二化螟味觉受体基因鉴定、克隆与表达模式分析

doi:10.3864/j.issn.0578-1752.2023.13.006

Abstract

Abstract:

【Background】The rice striped stem borer, Chilo suppressalis, is one of the important rice pests in China. The gustatory receptors (GRs) play important roles in insect feeding, oviposition, and so on.【Objective】Based on transcriptomic and genomic data, the gustatory receptor gene sequences of C. suppressalis were identified and cloned in this study. Then, their expression characteristics at different developmental stages and in different tissues of adults were clarified. These results will lay a foundation for further study on the function of gustatory receptor genes in C. suppressalis.【Method】Combined with the transcriptome data of C. suppressalis and the GR sequences of other insects, the GR genes in C. suppressalis were identified by multiple sequence alignment. The complete open reading frame (ORF) sequences of the GR genes in C. suppressalis were cloned by RT-PCR, and the molecular biological characteristics and domains of the amino acid sequences encoded by the GR genes in C. suppressalis were analyzed by bioinformatics analysis tools. The phylogenetic tree of GR gene protein sequences of C. suppressalis and other insect GR genes was constructed based on the maximum likelihood method. The expression patterns of GR genes at different developmental stages (1-6 instar larvae, female and male adults) and in different adult tissues (antennae, head, wing, abdomen and leg) of C. suppressalis were determined by real-time quantitative PCR (RT-qPCR).【Result】Five gustatory receptor genes were identified and named as CsupGR1-5. The complete ORFs of these GR genes were between 1 122 and 1 428 bp in length, encoding 373-475 amino acids. Among them, CsupGR2, CsupGR4, CsupGR5 had seven transmembrane domains, CsupGR1, CsupGR3 had eight transmembrane domains. Phylogenetic analysis showed that CsupGR1 and CsupGR2 were closely related to DmelGR63a in Drosophila melanogaster, PxylGR7 and PxylGR8 in Plutella xylostella, belonging to the CO₂ receptor family. CsupGR3 was closely related to DmelGR43a in D. melanogaster, BmorGR9 and BmorGR10 in Bombyx mori, belonging to the fructose/inositol receptor family. CsupGR4 and CsupGR5 were closely related to DmelGR64 in D. melanogaster and BmorGR5-8 in B. mori, belonging to the sugar receptor family. The expression profile analysis showed that the five GR genes were all expressed at different developmental stages in C. suppressalis. Among them, CsupGR1 and CsupGR4 were highly expressed in male adults, CsupGR2 had the highest expression level in the 1st instar larvae and male adults, CsupGR3 was highly expressed in adults, and CsupGR5 had the highest expression level in the 4th instar larvae. Tissue expression profile analysis showed that these five GR genes were expressed in all tissues of male and female adults. CsupGR1 and CsupGR5 were highly expressed in the head of adults, and CsupGR2-4 were highly expressed in the antennae of adults.【Conclusion】The five GR genes of C. suppressalis identified in this study have the typical characteristics of insect gustatory receptors and are highly expressed in the antennae or heads of adults. It is speculated that these five genes may be related to the recognition and adaptation of C. suppressalis to host plants.

Key words: Chilo suppressalis, gustatory receptor (GR), gene identification, sequence analysis, expression profile

HUANG YuXuan, SHEN Chen, JU JiaFei, YANG Lei, LUO GuangHua, FANG JiChao. Identification, Cloning and Expression Profiles of Gustatory Receptor Genes in Chilo suppressalis[J].Scientia Agricultura Sinica, 2023, 56(13): 2504-2517.

