枸杞棉蚜抗、感吡虫啉品系转录组比较分析

doi:10.7668/hbnxb.20195087

摘要/Abstract

摘要：

为挖掘枸杞棉蚜对杀虫剂吡虫啉产生抗药性的候选基因,以枸杞棉蚜对吡虫啉的室内抗药性品系和相对敏感性品系为材料,利用Illumina HiSeq 2500高通量测序技术获得2个品系的转录组数据,通过NCBI数据库进行基因注释,在转录水平上对2个品系的差异基因GO功能及KEGG代谢途径等生物信息学进行分析,采用qRT-PCR技术检测其中8个候选差异基因(CYP6a2、CYP6a13、CYP6k1、CYP6j1、CYP4c1、AChE2、CarE和ALP3)的相对表达情况,并分析了抗药性相关基因的进化关系。经测序及序列拼接,共获得70 101条Unigene(单基因簇),平均长度为654.37 bp,在NR、GO和KEGG数据库中分别注释到29 131,27 861,2 993条Unigene。通过对两品系的差异基因进行NR注释分析,共发现289个差异基因,其中,22个可能与抗药性相关,包括9个解毒酶系基因(CYP6a13、CYP6k1、CYP6j1、CYP4c1和ALP3各1个,CYP6a2和CarE各2个),8个表皮蛋白基因(CP)及其前体(Cuticular protein precursor,CPP),1个靶标酶系基因(Alkaline phosphatase,AChE2),2个转录因子基因(WRKY1、LZTFL1,Leucine zipper transcription factor-like protein 1 gene),1个胰脂肪酶相关蛋白2基因(PLRP2)和1个多抗相关蛋白基因(Multidrug resistance-associated protein,MRP)。定量结果表明,差异基因CYP6a2、CYP6a13、CYP6k1、CYP6j1、CarE和ALP3在抗药性品系的表达量均显著高于敏感性品系。对抗药性相关重要基因CYP、GST、ALP和CP构建系统进化树,通过分析基因的遗传关系,发现枸杞棉蚜与大豆蚜、豌豆蚜的亲缘关系相对较近。获得了枸杞棉蚜对吡虫啉的抗药性与敏感性品系转录组的整体表达模式,筛选到抗药性相关差异表达基因,发现2个品系中87.50%的候选差异基因在转录水平和mRNA水平的表达量变化一致。

关键词: 枸杞棉蚜, 吡虫啉, 抗药性, 敏感性, 转录组, 差异表达基因

Abstract:

To explore candidate genes for resistance of Aphis gossypii to the insecticide imidacloprid,the transcriptome data of the two strains were obtained and compared with imidacloprid indoor resistant and relatively sensitive lines of Aphis gossypii by using Illumina HiSeq 2500 high-throughput sequencing technology.The gene annotation was performed using the NCBI database,and bioinformatics analysis of the differential genes at the transcriptional level included GO function,KEGG metabolic pathway,and other analyses.The relative expression of eight candidate differentially expressed genes(CYP6a2,CYP6a13,CYP6k1,CYP6j1,CYP4c1,AChE2,CarE and ALP3)was detected using qRT-PCR technology,and the evolutionary relationships of resistance related genes were analyzed.After sequencing and sequence splicing,a total of 70 101 Unigenes were obtained,with an average length of 654.37 bp.29 131,27 861 and 2 993 Unigenes were annotated in NR,GO and KEGG databases,respectively.According to the NR annotation analysis of the differential genes of insecticide resistance and sensitivity strains,a total of 22 differential genes that may be related to insecticide resistance were found,including 9 detoxification enzyme genes(CYP6a13,CYP6k1,CYP6j1,CYP4c1 and ALP3 each,two CYP6a2,two CarE),8 cuticle protein genes(CP)and their precursors(CPP),target enzyme genes(AChE2),2 transcription factors(WRKY1,leucine zipper transcription factor-like protein 1 gene,LZTFL1),one pancreatic lipase-related protein 2 gene(PLRP2)and one multidrug resistance-associated protein gene(MRP).The results of qRT-PCR indicated that the expression levels of CYP6a2,CYP6a13,CYP6k1,CYP6j1,CarE and ALP3 genes in insecticide resistance strains were significantly higher than those in sensitive strains.The phylogenetic tree analysis of CYP,ALP,GST and CP genes obtained by NR annotation indicated that the genetic relationship between the Aphis gossypii and Aphis glycines and Acyrthosiphon pisum was relatively close.We found that 87.50% of the candidate differentially expressed genes in the two strains showed consistent changes in expression levels at the transcriptional and mRNA levels.

