瓜型棉蚜分别采自中国农业科学院棉花研究所东场试验田（114°42ʹ E,36°11ʹ N）的棉花和黄瓜植株上,通过分子鉴定技术鉴定其专化型（Zhang et al.,2018）,鉴定后的棉蚜单头饲养于温度为（26±1）℃,相对湿度为70%-80%,光周期L:D=16:8的人工气候箱内,最终获得遗传背景一致的品系。
1.3 试验处理
将黄瓜型棉蚜和棉花型棉蚜的成蚜分别转接到离体的黄瓜叶或棉花叶片上,将叶片的梗部插入1.8%琼脂的保湿培养皿中,盖上盖子后置于人工气候室中,24 h后移除成蚜,留下若蚜在叶片上生长。每隔2 d更换一次新鲜叶片以保证蚜虫的正常生长发育。连续收集棉花型棉蚜和黄瓜型棉蚜2、3、4龄的若蚜和1、3、5、7日龄的成蚜。每个时期收集20头作为一个处理,共3个重复。将收集的样品储存在﹣80 ℃备用。
1.4 棉蚜总RNA的提取及cDNA的合成
棉蚜的总RNA的提取步骤按照TRIzol试剂（Ambion公司）说明书进行。使用NanoDrop 2000C及琼脂糖凝胶电泳检测RNA浓度、纯度及其完整性。
利用TaKaRa公司的PrimeScriptTM RT reagent Kit（Perfect Real Time）反转录试剂盒将上述不同棉蚜的RNA反转录成单链cDNA。
1.5 棉蚜GST基因的克隆
根据本实验室所建立的苗蚜和伏蚜转录组数据库信息（Li et al.,2013）中选出表达量相对较高的棉蚜3个GST基因,运用Primer Premier 5.0软件设计验证引物。AgoGST-s1的上游引物为5ʹ- TGATAATGGCACTTAACTACTA-3ʹ,下游引物为5ʹ-GGAGATAAGCGAATAAGGGTCATA-3ʹ;AgoGST-d1的上游引物为5ʹ-GCCGATCGACTTC TACTACACTCCC-3ʹ,下游引物为5ʹ-TCAAAAC ACGAAACGCAAAATCTG-3ʹ;AgoGST-d2的上游引物为5ʹ-GTTGCGAAAACTACAAAATGACTAT- 3ʹ,下游引物为5ʹ-TTTAATCTAGCAGTTTTGAA AACAG-3ʹ。PCR反应后将扩增产物用2%的琼脂糖凝胶电泳检测后,切胶回收并纯化,按照pEASY-T3 Cloning Kit试剂盒（全式金）的说明书将其连接到pEASY-T3载体上,然后转化Trans1-T1 Phage Resistant化学感受态细胞,挑取单克隆进行测序。
1.6 生物信息学分析
运用网站（
1.7 荧光定量PCR
使用Beacon Designer 7软件设计荧光定量引物,引物由生工生物工程（上海）股份有限公司合成。将cDNA模板稀释成5个浓度梯度,进行标准曲线的制作,并计算各引物的扩增效率（表1）。选取棉蚜的甘油三磷酸脱氢酶GAPDH作为内参基因（GenBank登录号：KP676380.1）（Cao et al.,2014）（表1）。荧光定量PCR反应使用GoTaq qPCR Master Mix（Promega公司）试剂盒进行。反应体系为qPCR MasterMix 10 μL、cDNA模板1.0 μL、上下游引物各0.8 μL、无核酸酶水7.4 μL,混匀离心,放入荧光定量PCR仪中扩增。反应条件为95 ℃变性2 min;接着进行40个循环,条件为95 ℃ 15 s、60 ℃ 1 min。定量PCR仪器为Eppendorf公司的Mastercycler ep realplex荧光定量PCR仪。表达量的计算根据Ct值采用2–ΔΔCt法计算基因的相对表达水平（Livak and Schmittgen,2001;Broeders et al.,2014）,不同寄主型蚜虫各个不同龄期的GST基因的相对表达水平均表示为棉花型若蚜2龄AgoGST-s1基因（设为1倍）的相对倍数。
表1 实时荧光定量PCR引物
Table 1
| 基因 Gene | 引物序列 Primer sequence（5ʹ-3ʹ） | 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 |
2 结果与分析
2.1 棉蚜3个GSTs基因克隆和序列特征分析
图1
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
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.
