桃蛀果蛾雌雄虫不同发育时期嗅觉基因的表达水平差异

doi:10.13304/j.nykjdb.2023.0338

摘要/Abstract

摘要：

桃蛀果蛾是严重隐蔽危害的蛀果害虫，亟待开发环境友好型防治方法，昆虫行为调控技术被认为是新型绿色的害虫防控手段，明晰嗅觉识别机制是开发行为调控剂的基础。基于桃蛀果蛾触角转录组得到242个嗅觉基因，基于序列比对和近缘物种相似基因的功能背景，选取了13个嗅觉基因，采用RT-qPCR进一步分析其潜在识别功能。结果表明，13个嗅觉基因中有6个基因在雌雄虫不同发育时期的表达水平存在显著差异（ P<0.05），推测这6个基因与成虫期嗅觉识别行为相关；根据雌雄成虫初羽化、成熟未交尾和交尾后基因表达水平的差异推测， OBP11可能在识别性信息素和交配行为中发挥重要作用，OR45可能与雌虫识别寄主植物和产卵过程相关，IR5可能与交配和产卵行为相关。以上研究结果为解析桃蛀果蛾嗅觉识别机制奠定了基础，同时也为开发新型昆虫行为调控剂、构建环境友好的害虫治理策略提供了思路。

关键词: 桃蛀果蛾, 转录组, 嗅觉基因, 触角, RT-qPCR

Abstract:

Carposina sasakii Matsumura is a notable fruit pest that damages many types of fruit trees. Behavior regulation technology has gradually gained attraction as a potential pest control method that is both environmentally conscious and effective. The insect behavioral mechanism of chemosensory perception is the basis of behavior regulation. A total of 242 olfactory genes were identified in the antenna transcriptome of the C. sasakii. Based on sequence alignment and functional background of similar genes in related species， 13 olfactory genes were selected and their potential recognition functions were analyzed using RT-qPCR. The results showed that 6 genes differentially expressioned between males and females at different developmental stages （ P<0.05）， which corresponded to the behavioral phenotypes of adults. According to the difference expression levels between male and female adults during newly emerged， mature unmated and mature mated， OBP11 might play a role in detecting pheromones and mating behavior， OR45 might correspond to host-seeking and oviposition activities and IR5 was associated with mating and oviposition behavior. Above results laid theoretical molecular foundation for the identification of C. sasakii pheromone and olfactory-related genes and supported the implementation of behavioral regulators against the olfactory perception mechanism of C. sasakii as a more environmentally sustainable pest control strategy.

Key words: Carposina sasakii, transcriptome, chemosensory genes, antennae, RT-qPCR

中图分类号:

张倩, 门丽娜, 李一然, 刘巧, 胡晓雯, 张宇宏, 张志伟, 张伟. 桃蛀果蛾雌雄虫不同发育时期嗅觉基因的表达水平差异[J]. 中国农业科技导报, 2024, 26(8): 151-162.

Qian ZHANG, Lina MEN, Yiran LI, Qiao LIU, Angie DENG, Xiaowen HU, Yuhong ZHANG, Zhiwei ZHANG, Wei ZHANG. Differential Expression Paradigm of Chemoreceptor Genes Between Males and Females at Different Developmental Stages of Carposina sasakii Matsumura[J]. Journal of Agricultural Science and Technology, 2024, 26(8): 151-162.