Key words: Aphis gossypii, Imidacloprid, Insecticide resistance, Insecticide sensitive, Transcriptome, Differentially expressed gene

王新霞, 张慧斌, 刘雲祥, 刘思雨, 来有鹏, 李秋荣. 枸杞棉蚜抗、感吡虫啉品系转录组比较分析[J]. 华北农学报, 2025, 40(1): 177-188. doi: 10.7668/hbnxb.20195087.

WANG Xinxia, ZHANG Huibin, LIU Yunxiang, LIU Siyu, LAI Youpeng, LI Qiurong. Comparative Transcriptome Analysis of Resistant and Sensitive Strains to Imidacloprid of Aphis gossypii(Hemiptera:Aphisidae)[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(1): 177-188. doi: 10.7668/hbnxb.20195087.

http://www.hbnxb.net/CN/Y2025/V40/I1/177

图/表 11

表1 qRT-PCR扩增内参及候选差异基因的引物序列

Tab.1 Sequences of primers for qRT-PCR amplification of candidate differential protein genes and internal reference genes

基因名称 Gene name	引物名称 Primer name	序列(5'—3') Sequence(5'—3')	退火温度/℃ Annealing temperature
18S rRNA	上游引物	ATTGACGGAAGGGCACC	57
	下游引物	CGCTCCACCAACTAAGAACG
CYP6a2	上游引物	ACCTTGGAGGAACCGATACAC	58
	下游引物	TAAGATGTTGACAGACGGATGAGT
CYP6a13	上游引物	CTTTTGGAATGTTTGCTGCTG	58
	下游引物	GGAGGATACTTTCGGAGTGTTT
AChE2	上游引物	TCGGTTTCACTACACTTGCTATCT	58
	下游引物	TGCTTTTGCTAGTTTAAGTCCTCT
CarE	上游引物	AGGCATACCCTACGCTCAAC	57
	下游引物	TTCTGCTGTGGCACCGATA
ALP3	上游引物	GAATCGGAAGTGGGAATGC	57
	下游引物	TGCTTGTCGTCTATCCACTCG
CYP6k1	上游引物	AGAACTCGGACATTGAAGAT	61
	下游引物	TGAACCAGCAGTGAACAT
CYP6j1	上游引物	ACCCAACTCAGATTTCACCCT	58
	下游引物	TTCCGAACCGCATACCAA
CYP4c1	上游引物	TAAACATTCAGCAGGTGGGC	59
	下游引物	TGCGGTTCCATTTGTAGTGAC

图1 比较组表达差异火山图横轴为基因在不同组样本间的表达差异倍数Fold Change(log₂(B/A))值,纵轴为代表基因表达量变化的统计学显著程度P value,P value越小,-log₁₀(P value)越大,差异越显著。图中每个点代表一个基因,其中,红色表示上调基因,绿色表示下调基因,黑色表示非差异基因。

Fig.1 Comparison group expression differences volcano diagram The horizontal axis represents the Fold Change(log₂(B/A))value of gene expression differences between different groups of samples,while the vertical axis represents the statistical significance level P value of gene expression changes.The smaller the P value,the greater the -log₁₀(P value),and the more significant the difference.Each point in the graph represents a gene,where red represents upregulated genes,green represents downregulated genes,and black represents non differentially expressed genes.