AgoGST-s1基因包括612 bp的ORF,编码203个氨基酸残基,预测蛋白质分子量为23.14 ku,等电点为5.17。经过PROSITE和Conserved Domains保守区域分析得知,AgoGST-s1包含Sigma家族特有的催化残基位点“Y8”,N端结构域位于氨基酸序列第4-73位,C端结构域位于第83-185位,其中分别含有GSH结合位点（8Y-14L-50K-51V-52P-63Q-64S）和疏水底物结合位点（96H-99R-100Q-103A-104G-159V-162Y）。
AgoGST-d1基因包括651 bp的ORF,编码216个氨基酸,预测蛋白质分子质量为24.17 ku,等电点为6.51。该基因含有Delta家族特有的典型序列“T*AD”和保守的N端、C端结构域,N端结构域位于氨基酸序列第3-76位,C端结构域位于第90-207位,分别具有GSH结合位点（11S-52H-53C-54V-66E-67S）和疏水底物结合位点（103V-107Y-108P-111A-112D-115Y-119F- 164S-167T-206F）。
AgoGST-d2基因包括666 bp的ORF,编码221个氨基酸,预测相对分子质量为24.89 ku,等电点为6.90。同样含有Delta家族典型的序列“T*AD”,保守的N端和C端结构域分别位于氨基酸序列第3-76位第96-212位,具有GSH和结合位点（11S-52H-53T-54I-66E-67S）和疏水底物结合位点（109L-113Y-114R-117S-118D-121G- 125A-169T-172C-211F）。
图3
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.
2.2 3个GST基因在棉蚜不同发育阶段的表达
2.2.1 棉花型棉蚜的GST基因的表达 利用荧光定量PCR技术检测了3个GST基因在棉花型棉蚜若蚜不同龄期和成蚜不同日龄的相对表达量,结果见图4。AgoGST-s1、AgoGST-d1和AgoGST-d2这3个基因不同时期的表达谱基本一致,除若蚜3龄时相对表达量最低,其余基本呈现出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和AgoGST-d2的变化幅度较大;其中AgoGST-d1、AgoGST-d2和AgoGST-s1表达差异最大时期分别出现在成蚜3日龄和成蚜7日龄,分别比AgoGST-s1高31倍和27倍,差异最小时期同时出现在若蚜2龄,但也分别比AgoGST-s1高出14倍。
图4
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).
2.2.2 黄瓜型棉蚜GST基因的表达 3个GST基因在黄瓜型棉蚜不同发育时期的相对表达量见图4。AgoGST-s1、AgoGST-d1和AgoGST-d2基因在黄瓜型棉蚜不同发育阶段中均表达,且除成蚜3日和5日龄,都表现为表达量随龄期增加而上升。AgoGST-d1和AgoGST-d2在黄瓜型棉蚜的表达量明显高于AgoGST-s1;其中AgoGST- d1、AgoGST-d2和AgoGST-s1表达差异最大时期同时出现在成蚜5日龄,分别比AgoGST-s1高138倍和284倍,差异最小时期分别出现在若蚜3龄和成蚜7日龄,但也分别比AgoGST-s1高出23倍。AgoGST-s1表达最低和最高分别在成蚜5日龄、成蚜7日龄,两者间超过20倍。AgoGST-d1基因表达量最低和最高时期分别出现在成蚜3日龄、成蚜7日龄,两者间相差近20倍。AgoGST-d2基因表达量最低和最高时期分别出现在若蚜2龄、成蚜7日龄,两者间相差近4倍。
2.3 棉花型棉蚜和黄瓜型棉蚜GST基因的表达量对比
进一步分析AgoGST-s1、AgoGST-d1和AgoGST-d2在两种寄主专化型相同龄期的表达情况可以发现（图4）,3个基因的相对表达量差异明显且基本都达到显著水平,在大部分龄期都是黄瓜型蚜虫中的表达量高于棉花型蚜虫。在成蚜1日龄和成蚜7日龄,两种寄主型间基因表达差异相对较大,AgoGST-s1在黄瓜型棉蚜中的表达量分别是棉花型棉蚜中表达量的2倍、3倍,AgoGST-d1表达量分别是棉花型棉蚜的5倍、7倍,AgoGST-d2表达量分别是棉花型棉蚜的5倍、2倍。
3 讨论