图/表 9

参考文献 43

1	LIU B, ZHAN G P, REN L L, et al.. Toxicity of pure phosphine to Carposina sasakii Matsumura (Lepidoptera: Carposinadae) [J]. Plant Prot., 2016, 42( 6): 191- 196.
2	KIM D S, LEE J H, YIEM M S. Temperature-dependent development of Carposina sasakii (Lepidoptera: Carposinidae) and its stage emergence models [J]. Environ. Entomol., 2001, 30( 2): 298- 305.
3	HUA B Z, ZENG X H, ZHANG H. Influences of apple maturity on the development and diapause of Carposina sasakii Matsumura [J]. J. Northwest A&F Univ., 1996, 24( 6): 42- 45.
4	FENG D D, XUE Q Q, MEN L N, et al.. Demography and mass-rearing of Carposina sasakii Matsumura (Lepidoptera: Carposinidae) reared on golden delicious and red fuji apples in the laboratory [J]. J. Asia-Pac. Entomol., 2020, 23( 4): 1194- 1201.
5	ZHANG Z W, LI X W, XUE Y H, et al.. Increased trapping efficiency for the peach fruit moth Carposina sasakii (Matsumura) with synthetic sex pheromone [J]. Agric. For. Entomol., 2017, 19( 4): 424- 432.
6	YANG H B, DONG J F, SUN Y L, et al.. Antennal transcriptome analysis and expression profiles of putative chemosensory soluble proteins in Histia rhodope Cramer (Lepidoptera: Zygaenidae) [J/OL]. CBP, Part D: Genomics Proteomics, 2020, 33: 100654 [ 2023-03-25]. .
7	HU P, WANG J Z, CUI M M, et al.. Antennal transcriptome analysis of the asian longhorned beetle Anoplophora glabripennis [J/OL]. Sci. Rep., 2016, 6: 26652 [ 2023-03-25]. .
8	CHOO Y M, XU P X, HWANG J K, et al.. Reverse chemical ecology approach for the identification of an oviposition attractant for culex quinquefasciatus [J]. Proc. Natl. Acad. Sci. USA, 2018, 115: 714- 719.
9	KEPCHIA D, MOLIVER S, CHOHAN K, et al.. Inhibition of insect olfactory behavior by an airborne antagonist of the insect odorant receptor co-receptor subunit [J/OL]. PLoS One, 2017, 12( 8):e 0183009 [ 2023-03-25]. .
10	CAO S, SUN D D, LIU Y, et al.. Mutagenesis of odorant coreceptor orco reveals the distinct role of olfaction between sexes in Spodoptera frugiperda [J/OL]. J. Integr. Agric., 2022, 11: 004 [ 2023-03-25]. .
11	GRABHERR M G, HAAS B J, YASSOUR M, et al.. Full length transcriptome assembly from RNA seq data without a reference genome [J]. Nat. Biotechnol., 2011, 29: 644- 652.
12	TRAPNELL C, WILLIAMS B A, PERTEA G, et al.. Transcript assembly and quantification by RNA seq reveals unannotated transcripts and isoform switching during cell differentiation [J]. Nat. Biotechnol., 2010, 28( 5): 511- 515.
13	LI B, DEWEY C N. RSEM: accurate transcript quantification from RNA-seq data with or without a reference genome [J/OL]. BMC Bioinf., 2011, 12: 323 [ 2023-03-25]. .
14	WANG E H, WANG X Q, LI H T, et al.. Effective extraction of total RNA from Carposina sasakii and the clone of the gene β⁃ actin [J]. J. Shenyang Agric. Univ., 2014, 45( 4): 403- 407.
15	SCHMITTGEN T D, LIVAK K J. Analyzing real-time PCR data by the comparative C(T) method [J]. Nat. Protocol, 2008, 3( 6): 1101- 1108.
16	LAN X N, XIANG S S, ZHU H. Research progress of the types and functions of insect antennal sensilla [J]. J. Environ. Entomol., 2023, 45( 5): 1197- 1216..
17	CHENG J, WANG C Y, LYU Z H, et al.. Candidate olfactory genes identified in Heortia vitessoides (Lepidoptera: Crambidae) by antennal transcriptome analysis [J]. CBP, Part D: Genomics Proteomics, 2019, 29: 117- 130.
18	BRITO N F, MOREIRA M F, MELO A C. A look inside odorant-binding proteins in insect chemoreception [J]. J. Insect Physiol., 2016, 95: 51- 65.
19	LEAL W S. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes [J]. Annu. Rev. Entomol., 2013, 58: 373- 391.
20	LIU N Y, ZHANG T, YE Z F, et al.. Identification and characterization of candidate chemosensory gene families from Spodoptera exigua developmental transcriptomes [J]. Int. J. Biol. Sci., 2015, 11( 9): 1036- 1048.
21	TIAN Z Q, SUN L N, LI Y Y, et al.. Antennal transcriptome analysis of the chemosensory gene families in Carposina sasakii (Lepidoptera: Carposinidae) [J/OL]. BMC Genomics, 2018, 19: 544 [ 2023-03-25]. .
22	ZHOU S S, SUN Z, MA W H, et al.. De novo analysis of the Nilaparvata lugens (Stål) antenna transcriptome and expression patterns of olfactory genes [J]. CBP, Part D: Genomics Proteomics, 2014, 9: 31- 39.
23	ANTONY B, JOHNY J, ALDOSARI S A. Silencing the odorant binding protein RferOBP1768 reduces the strong preference of palm weevil for the major aggregation pheromone compound ferrugineol [J/OL]. Front. Physiol., 2018, 9: 252 [ 2023-03-25]. .
24	ZHANG Y Y, GUO J M, WEI Z Q, et al.. Identification and sex expression profiles of olfactory-related genes in Mythimna loreyi based on antennal transcriptome analysis [J/OL]. J. Insect Sci., 2022, 25( 3): 101934 [ 2023-03-25]. .
25	LIU P J, ZHANG X F, MENG R J, et al.. Identification of chemosensory genes from the antennal transcriptome of Semiothisa cinerearia [J/OL]. PLoS One, 2020, 15( 8):e 0237134 [ 2023-03-25]. .
26	PELOSI P, IOVINELLA I, FELICIOLI A, et al.. Soluble proteins of chemical communication: an overview across arthropods [J/OL]. Front. Physiol., 2014, 5: 320 [ 2023-03-25]. .
27	PELOSI P, IOVINELLA I, ZHU J, et al.. Beyond chemoreception: diverse tasks of soluble olfactory proteins in insects [J]. Biol. Rev. Cambridge Philos. Soc., 2018, 93: 184- 200.
28	ZHANG Y, FENG K, MEI R L, et al.. Analysis of the antennal transcriptome and identification of tissue-specific expression of olfactory-related genes in Micromelalopha troglodyta (Lepidoptera: Notodontidae) [J/OL]. J. Insect Sci., 2022, 22( 5): 8 [ 2023-03-25]. .
29	HA T S, SMITH D P. Odorant and pheromone receptors in insects [J/OL]. Front. Cell. Neurosci., 2009, 3: 10 [ 2023-03-25]. .
30	WICHER D, MIAZZI F. Functional properties of insect olfactory receptors: ionotropic receptors and odorant receptors [J]. J. Cell Tissue Res., 2021, 383( 1SI): 7- 19.
31	HU P, TAO J, CUI M M, et al.. Antennal transcriptome analysis and expression profiles of odorant binding proteins in Eogystia hippophaecolus (Lepidoptera: Cossidae) [J/OL]. BMC Genomics, 2016, 17: 651 [ 2023-03-25]. .
32	NIE H, XU S P, XIE C Q, et al.. Comparative transcriptome analysis of Apis mellifera antennae of workers performing different tasks [J]. Mol. Genet. Genomics, 2018, 293: 237- 248.
33	LIU Y P, DU L X, ZHU Y, et al.. Identification and sex-biased profiles of candidate olfactory genes in the antennal transcriptome of the parasitoid wasp Cotesia vestalis [J/OL]. CBP, Part D: Genomics Proteomics, 2020, 34: 100657 [ 2023-03-25]. .
34	CLAUDIANOS C, LIM J, YOUNG M, et al.. Odor memories regulate olfactory receptor expression in the sensory periphery [J]. Eur. J. Neurosci., 2014, 39: 1642- 1654.
35	WANG S N, PENG Y, LU Z Y, et al.. Identification and expression analysis of putative chemosensory receptor genes in microplitis mediator by antennal transcriptome Screening [J]. Int. J. Biol. Sci., 2015, 11( 7): 737- 751.
36	XIA Q Y, ZHOU Z Y, LU C, et al.. A draft sequence for the genome of the domesticated silkworm ( Bombyx mori) [J]. Science, 2004, 306( 5703): 1937- 1940.
37	ZHAO H T, GAO P F, ZHANG G X, et al.. Expression and localization analysis of the odorant receptor gene Orco in drones antennae of Apis cerana cerana [J]. Sci. Agric. Sin., 2015, 48( 4): 796- 803.
38	SAXENA K N, GOYAL S. Host-plant relations of the citrus butterfly Papilio demoleus L.: Orientational and ovipositional responses [J]. Entomol. Exp. Appl., 1978, 24( 1): 1- 10.
39	RYTZ R, CROSET V, BENTON R. Ionotropic receptors (IRs): chemosensory ionotropic glutamate receptors in Drosophila and beyond Insect [J]. Insect Biochem. Mol. Biol., 2013, 43( 9): 888- 897.
40	HU J, WANG X Y, TAN L S, et al.. Identification of chemosensory genes, including candidate pheromone receptors, in Phauda flammans (Walker) (Lepidoptera: Phaudidae) through transcriptomic analyses [J/OL]. Front. Physiol., 2022, 13: 907694 [ 2023-03-25]. .
41	POUDEL S, KIM Y, KIM Y T, et al.. Gustatory receptors required for sensing umbelliferone in Drosophila melanogaster [J]. Insect Biochem. Mol. Biol., 2015, 66: 110- 118.
42	SCOTT K, BRADY R J, CRAVCHIK A, et al.. A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila [J]. Cell, 2001, 104: 661- 673.
43	CHANG X Q, NIE X P, ZHANG Z, et al.. De novo analysis of the oriental armyworm Mythimna separata antennal transcriptome and expression patterns of odorant-binding proteins [J]. CBP, Part D: Genomics Proteomics, 2017, 22: 120- 130.