图2 枸杞棉蚜抗感品系DEGs的GO功能分类浅色代表差异基因,深色代表所有基因。1.多生物体过程;2.节律过程;3.多细胞生物过程;4.生殖过程;5.生殖;6.细胞过程;7.细胞杀伤;8.发育过程;9.解毒;10.生物过程的负调控;11.代谢过程;12.细胞成分组织或生物发生;13.刺激反应;14.生物过程调节;15.行为;16.生物调节;17.生长;18.免疫系统过程;19.生物过程的正向调节;20.移动;21.定位;22.信号;23.定位的建立;24.生物附着;25.生物相;26.细胞聚集;27.细胞器;28细胞器部分;29.膜封闭管腔;30.超分子纤维;31.胞外区部分;32.病毒粒子部分;33.病毒粒子;34.含蛋白复合物;35.细胞外区;36.其他生物体;37.其他生物体部分;38.细胞部分;39.细胞外基质;40.细胞;41.膜;42.膜部分;43.细胞连接;44.类核细胞外;45.矩阵成分;46.突触部分;47.突触;48.共质体;49.结构分子活性;50.酶调节活性;51.结合活性;52.分子功能调节剂;53.催化活性;54.电子转移活性;55.转录因子活性,蛋白结合;56.转运蛋白活性;57.信号转换器活动;58.形态发生活性;59.抗氧化活性;60.通道调节活动;61.金属伴侣活性;62.受体调节活性;63.蛋白标签;64.趋化活性;65.翻译调节器活动;66.化学驱避活性;67.营养储存库活动;68.分子传感器活性。

Fig.2 GO function categorization of DEGs between ApRS and ApSS The light color represents the differential genes,and dark on behalf of all genes.1.Multi-organism process; 2.Rhythmic process; 3.Multicellular organismal process; 4.Reproductive process; 5.Reproduction; 6.Cellular process; 7.Cell killing; 8.Developmental process; 9.Detoxification; 10.Negative regulation of biological process; 11.Metabolic process; 12.Cellular component organization or biogenesis; 13.Response to stimulus; 14.Regulation of biological process; 15.Behavior; 16.Biological regulation; 17.Growth; 18.Immune system process; 19.Positive regulation of biological process; 20.Locomotion; 21.Localization; 22.Signaling; 23.Establishment of localization; 24.Biological adhesion; 25.Biological phase; 26.Cell aggregation; 27.Organelle; 28.Organelle part; 29.Membrane enclosed lumen; 30.Supramolecular fiber; 31.Extracellular region part; 32.Virion part; 33.Virion; 34.Protein containing complex; 35.Extracellular region; 36.Other organism; 37.Other organism part; 38.Cell part; 39.Extracellular matrix; 40.Cell; 41.Membrane; 42.Membrane part; 43.Cell junction; 44.Nucleoid extracellular; 45.Matrix component; 46.Synapse part; 47.Synapse; 48.Symplast; 49.Structural molecule activity; 50.Enzyme regulator activity; 51.Binding; 52.Molecular function regulator; 53.Catalytic activity; 54.Electron transfer activity; 55.Transcription factor activity,protein binding; 56.Transporter activity; 57.Signal transducer activity; 58.Morphogen activity; 59.Antioxidant activity; 60.Channel regulator activity; 61.Metallochaperone activity; 62.Receptor regulator activity; 63.Protein tag; 64.Chemoattractant activity; 65.Translation regulator activity; 66.Chemorepellent activity; 67.Nutrient reservoir activity; 68.Molecular transducer activity.

