A method for screening a potato beetle olfactory receptor gene

Abstract

The application discloses a screening method of a leucinodes orthonetria olfactory receptor gene, which can effectively screen the olfactory receptor gene sensitive to target host volatiles, and is convenient for in-depth study of the olfactory molecular mechanism of the leucinodes orthonetria. Through the screening method, the olfactory molecular mechanism of the target host volatiles in promoting the mating behavior of the leucinodes orthonetria can be further clarified, a scientific basis for research and development of a technology based on regulation of the mating behavior of the leucinodes orthonetria is provided, and theoretical support for green prevention and control of the leucinodes orthonetria is provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of agricultural pest control, in particular to a screening method of Leptinotarsa decemlineata olfactory receptor gene. BACKGROUND

[0002] Leptinotarsa decemlineata (Say) is one of the most important potato pests in the world, which has been listed in the "List of Quarantine Harmful Organisms of the People's Republic of China" and the "Top 20 Most Dangerous Invasive Species in China". Its harm directly threatens the biological safety, ecological safety and food safety of our country.

[0003] In the natural ecosystem, insects rely on a complex and sensitive olfactory system to support their key navigation in behaviors such as seeking mates, mating, oviposition, foraging and avoiding enemies. At present, the olfactory molecular mechanism of insect mating behavior mainly focuses on the role of pheromones. However, the role of host plant volatiles in insect mating behavior is also important. Studies have shown that host plant volatiles have a significant promoting effect on the mating behavior of Leptinotarsa decemlineata, which has preliminarily confirmed that the behavior is affected by the olfactory pathway.

[0004] In order to further reveal the molecular mechanism of the olfactory system of Leptinotarsa decemlineata, the present application provides a screening method of Leptinotarsa decemlineata olfactory receptor gene, which is used to identify and screen olfactory receptor genes sensitive to target host volatiles, and provides a new technical means for controlling the invasion and spread of Leptinotarsa decemlineata. SUMMARY

[0005] The purpose of the present application is to provide a screening method of Leptinotarsa decemlineata olfactory receptor gene to solve the problems in the background art.

[0006] In order to solve the above technical problems, the present application provides the following technical scheme: a screening method of Leptinotarsa decemlineata olfactory receptor gene, the screening method comprising the following steps:

[0007] S1: selecting sexually mature Leptinotarsa decemlineata adults with consistent development, 10 males and 10 females, placing them in a semi-closed space, adding 5ml of Leptinotarsa decemlineata pheromone with a concentration of 5%, stimulating for 5 hours, and then immediately taking the antennal tissues to obtain the treated group of antennal samples; at the same time, taking the antennal tissues of wild type Leptinotarsa decemlineata without pheromone stimulation as wild type antennal samples; performing transcriptome sequencing on the treated group of antennal samples and the wild type antennal samples respectively to obtain their respective gene expression profiles, and verifying by RT-PCR; comparing the expression levels of the same gene in the wild type antennal samples and the treated group of antennal samples, and screening out the genes with decreased expression levels after stimulation as candidate olfactory receptor genes for sensing target host volatiles;

[0008] S2. Analyze the expression distribution of olfactory genes in the antennae of potato beetle larvae and male and female adults through transcriptome sequencing. Identify olfactory receptor genes commonly expressed in different stages of the potato beetle. Perform phylogenetic tree analysis and select a relatively conserved gene from each evolutionary branch as a candidate olfactory receptor gene for sensing target host volatiles.

[0009] S3, combining the candidate olfactory receptor genes for sensing target host volatiles obtained in S1 and S2 to obtain olfactory receptor genes for sensing target host volatiles;

[0010] S4. Verify the olfactory receptor gene that senses the target host volatiles obtained in step S3 through a heterologous expression system and electrophysiological response test to confirm its responsiveness to the target host volatiles.

[0011] Compared with existing technologies, the present invention achieves the following beneficial effects: It proposes a method for screening olfactory receptor genes for the potato beetle, effectively identifying olfactory receptor genes sensitive to target host volatiles, facilitating in-depth research on the molecular mechanisms of olfaction in the potato beetle. This screening method further elucidates the molecular mechanisms of olfaction in the potato beetle through the role of target host volatiles in promoting mating behavior, providing a scientific basis for the development of technologies for regulating potato beetle mating behavior and theoretical support for achieving green control of the potato beetle. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0013] Figure 1 This is a diagram showing the changes in expression of different olfactory genes in adult potato beetles after pheromone stimulation (WT represents the wild type, T represents the treatment group, ★ indicates significant differences at the 0.05 level; ★★ indicates significant differences at the 0.01 level);

[0014] Figure 2 This is a diagram showing the distribution of olfactory receptor gene expression in different tissues of the potato beetle (A in the figure shows the distribution of male adults, B shows the distribution of female adults, and C shows the distribution of larvae);

[0015] Figure 3 This is a phylogenetic tree constructed by the olfactory receptor genes expressed in different stages of the potato beetle (★ in the figure indicates relatively conserved genes);

[0016] Figure 4 This is the response spectrum of the potato beetle olfactory receptor gene LdecOR11 to some compounds;

[0017] Figure 5 This is the action potential diagram of the potato beetle olfactory receptor gene LdecOR11 after being stimulated by paraffin oil and CPB1. DETAILED DESCRIPTION