昆虫谷胱甘肽转移酶基因GST被划分为Delta、Epsilon、Omega、Sigma、Theta和Zeta 6个家族及1个“未分类”亚族（Ketterman et al.,2011;尤燕春等,2013;Liu et al.,2017）。本研究从棉蚜的转录组数据库中获得3个高表达GST基因AgoGST-d1、AgoGST-d2和AgoGST-s1,通过分析全长克隆获得的基因序列和构建系统发育进化树后,明确这3个基因分别属于Delta和Sigma家族。Delta是昆虫特有的家族（Enayati et al.,2005）,Ranson等（2002）通过功能验证发现Delta家族对外源物质解毒代谢起着重要的作用,能够增强昆虫对环境的适应性。Sigma家族则广泛存在于生物体中,Ranson等（2001）报道在对外源物质DDT有抗性的冈比亚按蚊A.gambiae中,Sigma家族基因mRNA的水平比敏感品系高出5倍。不管是昆虫特有的Delta、Epsilon家族还是生物体都有的其他家族GST基因,在内源和外源化合物代谢等方面都起着重要作用。本研究发现的3个GSTs基因分属不同的家族,在两种寄主专化型棉蚜各个发育阶段中均有表达,并且相对表达量在不同龄期的变化明显,而且Delta家族的两个基因AgoGST-d1和AgoGST-d2表达量远高于Sigma家族的AgoGST- s1,推测在不同寄主型棉蚜生长发育过程中,AgoGST-d1、AgoGST-d2和AgoGST-s1在不同程度上参与了植物外源次生物质的解毒代谢。
许多植物次生物质是GSTs的底物,植食性昆虫的GSTs参与对取食过程中遇到的植物次生物质的代谢（Wadleigh and Yu,1987）,同时GSTs活性也受到植物次生物质或杀虫剂的调节（Lee,1991;Yu,1999;汤方等,2005;金燕璐等,2018）。GSTs活性增加有两种方式,一种是在昆虫体内原有的谷胱甘肽-S-转移酶一种或多种同工酶上过量表达,另一种是合成新的同工酶,目前更多的研究表明昆虫GSTs活性的增加是基因的过量表达引起的（Hayaoka and Dauterman,1983;Reidy et al.,1990）。在本研究中棉蚜AgoGST-s1、AgoGST-d1和AgoGST-d2基因在两种寄主专化型棉蚜中的相对表达量基本都是随着龄期的增加表达呈现上升的趋势,除黄瓜型棉蚜3日龄和5日龄成蚜中AgoGST-s1表达量偏低外,成蚜期3个基因的表达量普遍高于若蚜期。翟颖婷（2019）研究发现,在生物型HM29麦长管蚜Sitobion avenae的人工饲料中分别添加浓度为0.04%没食子酸饲喂后,SaGST3基因和SaGST4基因的表达量分别是对照组的2.0倍和1.4倍,添加浓度为0.03%豆香素饲喂后,SaGST3基因和SaGST4基因的表达量分别是对照组的1.4倍和1.2倍,同时分别检测到GSTs活性增加。Yang等（2017）报道在人工饲料中添加2 μg/mL的阿魏酸饲喂褐飞虱后,NIGSTD1基因表达量是对照组的1.3倍,NIGSTE1基因的表达量是对照组的2.5倍,同时GSTs活性增加。棉蚜随着生长发育的进行或在特定阶段生长发育较快、繁殖能力较强,所需的营养物质和能量较多,伴随着营养物质进入棉蚜体内的次生物质也相应增多,所以通过GST基因表达量的提升,增强谷胱甘肽转移酶的活性,以促进棉蚜对外源物质的解毒或转化利用,进而增强棉蚜在寄主植物上的适应能力。
寄主专化性是指昆虫对特定植物种类和对植物取食的部位有着长期和专一的嗜好性（刘向东等,2004）。随着这种特性的定向发展,昆虫往往又会分化成不同的寄主专化型来适应寄主及环境变化（Wadleigh and Yu,1988）,这种适应性的分化是渐进的,其演化速度可能受当地种植模式、地理条件和气候条件等多种因素的影响,并最终可能在合适的条件下形成严格的寄主专化型（袁立兵等,2008）。南京地区的棉蚜被分为棉花型棉蚜和黄瓜型棉蚜（刘向东等,2003）。王丽（2015）采用气相色谱-飞行时间质谱联用仪（Gas chromatography time-of-flight mass spectrometry,GC-TOF/MS）进一步明确黄瓜、棉花及西葫芦苗期叶片等寄主植物的次生化合物组成不同很可能与棉蚜寄主专化型形成有关。
有研究表明萜烯类化合物（棉酚）、黄酮类化合物（槲皮素、芸香苷、儿茶素）、单宁类化合物（单宁酸）是棉花防御害虫棉蚜A. gossypii和棉铃虫Helicoverpa armigera的主要次生物质（Hedin et al.,1992;汤德良,1999;武予清等,2000;Mao et.al.,2007;Gao et al.,2008;刘泽辉等,2008）。黄酮类化合物、单宁类化合物、酚类和木质素则是黄瓜植株中的重要次生物质。寄主植物和害虫之间存在明显的协同进化关系,植物体内的次生物质和害虫体内的解毒代谢系统间相互影响和适应,进而影响昆虫的寄主适应性。研究发现,将B型烟粉虱Bemisia tabaci从原始适宜寄主棉花上转接到不适宜寄主辣椒上后,GST7基因的表达量上升了2倍,表现出显著差异（何超等,2017）。辣椒型桃蚜Myzus persicae从适宜寄主辣椒上转移到不适宜寄主蚕豆上后,体内GSTd4基因的表达水平下调60%（朱经云,2017）。Yu（1982）研究发现分别取食芥末和大豆两种不同寄主植物后,草地贪夜蛾Spodoptera frugiperda体内的谷胱甘肽-S-转移酶活性相差10倍。