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References 49

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	引物用途 Usage of primers
CsGR1-F	ATGTCCTATTATACACATAA	开放阅读框扩增Amplification of ORF
CsGR1-R	TTAATGTTTCCTTTTCTTCT
CsGR2-F	ATGATGATTCCTGATCATTT
CsGR2-R	TCATGAGAGGGCTGCTTGAG
CsGR3-F	ATGTTTGTGGAGAAGCCGGG
CsGR3-R	TTACTGTTCCGAAAGCTGGA
CsGR4-F	GGCTTCGCCCAAGGATCTCC
CsGR4-R	TCAAGCGACAGCGATTACCG
CsGR5-F	ATGAAAACTCAAATCCTGGG
CsGR5-R	TTACTTAATAAATTGCAGCA
CsqGR1-F	AACGCGATAGCACCTTTGGA	实时荧光定量PCR RT-qPCR
CsqGR1-R	AGAAGGTCTGGTGATCGGGA
CsqGR2-F	TGCCGTCATGCCTATCACAA
CsqGR2-R	ACGCTCGTATCCAACGAACA
CsqGR3-F	CGGGGTTCTGCTGTTACTCC
CsqGR3-R	CAGACCACACCACAACACCT
CsqGR4-F	CCAAGTTCACGGGGATACGG
CsqGR4-R	GAAATGGTGGCGGCAAGAAA
CsqGR5-F	GTCAGCAGCGTTGGTCAAAT
CsqGR5-R	GAGCGATACTGACCCTGACG
EF1-F	AAAATGGACTCGACTGAACCCC
EF1-R	TCTCCGTGCCAACCAGAAATA

基因 Gene	登录号 Accession number	开放阅读框长度 ORF length (bp)	氨基酸数量 Number of AA	跨膜结构域数量 Number of TMD	最佳比对结果BLAST best hit
基因 Gene	登录号 Accession number	开放阅读框长度 ORF length (bp)	氨基酸数量 Number of AA	跨膜结构域数量 Number of TMD	物种 Species	登录号 Accession number	E值 E-value	相似度 Similarity (%)
CsupGR1	OQ834263	1428	475	8	亚洲玉米螟O. furnacalis	XP_028179334.1	0	80.62
CsupGR2	OQ834264	1302	433	7	粉纹夜蛾T. ni	XP_026735279.1	0	86.95
CsupGR3	OQ834265	1317	438	8	棉红铃虫P. gossypiella	XP_049878938.1	3.00E-63	31.45
CsupGR4	OQ834266	1122	373	7	黄野螟H. vitessoides	UVB79175.1	7.00E-174	73.70
CsupGR5	OQ834267	1269	422	7	亚洲玉米螟O. furnacalis	XP_028178841.1	0	66.91

		CsupGR1	CsupGR2	CsupGR3	CsupGR4	CsupGR5
分子式Molecular formula		C₂₄₉₁H₃₉₀₈N₆₁₀O₆₇₆S₂₆	C₂₂₈₇H₃₅₄₇N₅₆₇O₆₁₈S₂₄	C₂₂₃₉H₃₅₉₆N₅₆₆O₆₀₈S₂₃	C₁₈₉₉H₂₉₆₈N₄₇₄O₅₁₃S₂₀	C₂₂₇₃H₃₅₅₈N₅₆₀O₅₈₉S₂₄
分子质量Molecular weight (Da)		54051.67	49643.19	48909.99	41288.52	48924.09
等电点Theoretical pI		8.75	7.51	9.28	7.09	9.31
氨基酸含量 Amino acid content	Maximum	Leu 11.2%	Leu 11.8%	Leu 15.8%	Leu 12.3%	Leu 14.7%
	Minimum	Trp 1.1%	Trp 1.4%	Trp 0.9%	Asp 0.8%	Trp 1.2%
	Nonentity	Pyl, Sec	Pyl, Sec	Pyl, Sec	Pyl, Sec	Pyl, Sec
正电荷残基Positively charged residues		40	40	43	21	36
负电荷残基Negatively charged residues		34	39	29	21	23
吸收率Absorptivity		1.205	1.370	1.041	0.884	1.294
不稳定指数Instability index		33.75	33.17	34.53	34.79	38.05
脂肪族指数Aliphatic index		109.18	110.35	120.59	114.48	119.36
疏水性系数的总平均值 Grand average of hydrophobicity coefficient		0.327	0.337	0.522	0.656	0.489

Identification, Cloning and Expression Profiles of Gustatory Receptor Genes in Chilo suppressalis

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