表2 枸杞棉蚜抗感品系差异基因的GO富集分析结果

Tab.2 GO enrichment of DEGs between ApRS and ApSS

品系名称 Name of strains	分子功能 Molecular function	生物学过程 Biological process	细胞组分 Cellular component
抗药性品系	GO:0042302角质层的结构成分	GO:0014075胺反应	GO:0000323裂解液泡
ApRS	GO:0004197半胱氨酸型内肽酶活性	GO:0071260细胞对机械刺激的反应	GO:0005764溶酶体
	GO:0030984激肽原结合	GO:0070670对白细胞介素-4的反应	GO:0016324顶端质膜
	GO:0043621蛋白质自结合	GO:0009612机械刺激反应	GO:0042383肌膜
	GO:0008234半胱氨酸型肽酶活性	GO:0045471乙醇的反应	GO:0005773液泡
	GO:0015020葡糖羧酸基转移酶	GO:0007519骨骼肌组织发育	GO:0009897质膜外侧
	GO:0042277肽结合	GO:0071214细胞对非生物刺激的反应	GO:0045177细胞顶端
	GO:0008194 UDP糖基转移酶活性	GO:0104004细胞对环境刺激的反应	GO:0042470黑素体
	GO:0005198结构分子活性	GO:0009749葡萄糖反应	GO:0048770色素粒
	GO:0033218酰胺结合	GO:0009611伤害反应	GO:0098552薄膜侧面
	GO:0070011肽酶活性,作用于L-氨基酸肽	GO:0009746对己糖的反应	GO:0044421胞外区部分
	GO:0008233肽酶活性	GO:0060538骨骼肌器官发育	GO:0005576胞外区
	GO:0016758转移酶活性,转移己糖基	GO:0034284单糖的反应	GO:0005615胞外区
	GO:0004175内肽酶活性	GO:0097305酒精反应	GO:0098590质膜区
	GO:0008239二肽基肽酶活性	GO:0009743碳水化合物反应	GO:0044425膜部分
	GO:0016757转移酶活性,转移糖基	GO:0009636对有毒物质的反应	GO:0005968蛋白-香叶基转移酶复合物
	GO:0004177氨肽酶活性	GO:0014706横纹肌组织发育	GO:0005739线粒体
	GO:0003968 RNA导向的5'—3'RNA聚合酶活性	GO:0060537肌肉组织发育	GO:0035060布拉复合体
	—	GO:0043434肽激素反应	GO:0044444细胞质部分
	—	GO:0034097对细胞因子的反应	GO:0031982囊泡
敏感性品系	GO:0003676核酸结合	GO:0090304核酸代谢过程	GO:0005634细胞核
ApSS	GO:0003968 RNA导向的5'—3'RNA聚合酶活性	GO:0039694病毒核糖核酸基因组复制	GO:0044428核部分
	—	GO:0006139含碱基化合物代谢过程	GO:0043229胞内细胞器
	—	GO:0046483杂环代谢过程	GO:0031981核腔
	—	GO:0006725芳香族化合物的细胞代谢过程	GO:0043226细胞器
	—	GO:0019079病毒基因组复制	GO:0044446胞内细胞器部分
	—	GO:1901360有机环状化合物代谢过程	GO:0043227膜结合细胞器
	—	GO:0006259DNA代谢过程	GO:0044422细胞器部分
	—	—	GO:0043231细胞内膜结合细胞器
	—	—	GO:0070013细胞器内腔
	—	—	GO:0031974膜封闭腔
	—	—	GO:0043233细胞器腔
	—	—	GO:0005730核仁
	—	—	GO:0043232细胞内无膜细胞器
	—	—	GO:0043228无膜细胞器
	—	—	GO:0015630微管细胞骨架
	—	—	GO:0044424胞外部分