与取食人工饲料的对照组相比,取食防风草和欧芹两种寄主植物后,草地贪夜蛾S. frugiperda体内的谷胱甘肽-S-转移酶的活性分别升高39倍和19倍（Yu,1984）。本研究发现相同龄期两种寄主专化型棉蚜GSTs基因表达量的差异明显,黄瓜型棉蚜中普遍表达较高,如在若蚜4龄、成蚜1日龄、成蚜7日龄时,黄瓜型棉蚜中AgoGST-s1相对表达量分别是棉花型棉蚜的2倍、2倍、3倍;AgoGST-d1相对表达量分别是棉花型棉蚜的3倍、5倍、7倍;AgoGST-d2相对表达量是黄瓜型棉蚜的2倍、5倍、2倍。棉花和黄瓜的主要植物次生物质不同,棉蚜通过调节体内相应代谢酶基因的表达,加强对不同寄主的适应能力。本研究中明确了棉花型棉蚜和黄瓜型棉蚜中这3个GST基因表达量间的明显差异,推测与其寄主专化性有关。但黄瓜和棉花两种寄主植物中哪些次生物质对棉蚜GST基因的调节起着主导作用,以及除谷胱甘肽-S-转移酶之外棉蚜体内还有哪些酶类等,参与对次生物质的代谢以进一步影响寄主专化型的形成,这些问题仍需要进一步的研究和验证。
参考文献
Guidelines for validation of qualitative real-time PCR methods
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Molecular Characterization of the Aphis gossypii olfactory receptor gene families
DOI:10.1371/journal.pone.0101187
URL
PMID:24971460
[本文引用: 1]
The cotton aphid, Aphis gossypii Glover, is a polyphagous pest that inflicts great damage to cotton yields worldwide. Antennal olfaction, which is extremely important for insect survival, mediates key behaviors such as host preference, mate choice, and oviposition site selection. In insects, odor detection is mediated by odorant receptors (ORs) and ionotropic receptors (IRs), which ensure the specificity of the olfactory sensory neuron responses. In this study, our aim is to identify chemosensory receptors in the cotton aphid genome, as a means to uncover olfactory encoding of the polyphagous feeding habits as well as to aid the discovery of new targets for behavioral interference. We identified a total of 45 candidate ORs and 14 IRs in the cotton aphid genome. Among the candidate AgoORs, 9 are apparent pseudogenes, while 19 can be clustered with ORs from the pea aphid, forming 16 AgoOR/ApOR orthologous subgroups. Among the candidate IRs, we identified homologs of the two highly conserved co-receptors IR8a and IR25a; no AgoIR retain the complete glutamic acid binding domain, suggesting that putative AgoIRs bind different ligands. Our results provide the necessary information for functional characterization of the chemosensory receptors of A. gossypii, with potential for new or refined applications of semiochemicals-based control of this pest insect.