Gene	Forward primer （5’-3’）	Reverse primer （5’-3’）
obp11	AAAATGCTGTCCCTCCTGCC	GGCATAAGTCAGAGCCACCT
pbp2	TGGATCCTGATGGCAAGCTG	GCCATCTTTGAAGCAGCGAG
pbp3	AAACGCTGTCTCCTTGTCGG	TCCGACCTCCTTGCTTACCA
csp8	GTCCGGCTCTGCAAGATGTA	AGAGGACGCCACAAATCTCG
or2	CGCCTCTTCTGAACCGTCAT	CACGCTCACTCTACTCGCAT
or5	GTCTCGCAGCTCCCATACAA	CATTACGGCCTCTACCGTGG
or14	TGAGCATAAGATCCCGACGC	TGCTTTTTCTCGTTCACCTGT
or21	AGCAGATGGAGAATCCAGCG	TCGCAACCATTCCCGTTGTA
or45	GGAACAAACGCAGACCAACA	GGGATGAAAGGTGGCAGGAG
or48	AAGCAGAGCAGTAAAGCGGA	TATGCCAGCGCCAAGAGATT
ir1	ACGCTTTGTTGGAGTGACCA	ATATTCGGGCACGTCAGGTC
ir5	CGGCAGAAAGGGAAGAGGTT	AAAGCCGGTGAGGACTAACG
ir7	ACCCTGTTAGCCGCATCAAA	CCCAAGGGTCACAATCGACA

