Cloning and tissue-specific expression patterns of a gene encoding an atypical odorant receptor co-receptor in the leaf beetle Diorhabda rybakowi （Coleoptera： Chrysomelidae）

doi:10.11686/cyxb2023265

Abstract

Abstract:

The leaf beetle Diorhabda rybakowi is one of the most important insect pests causing serious damage to the pioneer plant Nitraria spp.， which has windbreak and sand-fixation functions in the desert grasslands of Northwest China. The development and utilization of a chemical method based on insect perception of sex pheromones or host volatiles is a promising pest control strategy. In this study， we obtained an atypical odorant receptor co-receptor （Orco） gene sequence from the antennae of D. rybakowi adults using a molecular cloning method. We predicted the tertiary structure of the DrybOrco protein using homology modeling， constructed a phylogenetic tree， and detected the transcript levels of the DrybOrco gene in different tissues of male and female adults by real-time fluorescence quantitative PCR. It was found that the DrybOrco full-length gene sequence was 1918 bp long， with an open reading frame of 1440 bp encoding 479 amino acids. The putative protein had a predicted molecular weight of 53.94 kD， contained seven transmembrane domains， and was a hydrophobic membrane protein.The phylogenetic analysis revealed high conservation of Orco genes among 68 species of insects in six different orders （similarity >68%）. In the phylogenetic tree， these genes grouped into three branches， with genes from the same insect order clustered on the same branch. The DrybOrco genewas clustered on the branch with sequences from other coleoptera species， and showed the highest nucleotide sequence similarity （92.28%） to the gene from Diabrotica virgifera. The qRT-PCR results showed that there were high transcript levels of the DrybOrco gene in the adult antennae， and with significantly higher levels （2.27 times higher） in the antennae of males than in the antennae of females. Low levels of DrybOrco transcripts were also detected in the legs of male adults and the wings of female adults， but its transcripts were barely detectable or absent from the other sampled tissues. The results of this study reveal the sequence characteristics and transcript profiles of an Orco gene in D. rybakowi adults， and details of the structure and characteristics of the putative protein. These findings provide a theoretical basis for elucidating the physiological function of the DrybOrco protein in chemosensory processes， and for further detailed studies on the olfactory molecular mechanism of host specialization in D. rybakowi.

Key words: Diorhabda rybakowi, olfactory, atypical odorant receptor co-receptor, cloning, tissue expression

Bo-xin XI, Xiao-ning CUI, Su-qin SHANG, Gui-xin HU, Yan WANG, Chang-ning LI, Bin PENG, Xue-qiang SHI. Cloning and tissue-specific expression patterns of a gene encoding an atypical odorant receptor co-receptor in the leaf beetle Diorhabda rybakowi （Coleoptera： Chrysomelidae）[J]. Acta Prataculturae Sinica, 2024, 33(5): 204-215.

Figures/Tables 6

References 66

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引物名称 Primer name	5′→3′引物序列 5′→3′ primer sequence	产物大小 Product size （bp）	试验用途 Use of experiment
Orco-F	GATTAGTGAGTTTAAGATGTCGCA	1918	克隆Cloning
Orco-R	TGGTGTTGATGGAAGTTGG	1918	克隆Cloning
RPLS18-F	GACAAACGTGCGGGAGAA	102	荧光定量PCR Rreal-time PCR
RPLS18-R	CTGTTCAAAAACCAGTCGGG	102	荧光定量PCR Rreal-time PCR
RPL13A-F	CCGTTCCATTTTAGAGCTCCCT	158	荧光定量PCR Real-time PCR
RPL13A-R	CCAGGTACAACAACACGTTTCC	158	荧光定量PCR Real-time PCR
DOrco-F	GGATCCTGAAAACGAGAACG	157	荧光定量PCR Real-time PCR
DOrco-R	CAGATTCGCGTGCATTAGAG	157	荧光定量PCR Real-time PCR

引物名称 Primer name	5′→3′引物序列 5′→3′ primer sequence	产物大小 Product size （bp）	试验用途 Use of experiment
Orco-F	GATTAGTGAGTTTAAGATGTCGCA	1918	克隆Cloning
Orco-R	TGGTGTTGATGGAAGTTGG	1918	克隆Cloning
RPLS18-F	GACAAACGTGCGGGAGAA	102	荧光定量PCR Rreal-time PCR
RPLS18-R	CTGTTCAAAAACCAGTCGGG	102	荧光定量PCR Rreal-time PCR
RPL13A-F	CCGTTCCATTTTAGAGCTCCCT	158	荧光定量PCR Real-time PCR
RPL13A-R	CCAGGTACAACAACACGTTTCC	158	荧光定量PCR Real-time PCR
DOrco-F	GGATCCTGAAAACGAGAACG	157	荧光定量PCR Real-time PCR
DOrco-R	CAGATTCGCGTGCATTAGAG	157	荧光定量PCR Real-time PCR

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[10]	SHAO Lin-Hui, ZHENG Xing-Wei, LI Cong. Cloning and expression analysis of a U-box gene of E3 ubiquitin ligase from Medicago truncatula [J]. Acta Prataculturae Sinica, 2016, 25(7): 62-72.
[11]	CHEN Chun-Yan, MA Hui-Ling, DONG Wen-Ke. Cloning and Expression Analysis of a Leucoanthocyanidin Reductase (LAR) Gene from Onobrychis viciifolia cv.Gansu [J]. Acta Prataculturae Sinica, 2015, 24(6): 177-187.
[12]	GUO Yu-peng. A study on advances in plant photorespiration [J]. Acta Prataculturae Sinica, 2014, 23(4): 322-329.
[13]	LUO Jun,WANG Yin-quan,WEN Sui-chao,LI Jing,ZHANG Jin-lin,XIA Qi. Cloning and tissue-specific expression analysis of phenylalanine ammonia-lyase gene fragment in Angelica sinensis [J]. Acta Prataculturae Sinica, 2014, 23(4): 130-137.
[14]	WANG Jia,ZHENG Lin-lin,GU Tian-pei,WANG Xue-feng,WANG Ying-chun. Cloning and expression analysis of two WRKY transcription factors from the rare recretohalophyte Reaumuria trigyna [J]. Acta Prataculturae Sinica, 2014, 23(4): 122-129.
[15]	ZHANG Yang, GUO Hai-jun, LIU Long-biao, WANG Sa, LIANG Ying, NIE Yu-zhe, LI Yu-hua. Cloning and expression of PtGAPDH from Puccinellia tenuiflora [J]. Acta Prataculturae Sinica, 2014, 23(2): 207-214.