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0019] The present invention provides a technical solution: a method for screening potato beetle olfactory receptor genes, the screening method comprising the following steps:

[0020] S1: Select 10 sexually mature adult potato beetles of the same developmental status, 10 males and 10 females, place them in a semi-enclosed space, add 5 ml of a prepared 5% potato beetle pheromone, and immediately collect antennal tissues after 5 hours of stimulation to obtain antennal samples of the treatment group. Simultaneously, collect antennal tissues of wild-type potato beetles that have not been stimulated with pheromones as wild-type antennal samples. Perform transcriptome sequencing on antennal samples of the treatment group and wild-type antennal samples to obtain their respective gene expression profiles, which are then verified by RT-PCR. Compare the expression levels of the same genes in antennal samples of the wild-type and treatment groups, and screen genes whose expression levels decrease after stimulation as candidate olfactory receptor genes for the target host volatiles.

[0021] FPKM values ​​were used as a measure of gene expression in transcriptome data, and the t-test was used for significant difference analysis. Each sample was repeated at least three times. Figure 1 As shown, a total of six olfactory receptor genes, including LdecOR1, LdecOR7, LdecOR11, LdecOR14, LdecOR23, and LdecOR40, with decreased transcriptional expression levels were screened in the antennae of male and female adults as candidate olfactory receptor genes for sensing target host volatiles;

[0022] S2. Analyze the expression distribution of olfactory genes in the antennae of potato beetle larvae and male and female adults through transcriptome sequencing. Identify olfactory receptor genes commonly expressed in different stages of the potato beetle. Perform phylogenetic tree analysis and select a relatively conserved gene from each evolutionary branch as a candidate olfactory receptor gene for sensing target host volatiles.

[0023] like Figure 2 and Figure 3As shown, the three insect stages express a total of 38 olfactory receptor genes. Phylogenetic tree analysis revealed 13 relatively conserved genes as candidate olfactory receptor genes for sensing target host volatiles.

[0024] S3, combining the candidate olfactory receptor genes for sensing target host volatiles obtained in S1 and S2 to obtain olfactory receptor genes for sensing target host volatiles;

[0025] S4. Verify the olfactory receptor gene for the target host volatiles obtained in step S3 by using a heterologous expression system and electrophysiological response testing to confirm its responsiveness to the target host volatiles;

[0026] The specific method of S4 is as follows: full-length primers were designed to amplify the full length of the olfactory receptor gene, the target fragment was cloned into pGEM-T, LdecOR was ligated into pJFRC81 to construct the Drosophila expression vector pJFRC81-UAS-OR; the purified pJFRC81-UAS-OR vector plasmid was injected into fresh Drosophila embryos collected within 1 hour (genotype: nos-φC31 integrase; attP2), and the UAS-OR element was site-specifically inserted into the attP2 site of chromosome 3 using φC31 recombinase. Through standard genetic hybridization methods, a stable Drosophila strain W; Sp / Cyo; UAS-OR / TM6B with successful insertion of the UAS-OR element was obtained; the Drosophila OR22abGal4 null neuron system was used to specifically express OR in ab3A neurons, and the experimental strain was W; OR22abGal4 / OR22abGal4; UAS-OR / UAS-OR. The SSR technology was used to record the response characteristics of the transgenic fruit fly ab3A neurons to target host volatiles with different concentration gradients.

[0027] LdecOR11 was selected as the olfactory receptor gene for verification and stimulated with a variety of compounds. The test results are as follows: Figure 4 As shown, the action potential diagram after being stimulated by paraffin oil and CPB1 is as follows Figure 5 shown.

[0028] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for screening potato beetle olfactory receptor genes, characterized by: The screening method comprises the following steps: S1. Select 10 sexually mature adult potato beetles of the same developmental status, 10 males and 10 females, place them in a semi-enclosed space, add 5 ml of a prepared 5% potato beetle pheromone, and immediately collect antennal tissue from them after 5 hours of stimulation to obtain antennal samples of the treatment group; simultaneously, collect antennal tissue of wild-type potato beetles that have not been stimulated with the pheromone as wild-type antennal samples; perform transcriptome sequencing on the antennal samples of the treatment group and the wild-type antennal samples to obtain their respective gene expression profiles, and verify them by RT-PCR; compare the expression levels of the same genes in the wild-type antennal samples and the antennal samples of the treatment group, and screen out genes whose expression levels decrease after stimulation as candidate olfactory receptor genes that sense volatiles of the target host; S2. Analyze the expression distribution of olfactory genes in the antennae of potato beetle larvae and male and female adults through transcriptome sequencing. Identify olfactory receptor genes commonly expressed in different stages of the potato beetle. Perform phylogenetic tree analysis and select a relatively conserved gene from each evolutionary branch as a candidate olfactory receptor gene for sensing target host volatiles. S3, combining the candidate olfactory receptor genes for sensing target host volatiles obtained in S1 and S2 to obtain olfactory receptor genes for sensing target host volatiles; S4. Verify the olfactory receptor gene that senses the target host volatiles obtained in step S3 through a heterologous expression system and electrophysiological response test to confirm its responsiveness to the target host volatiles.

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