Study on the host specialisation of Myzus persicae in south china
烟蚜寄主专化性的研究
Insect glutathione transferases and insecticide resistance
Morphological adaptation of aphid species on different host plant leaves
Morphological adaptation of insects to their host plants is an important part of the relationship between the two types of organisms. This paper dealt with herbivorous aphids to study their morphological adaptation on different host plant leaves. Twenty-six aphid species which feed on the leaves of 7 genera host plants belonging to 4 families (Poaceae, Salicaceae, Fagaceae, and Pinaceae) were studied. Based on alate viviparous females and apterous viviparous females, 37 morphological characters were compared and measured by using light microscope. Then the characters were statistically analysed. Based on the clone means, with the variables such as ultimate rostral segments, tarsi and claws which are related to aphid feeding behavior and adhesion on the surfaces of plants, three clustering dendrogram (alate viviparous female, apterous viviparous female, all morph) were output and mapped with host plant families and genera. The results indicated that some characters of the aphids, such as body, siphunculi and antennae, were different among different aphid families, but some of them, such as the ultimate rostral segments, tarsal segments Ⅰ, tarsal segments Ⅱ and claws, varied among different host plant families. The variations of morphologies within species were within a range with their CV (coefficient of variation) between 1.89% and 26.08%. The clustering analysis showed that the aphids were divided into the clusters which were corresponding to certain host plant family/genera; and the positions of Doraphis populi, Epipemphigus imaicus and Pemphigus matsumurai were special, because the first is the secondary host-plant morph and the latter two are the gall makers. The results suggest that the distinct separation of the aphid species with respectively different morphological character variations is corresponding to different host plants whose surface structures are diverse. These prove morphological adaptation of aphids to host plants.]]>
不同寄主植物叶片上蚜虫的形态适应
Interactive effects of elevated CO2 and cotton cultivar on tri-trophic interaction of Gossypium hirsutum, Aphis gossypii and Propylaea japonica
DOI:10.1603/0046-225X(2008)37[29:IEOECA]2.0.CO;2
URL
PMID:18348793
[本文引用: 1]
Information on the effects of enriched CO2 on both the chemical composition of plants and the consequences of such changes for performance of a herbivore and its predator is an important first step in understanding the responses of plants and insects to global environmental change. We examined interactions across three trophic levels, cotton, Gossypium hirsutum, an aphid herbivore, Aphis gossypii Glover, and a coccinellid predator, Propylaea japonica (Thunberg), as affected by elevated CO2 concentrations and crop cultivars. Plant carbon:nitrogen (C:N) ratios, condensed tannin, and gossypol content were significantly higher, and nitrogen content was significantly lower in plants exposed to elevated CO2 levels compared with that in plants exposed to ambient CO2. Cotton aphid survivorship significantly increased and free fatty acid content decreased with increased CO2 concentrations. No significant differences in survival and lifetime fecundity of P. japonica were observed between cultivars and CO2 concentration treatments. However, stage-specific larval durations of the lady beetle were significantly longer when fed aphids from elevated CO2 concentrations. Our results indicate that high gossypol in the cotton host plant had an antibiotic effect on A. gossypii and produced a positive effect on growth and development of P. japonica at the third trophic level. However, elevated CO2 concentrations showed a negative effect on P. japonica. We speculate that A. gossypii may become a more serious pest under an environment with elevated CO2 concentrations because of increased survivorship of aphid and longer development time of lady beetle.
