Method for screening olfaction receptor genes of potato beetles

Abstract

The invention discloses a method for screening olfactory receptor genes of potato beetles, which can effectively screen olfactory receptor genes sensitive to target host volatile matters and is convenient for deeply researching an olfactory molecular mechanism of the potato beetles. By means of the screening method, the olfactory molecular mechanism of the target host volatile matter in promoting the copulation behavior of the potato beetles can be further clarified, a scientific basis is provided for research and development of a technology based on regulation and control of the copulation behavior of the potato beetles, and theoretical support is provided for green prevention and control of the potato beetles.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural pest control, and in particular to a method for screening potato beetle olfactory receptor genes. Background Art

[0002] The potato beetle (Leptinotarsa ​​decemlineata (Say)) is one of the major potato pests worldwide and has been included in the "List of Pests for Plant Quarantine for Entry and Exit of the People's Republic of China" and the list of "China's 20 Most Dangerous Alien Invasive Species". Its harm directly threatens my country's biological security, ecological security and food security.

[0003] In natural ecosystems, insects rely on a complex and sensitive olfactory system to support their critical navigation in behaviors such as finding mates, mating and laying eggs, foraging, and avoiding enemies. At present, the research on the olfactory molecular mechanism of insect mating behavior mainly focuses on the role of pheromones. However, the role of volatiles from host plants in insect mating behavior is equally important. Studies have shown that volatiles from host plants have a significant promoting effect on the mating behavior of potato beetles, preliminarily confirming that this behavior is affected by the olfactory pathway.

[0004] In order to further reveal the molecular mechanism of the potato beetle olfactory system, the present application proposes a method for screening potato beetle olfactory receptor genes to identify and screen olfactory receptor genes that are sensitive to target host volatiles, providing a new technical means for controlling the invasion and spread of potato beetles. Summary of the invention

[0005] The object of the present invention is to provide a method for screening olfactory receptor genes of potato beetles to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solution: a method for screening potato beetle olfactory receptor genes, the screening method comprising the following steps:

[0007] S1: Select 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 the prepared potato beetle pheromone with a concentration of 5%, and immediately take their antennal tissues after stimulation for 5 hours to obtain antennal samples of the treatment group; at the same time, take the antennal tissues of wild-type potato beetles that have not been stimulated by pheromones as wild-type antennal samples; perform transcriptome sequencing on the antennal samples of the treatment group and the wild-type antennal samples, respectively, to obtain their respective gene expression profiles, and verify them by RT-PCR; compare the expression levels of the same gene 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;

[0008] S2. Analyze the expression distribution of olfactory genes in the antennae of potato beetle larvae and male and female adults through transcriptome sequencing, screen out the olfactory receptor genes commonly expressed in different stages of potato beetles, and perform phylogenetic tree analysis on them, and select a relatively conservative 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 respectively to obtain olfactory receptor genes for sensing target host volatiles;

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

[0011] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: the present invention proposes a method for screening olfactory receptor genes of potato beetles, which can effectively screen out olfactory receptor genes that are sensitive to target host volatiles, and facilitate in-depth research on the olfactory molecular mechanism of potato beetles. Through the screening method of the present invention, the olfactory molecular mechanism of target host volatiles in promoting the mating behavior of potato beetles can be further clarified, providing a scientific basis for the development of technologies based on regulating the mating behavior of potato beetles, and providing theoretical support for the realization of green prevention and control of potato beetles. 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 after pheromone stimulation of potato beetle adults (WT in the figure 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 insect tissues of potato beetles (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 The phylogenetic tree of olfactory receptor genes expressed in different stages of potato beetles (★ in the figure indicates relatively conservative 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 be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work 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 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 the prepared potato beetle pheromone with a concentration of 5%, and immediately take their antennal tissues after stimulation for 5 hours to obtain antennal samples of the treatment group; at the same time, take the antennal tissues of wild-type potato beetles that have not been stimulated by pheromones as wild-type antennal samples; perform transcriptome sequencing on the antennal samples of the treatment group and the wild-type antennal samples, respectively, to obtain their respective gene expression profiles, and verify them by RT-PCR; compare the expression levels of the same gene 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;

[0021] The FPKM value was used as a measure of gene expression in transcriptome data, and the t-test was used for difference significance analysis. Each sample was repeated at least three times. Figure 1 As shown, a total of 6 olfactory receptor genes with decreased transcriptional expression levels, including LdecOR1, LdecOR7, LdecOR11, LdecOR14, LdecOR23, and LdecOR40, 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, screen out the olfactory receptor genes commonly expressed in different stages of potato beetles, and perform phylogenetic tree analysis on them, and select a relatively conservative 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 38 olfactory receptor genes in total. Through phylogenetic tree analysis, 13 relatively conservative genes were screened out 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 respectively to obtain olfactory receptor genes for sensing target host volatiles;

[0025] S4, verifying the olfactory receptor gene for sensing the target host volatiles obtained in step S3 through a heterologous expression system and an electrophysiological response test to confirm its responsiveness to the target host volatiles;

[0026] The specific method of S4 is as follows: full-length primers are designed to amplify the full length of the olfactory receptor gene, the target fragment is cloned into pGEM-T, LdecOR is connected to pJFRC81 to construct the Drosophila expression vector pJFRC81-UAS-OR; the purified pJFRC81-UAS-OR vector plasmid is injected into fresh Drosophila embryos collected within 1 hour (genotype: nos-φC31 integrase; attP2), and the UAS-OR element is site-specifically inserted into the attP2 site of chromosome 3 using φC31 recombinase, and a stable Drosophila strain W; Sp / Cyo; UAS-OR / TM6B with successful insertion of the UAS-OR element is obtained through standard genetic hybridization methods; OR is specifically expressed in ab3A neurons using the Drosophila OR22abGal4 empty neuron system, and the experimental strain is 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 at 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 stimulation by paraffin oil and CPB1 is as follows Figure 5 shown.

[0028] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is 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 can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for screening potato beetle olfactory receptor genes, characterized in that: The screening method comprises the following steps: S1. Select 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 the prepared potato beetle pheromone with a concentration of 5%, and immediately take their antennal tissues after stimulation for 5 hours to obtain antennal samples of the treatment group; at the same time, take the antennal tissues of wild-type potato beetles that have not been stimulated by pheromones as wild-type antennal samples; perform transcriptome sequencing on the antennal samples of the treatment group and the wild-type antennal samples, respectively, to obtain their respective gene expression profiles, and verify them by RT-PCR; compare the expression levels of the same gene 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, screen out the olfactory receptor genes commonly expressed in different stages of potato beetles, and perform phylogenetic tree analysis on them, and select a relatively conservative 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 respectively to obtain olfactory receptor genes for sensing target host volatiles; S4. Verify the olfactory receptor gene for the target host volatiles obtained in step S3 through a heterologous expression system and an electrophysiological response test to confirm its responsiveness to the target host volatiles.

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