Gene	Forward primer （5’-3’）	Reverse primer （5’-3’）
obp11	AAAATGCTGTCCCTCCTGCC	GGCATAAGTCAGAGCCACCT
pbp2	TGGATCCTGATGGCAAGCTG	GCCATCTTTGAAGCAGCGAG
pbp3	AAACGCTGTCTCCTTGTCGG	TCCGACCTCCTTGCTTACCA
csp8	GTCCGGCTCTGCAAGATGTA	AGAGGACGCCACAAATCTCG
or2	CGCCTCTTCTGAACCGTCAT	CACGCTCACTCTACTCGCAT
or5	GTCTCGCAGCTCCCATACAA	CATTACGGCCTCTACCGTGG
or14	TGAGCATAAGATCCCGACGC	TGCTTTTTCTCGTTCACCTGT
or21	AGCAGATGGAGAATCCAGCG	TCGCAACCATTCCCGTTGTA
or45	GGAACAAACGCAGACCAACA	GGGATGAAAGGTGGCAGGAG
or48	AAGCAGAGCAGTAAAGCGGA	TATGCCAGCGCCAAGAGATT
ir1	ACGCTTTGTTGGAGTGACCA	ATATTCGGGCACGTCAGGTC
ir5	CGGCAGAAAGGGAAGAGGTT	AAAGCCGGTGAGGACTAACG
ir7	ACCCTGTTAGCCGCATCAAA	CCCAAGGGTCACAATCGACA

Sample	ReadSum	BaseSum	GC/%	Q20/%	Cycle Q20/%	Q30/%
1M	34 008 437	8 564 069 432	45.38	91.76	99.60	85.84
2M	31 812 028	8 012 439 316	44.85	91.98	99.60	86.20
3M	31 090 051	7 829 911 646	45.09	91.84	99.60	86.03
1F	31 277 367	7 875 117 126	45.14	91.85	99.60	85.99
2F	25 584 959	6 443 563 720	45.03	92.56	99.60	86.86
3F	25 864 927	6 513 965 846	44.78	91.67	99.60	85.55

Sample	ReadSum	BaseSum	GC/%	Q20/%	Cycle Q20/%	Q30/%
1M	34 008 437	8 564 069 432	45.38	91.76	99.60	85.84
2M	31 812 028	8 012 439 316	44.85	91.98	99.60	86.20
3M	31 090 051	7 829 911 646	45.09	91.84	99.60	86.03
1F	31 277 367	7 875 117 126	45.14	91.85	99.60	85.99
2F	25 584 959	6 443 563 720	45.03	92.56	99.60	86.86
3F	25 864 927	6 513 965 846	44.78	91.67	99.60	85.55

Length range/bp	Unigenes	Percentage of all unigenes/%
200~300	36 325	42.48
301~500	22 047	25.79
501~1 000	12 611	14.75
1 001~2 000	7 243	8.47
2 000+	7 275	8.51
Total number	85 501
Total length/bp	62 718 690
N50 length/bp	1 515
Mean length/bp	733.543 4