表2 枸杞棉蚜抗感品系差异基因的GO富集分析结果

Tab.2 GO enrichment of DEGs between ApRS and ApSS

品系名称 Name of strains	分子功能 Molecular function	生物学过程 Biological process	细胞组分 Cellular component
抗药性品系	GO:0042302角质层的结构成分	GO:0014075胺反应	GO:0000323裂解液泡
ApRS	GO:0004197半胱氨酸型内肽酶活性	GO:0071260细胞对机械刺激的反应	GO:0005764溶酶体
	GO:0030984激肽原结合	GO:0070670对白细胞介素-4的反应	GO:0016324顶端质膜
	GO:0043621蛋白质自结合	GO:0009612机械刺激反应	GO:0042383肌膜
	GO:0008234半胱氨酸型肽酶活性	GO:0045471乙醇的反应	GO:0005773液泡
	GO:0015020葡糖羧酸基转移酶	GO:0007519骨骼肌组织发育	GO:0009897质膜外侧
	GO:0042277肽结合	GO:0071214细胞对非生物刺激的反应	GO:0045177细胞顶端
	GO:0008194 UDP糖基转移酶活性	GO:0104004细胞对环境刺激的反应	GO:0042470黑素体
	GO:0005198结构分子活性	GO:0009749葡萄糖反应	GO:0048770色素粒
	GO:0033218酰胺结合	GO:0009611伤害反应	GO:0098552薄膜侧面
	GO:0070011肽酶活性,作用于L-氨基酸肽	GO:0009746对己糖的反应	GO:0044421胞外区部分
	GO:0008233肽酶活性	GO:0060538骨骼肌器官发育	GO:0005576胞外区
	GO:0016758转移酶活性,转移己糖基	GO:0034284单糖的反应	GO:0005615胞外区
	GO:0004175内肽酶活性	GO:0097305酒精反应	GO:0098590质膜区
	GO:0008239二肽基肽酶活性	GO:0009743碳水化合物反应	GO:0044425膜部分
	GO:0016757转移酶活性,转移糖基	GO:0009636对有毒物质的反应	GO:0005968蛋白-香叶基转移酶复合物
	GO:0004177氨肽酶活性	GO:0014706横纹肌组织发育	GO:0005739线粒体
	GO:0003968 RNA导向的5'—3'RNA聚合酶活性	GO:0060537肌肉组织发育	GO:0035060布拉复合体
	—	GO:0043434肽激素反应	GO:0044444细胞质部分
	—	GO:0034097对细胞因子的反应	GO:0031982囊泡
敏感性品系	GO:0003676核酸结合	GO:0090304核酸代谢过程	GO:0005634细胞核
ApSS	GO:0003968 RNA导向的5'—3'RNA聚合酶活性	GO:0039694病毒核糖核酸基因组复制	GO:0044428核部分
	—	GO:0006139含碱基化合物代谢过程	GO:0043229胞内细胞器
	—	GO:0046483杂环代谢过程	GO:0031981核腔
	—	GO:0006725芳香族化合物的细胞代谢过程	GO:0043226细胞器
	—	GO:0019079病毒基因组复制	GO:0044446胞内细胞器部分
	—	GO:1901360有机环状化合物代谢过程	GO:0043227膜结合细胞器
	—	GO:0006259DNA代谢过程	GO:0044422细胞器部分
	—	—	GO:0043231细胞内膜结合细胞器
	—	—	GO:0070013细胞器内腔
	—	—	GO:0031974膜封闭腔
	—	—	GO:0043233细胞器腔
	—	—	GO:0005730核仁
	—	—	GO:0043232细胞内无膜细胞器
	—	—	GO:0043228无膜细胞器
	—	—	GO:0015630微管细胞骨架
	—	—	GO:0044424胞外部分

表3 枸杞棉蚜抗感品系差异基因显著富集的KEGG途径

Tab.3 Significantly enriched KEGG pathways of DEGs in ApRS and ApSS

表达量变化趋势(抗药性/敏感性) The tendency of the expression quantity (ApRS/ApSS)	KEGG序号 KEGG ID	KEGG 通路 KEGG pathway	P值 P value
上调	ko04210	细胞凋亡	3.59×10^-7
Up-regulation	ko04142	溶酶体	9.76×10^-5
	ko01220	芳香化合物降解	0.012 663
	ko02024	群体感应	0.015 806
	ko04145	吞噬体	0.017 341
	ko00930	己内酰胺降解	0.018 940
	ko00430	牛磺酸和亚牛磺酸代谢	0.025 182
	ko00591	亚麻油酸代谢	0.040 634
下调	ko03450	异源末端连接	0.001 476
Down-regulation	ko03008	真核生物核糖体生物合成	0.003 058
	ko03015	mRNA监控通路	0.033 431
	ko04111	酵母细胞周期	0.034 403