The effect of phenobarbital induction on glutathione conjugation of diazinon in susceptible and resistant house flies
DOI:10.1016/0048-3575(83)90063-9 URL [本文引用: 1]
Comparson of the responses of glutathione- S-transferase genes in Bemisia tabaci B to different host plant transfers
谷胱甘肽-S-转移酶基因在B型烟粉虱寄主转换中的差异比较
Relationships of glands, cotton square terpenoid aldehydes, and other allelochemicals to larval growth of Heliothis virescens(Lepidoptera: Noctuidae)
DOI:10.1093/jee/85.2.359 URL [本文引用: 1]
Identifying and measuring the expression profiles, of Chilo suppressalis glutathione-S-transferase genes
二化螟谷胱甘肽-S-转移酶基因的鉴定与表达模式分析
Insect glutathione transferases
DOI:10.3109/03602532.2011.552911
URL
[本文引用: 1]
This article is an overview of the current knowledge of insect glutathione transferases. Three major topics are discussed: the glutathione transferase contributions to insecticide resistance, the polymorphic nature of the insect glutathione transferase superfamily, and a summary of the current structure-function studies on insect glutathione transferases.
Glutathione- S-transferase activities in phytophagous insects: Induction and inhibition by plant phototoxins and phenols
DOI:10.1016/0020-1790(91)90001-U URL [本文引用: 1]
Comparison of esterase and acetylcholinesterase of cotton aphid, Aphis gossypii Glover reared on different host plants
取食不同寄主棉蚜的羧酸酯酶和乙酰胆碱酯酶特性比较
The progress in the research of insect GST on insecticide detoxification and endogenous metabolism
昆虫谷胱甘肽硫转移酶农药解毒与内源代谢研究进展
Ecological adaption analysis of the cotton aphid ( Aphis gossypii) in different phenotypes by transcriptome comparison
DOI:10.1371/journal.pone.0083180
URL
PMID:24376660
[本文引用: 1]
BACKGROUND: The cotton aphid, Aphis gossypii Glover, is a destructive insect pest worldwide; it directly or indirectly damages (virus transmission) 300 species of host plants. Knowledge of their ecologically adaptive mechanisms at the molecular level may provide an essential and urgent method to effectively control this pest. However, no transcriptome information is available for the cotton aphid and sequence data are scarce. Therefore, we obtained transcriptome data. RESULTS: To facilitate such a study, two cotton aphid transcriptomes at different growth stages of cotton, seedling and summer, were sequenced. A total of 161,396 and 66,668 contigs were obtained and assembled into 83,671 and 42,438 transcripts, respectively. After combining the raw date for both transcriptomes, the sequences were reassembled into 66,695 transcripts, and 52,160 were annotated based on BLASTX analyses. Comparison of the transcriptomes revealed that summer presented less challenges for the cotton aphids than the seedling stage of cotton. In total, 58 putative heat shock protein genes and 66 candidate cytochrome p450 genes were identified with BLASTX. CONCLUSIONS: Our results form a basis for exploring the molecular mechanisms of ecological adaption in the cotton aphid. Our study also provides a baseline for the exploration of abiotic stress responses. In addition, it provides large-scale sequence information for further studies on this species.