表4 枸杞棉蚜对吡虫啉产生抗药性相关的差异表达基因

Tab.4 DEGs related to imidacloprid-resistance of Aphis gossyppi

序号 No.	单基因簇代码 Unigene No.	NR数据库注释信息 NR database annotation information	基因表达量Gene expression level
序号 No.	单基因簇代码 Unigene No.	NR数据库注释信息 NR database annotation information	Mean TPM (ApSS)	Mean TPM (ApRS)	log₂(Fold Change)	P value
1	DN14313_c0_g1	表皮蛋白12.5	232.66	1 144.08	2.30	9.33E-19
2	DN14592_c0_g3	碱性磷酸酶	0.35	0.15	-1.26	2.61E-03
3	DN15223_c0_g1	细胞色素P450 6a2	5.14	30.68	2.58	3.97E-15
4	DN15826_c1_g2	羧酸酯酶	9.29	1.64	-2.50	4.16E-03
5	DN15840_c1_g2	细胞色素P450 4c1	1.31	0.36	-1.88	5.14E-08
6	DN16071_c0_g1	细胞色素P450 6a13	1.09	4.73	2.11	3.77E-06
7	DN16226_c0_g4	表皮蛋白 CPG12样	300.69	602.05	1.00	7.91E-08
8	DN16730_c2_g6	表皮蛋白5前体	287.49	692.25	1.27	6.91E-08
9	DN17129_c3_g1	乙酰胆碱酯酶2	9.24	2.73	-1.76	7.65E-06
10	DN17367_c1_g2	胰脂肪酶相关蛋白2	21.06	148.19	2.81	1.20E-26
11	DN17671_c3_g4	细胞色素 P450 6a2	58.65	127.53	1.12	6.38E-04
12	DN17788_c3_g6	表皮蛋白28前体	2 297.53	5 538.42	1.27	2.81E-08
13	DN17827_c1_g1	细胞色素P450 6k1	62.22	397.81	2.68	3.29E-26
14	DN18057_c0_g6	表皮蛋白9	91.50	257.82	1.49	1.34E-11
15	DN18487_c0_g2	表皮蛋白7	4.21	58.24	3.79	3.14E-06
16	DN14492_c0_g1	WRKY 转录因子蛋白1	1.41	7.18	2.35	2.51E-03
17	DN13869_c0_g1	亮氨酸拉链转录因子样蛋白1	0.40	7.77	4.29	4.26E-06
18	DN18771_c0_g5	表皮蛋白前体	1 473.43	3 032.33	1.04	5.50E-08
19	DN18771_c0_g4	表皮蛋白7	91.22	185.41	1.02	7.74E-08
20	DN18913_c1_g1	羧酸酯酶	185.55	543.80	1.55	4.38E-03
21	DN18994_c1_g3	多药耐药相关蛋白	2.33	12.90	2.47	1.04E-03
22	DN19253_c0_g4	细胞色素 P450 6j1	11.32	38.38	1.76	1.85E-03

图3 8个Unigenes在枸杞棉蚜抗感品系体内的相对表达量每个基因的显著性差异,均为相同基因在处理组与对照组的表达量进行比较。图中数据为平均值±标准差,不同小写字母表示基因在敏感型品系和抗药型品系中差异显著(P<0.05)。

Fig.3 Relative expression of eight Unigenes in ApRS and ApSS The significant differences of each gene are compared between the expression levels of the same gene in the treatment group and the control group.The data in the figure are mean±s.Different lowercase letters indicate significant differences in genes between sensitive and resistant strains(P<0.05).

图4 基于氨基酸序列构建的枸杞棉蚜CYPs与其他已知昆虫细胞色素P450蛋白的系统发育树 Acra.豆蚜;Agly.大豆蚜;Agos.棉蚜;Apis.豌豆蚜;Dnox.麦双尾蚜;Mper.桃蚜;Msac.高粱蚜;Rmai.玉米蚜;Rpad.禾谷缢管蚜;Sfla.黄伪毛蚜。图5—7同。

Fig.4 Phylogenetic tree of AgosCYPs of Aphis gossypii and CYPs from other different insect species based on amino acid sequences Acra.Aphis craccivora; Agly.Aphis glycines; Agos.Aphis gossypii; Apis.Acyrthosiphon pisum; Dnox.Diuraphis noxia; Mper.Myzus persicae; Msac.Melanaphis sacchari; Rmai.Rhopalosiphum maidis; Rpad.Rhopalosiphum padi; Sfla.Sipha flava.The same as Fig.5—7.

图5 基于氨基酸序列构建的枸杞棉蚜GSTs与其他已知昆虫谷胱甘肽S-转移酶蛋白的系统发育树

Fig.5 Phylogenetic tree of AgosGSTs of Aphis gossypii and GSTs from other different insect species based on amino acid sequences

图6 基于氨基酸序列构建的枸杞棉蚜ALPs与其他已知昆虫碱性磷酸酶蛋白的系统发育树

Fig.6 Phylogenetic tree of AgosALPs of Aphis gossypii and ALPs from other different insect species based on amino acid sequences

图7 基于氨基酸序列构建的枸杞棉蚜CPs与其他已知昆虫表皮蛋白的系统发育树

Fig.7 Phylogenetic tree of AgosCPs of Aphis gossypii and CPs from other different insect species based on amino acid sequences

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