Characteristics of mRNA expression of GSTs1 gene in Spodoptera exigua and the effect of chlorantraniliprole on its expression
甜菜夜蛾GSTs1基因mRNA表达特征及氯虫苯甲酰胺对其表达量的影响
Identification and characterisation of seventeen glutathione- S-transferase genes from the cabbage white butterfly Pieris rapae
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URL
PMID:29183577
[本文引用: 1]
Insect glutathione S-transferases (GSTs) play essential roles in the detoxification of insecticides and other xenobiotic compounds. The cabbage white butterfly, Pieris rapae, is an economically important agricultural pest. In this study, 17 cDNA sequences encoding putative GSTs were identified in P. rapae. All cDNAs include a complete open reading frame and were designated PrGSTd1-PrGSTz2. Based on phylogenetic analysis, PrGSTs were divided into six classes (delta, epsilon, omega, sigma, theta and zeta). The exon-intron organizations of these PrGSTs were also analysed. Recombinant proteins of eight PrGSTs (PrGSTD1, PrGSTD2, PrGSTE1, PrGSTE2, PrGSTO1, PrGSTS1, PrGSTT1 and PrGSTZ1) were heterologously expressed in Escherichia coli, and all of these proteins displayed glutathione-conjugating activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Expression patterns in various larval tissues, at different life stages, and following exposure to sublethal doses of abamectin, chlorantraniliprole or lambda-cyhalothrin were determined by reverse transcription-quantitative PCR. The results showed that PrGSTe3, PrGSTs1, PrGSTs2, and PrGSTs4 were mainly transcribed in the fat body, while PrGSTe2 was expressed predominantly in the Malpighian tubules. Four genes (PrGSTe2, PrGSTo4, PrGSTs4 and PrGSTt1) were mainly expressed in fourth-instar larvae, while others were ubiquitously expressed in egg, larval, pupa and/or adult stages. Abamectin treatment significantly upregulated ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTt1). Chlorantraniliprole and lambda-cyhalothrin treatment significantly upregulated nine genes (PrGSTd1, PrGSTd2, PrGSTe1, PrGSTe2, PrGSTe3, PrGSTs1, PrGSTs3, PrGSTs4 and PrGSTz1) and ten genes (PrGSTd1, PrGSTd3, PrGSTe1, PrGSTe2, PrGSTo1, PrGSTo2, PrGSTs1, PrGSTs2, PrGSTs3 and PrGSTz2), respectively. These GSTs are potentially involved in the detoxification of insecticides.
Studies on the host biotypes and its cause of cotton aphid in Nanjing, China
南京地区棉蚜寄主专化型及其成因研究
Host biotypes and their formation causes in aphids
The possible causes of the formation of aphid host biotypes were reviewed. In ecological levels, the formation is mainly related with the selection capacity to host plants, the natural enemies, the symbionts (coexists ) and resistance to insecticides, and so on. The host biotypes have their genetic bases, including the mutation or change in enzymes and chromosomes, sexual reproduction, assortive mating and genetic differentiation of population. But the detailed causes for a certain host biotype in aphids were poorly understood at present.
蚜虫寄主专化型及其成因
Studies on content of cotton gossypol and characteristics of pest resistance
棉花棉酚含量与抗虫特性的研究
Analysis of relative gene expression data using real-time quantitative PCR and the 2 -ΔΔCT method
DOI:10.1006/meth.2001.1262
URL
PMID:11846609
[本文引用: 1]
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol
DOI:10.1038/nbt1352
URL
PMID:17982444
[本文引用: 1]
We identify a cytochrome P450 gene (CYP6AE14) from cotton bollworm (Helicoverpa armigera), which permits this herbivore to tolerate otherwise inhibitory concentrations of the cotton metabolite, gossypol. CYP6AE14 is highly expressed in the midgut and its expression correlates with larval growth when gossypol is included in the diet. When larvae are fed plant material expressing double-stranded RNA (dsRNA) specific to CYP6AE14, levels of this transcript in the midgut decrease and larval growth is retarded. Both effects are more dramatic in the presence of gossypol. As a glutathione-S-transferase gene (GST1) is silenced in GST1 dsRNA-expressing plants, feeding insects plant material expressing dsRNA may be a general strategy to trigger RNA interference and could find applications in entomological research and field control of insect pests.
Host-based genetic differentiation in the aphid Aphis gossypii Glover, evidenced from RAPD fingerprints
DOI:10.1046/j.1365-294x.1998.00421.x URL [本文引用: 1]
Characterization of microsatellite loci in the aphid species Aphis gossypii Glover
DOI:10.1046/j.1365-294x.1999.00876.x URL PMID:10327667 [本文引用: 1]
Evolution of supergene families associated with insecticide resistance
DOI:10.1126/science.1076781
URL
PMID:12364796
[本文引用: 1]
The emergence of insecticide resistance in the mosquito poses a serious threat to the efficacy of many malaria control programs. We have searched the Anopheles gambiae genome for members of the three major enzyme families- the carboxylesterases, glutathione transferases, and cytochrome P450s-that are primarily responsible for metabolic resistance to insecticides. A comparative genomic analysis with Drosophila melanogaster reveals that a considerable expansion of these supergene families has occurred in the mosquito. Low gene orthology and little chromosomal synteny paradoxically contrast the easily identified orthologous groups of genes presumably seeded by common ancestors. In A. gambiae, the independent expansion of paralogous genes is mainly a consequence of the formation of clusters among locally duplicated genes. These expansions may reflect the functional diversification of supergene families consistent with major differences in the life history and ecology of these organisms. These data provide a basis for identifying the resistance-associated enzymes within these families. This will enable the resistance status of mosquitoes, flies, and possibly other holometabolous insects to be monitored. The analyses also provide the means for identifying previously unknown molecules involved in fundamental biological processes such as development.
Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae
DOI:10.1042/bj3590295 URL [本文引用: 1]
Increased glutathione- S-transferase activity and glutathione content in an insecticide-resistant strain of Tribolium castaneum(Herbst)
DOI:10.1016/0048-3575(90)90035-Z URL [本文引用: 1]
Clone and expression analysis of salivary gland protein C002 in different host-specific types of Aphis gossypii Glover
棉蚜唾液蛋白C002基因克隆及在棉蚜不同寄主专化型中的表达分析
Insecticide resistance of the cotton aphid, Aphis gossypii Glover (Homoptera: Aphididae): Variation of esterase activity by host plants
DOI:10.1303/jjaez.34.37 URL [本文引用: 1]
The secondary metabolism of plant insect resistant
植物抗虫的次生代谢物质
2-tridecylketone and quercetin induce tissue-specific expression of glutathione- S-transferase in Helicoverpa armigera
2-十三烷酮和槲皮素诱导棉铃虫谷胱甘肽-S-转移酶组织特异性表达
Glutathione- S-transferases as antioxidant defence agents confer pyrethroid resistance in Nilaparvata lugens
DOI:10.1042/bj3570065 URL [本文引用: 1]
Glutathione transferase activity of fall armyworm larvae toward α, β-unsaturated carbonyl allelochemicals and its induction by allelochemicals
DOI:10.1016/0020-1790(87)90046-1 URL [本文引用: 2]
Detoxification of isothiocyanate allelochemicals by gluthione- S-transferase in three lepidopterous species
DOI:10.1007/BF01019352 URL [本文引用: 2]
Preliminary studies on the host biotypes and formation mechanism of cotton aphid in cotton areas of north China. Master dissertation.
华北棉区棉蚜寄主专化型及其形成机制初步研究
Identification of Aphis gossypii Glover (Hemiptera: Aphididae) biotypes from different host plants in north China
Mechanisms and cross-resistance of imidacloprid resistance in Frankliniella occidentalis
西花蓟马对吡虫啉的抗性机制及交互抗性研究
Analysis of flavonoid substance in cotton plants for resistance to pests by HPLC
棉株中抗虫物质黄酮类化合物的高效液相色谱分析
Interaction of ferulic acid with glutathione- S-transferase and carboxylesterase genes in the brown planthopper, Nilaparvata lugens
DOI:10.1007/s10886-016-0812-x URL [本文引用: 1]
The diversity and role of glutathione- S-transferase in insecticide resistance in insects
昆虫谷胱甘肽-S-转移酶的多样性及其介导的抗药性
Host plant induction of glutathione- S-transferase in the fall armyworm
DOI:10.1016/0048-3575(82)90092-X URL [本文引用: 1]
Interactions of allelochemicals with detoxication enzymes of insecticide-susceptible and resistant fall armyworms [ Spodoptera frugiperda, host plants]
DOI:10.1016/0048-3575(84)90010-5 URL [本文引用: 1]
Induction of new glutathione- S-transferase isozymes by allelochemicals in the fall armyworm
DOI:10.1006/pest.1999.2402 URL [本文引用: 1]
Fitness differentiation in Aphids gossypii populations to cotton and cucumber
不同棉蚜种群对棉花和黄瓜的适合度分化
Defense function analysis of three host secondary substances for different biotypes of Sitobion avenae. Master dissertation.
三种寄主次生物质对不同生物型麦长管蚜的防御功能分析
The biotypes and host shifts of cotton-melon aphis Aphis gossypii in northern China
DOI:10.1016/S2095-3119(17)61817-3 URL [本文引用: 1]
The regulation mechanisms of population density of Buchnera aphidicola in Aphis gossypii Glover. Doctoral dissertation.
棉蚜体内原生共生菌Buchnera aphidicola种群密度的调控机制
