CN-121574996-B - DcPDF gene and application of dsRNA thereof in prevention and control of diaphorina citri

Abstract

The invention discloses DcPDF genes and application of dsRNA thereof in controlling diaphorina citri. According to the invention, the expression level of the neuropeptide pigment dispersion factor gene DcPDF (the nucleotide sequence of which is shown as SEQ ID NO. 1) is reduced by feeding the dsRNA with the target nucleotide sequence shown as SEQ ID NO.13 to the diaphorina citri, so that the development of the ovary of the diaphorina citri can be obviously inhibited, and the spawning quantity can be obviously reduced. The invention provides a new high-efficiency target for green control of diaphorina citri based on RNA interference technology, is beneficial to realizing pesticide decrement and synergy and delays the development of pest drug resistance.

Inventors

• NIAN XIAOGE
• Zhang Songdou
• ZHANG ZHIXIAN

Assignees

• 广东省农业科学院植物保护研究所

Dates

Publication Date

20260508

Application Date

20260129

Claims (6)

1. 1. The application of inhibiting the expression of the gene DcPDF of the diaphorina citri in inhibiting the development of the egg nest of the female insect of the diaphorina citri or reducing the spawning quantity of the female insect of the diaphorina citri, wherein the nucleotide sequence of the DcPDF gene is shown as SEQ ID NO.1, the expression of the gene DcPDF of the diaphorina citri is reduced by feeding dsRNA of the targeted DcPDF gene, and the nucleotide sequence of a target area of the dsRNA of the targeted DcPDF gene is shown as SEQ ID NO. 13.
2. 2. The use according to claim 1, wherein the concentration of dsRNA targeting DcPDF genes is 500 ng/μl.
3. 3. The use of claim 1, wherein the method for preparing the dsRNA targeting DcPDF genes comprises the steps of: S1, extracting total RNA of diaphorina citri, and reversely transcribing the total RNA into cDNA; S2, designing a specific primer pair with a T7 sequence, and carrying out PCR amplification by taking the cDNA obtained in the step S1 as a template to obtain a DNA fragment containing a T7 promoter sequence, wherein the nucleotide sequence of an upstream primer of the primer pair for preparing the dsRNA of the target DcPDF gene is shown as SEQ ID NO.9, and the nucleotide sequence of a downstream primer of the primer pair for preparing the dsRNA of the target DcPDF gene is shown as SEQ ID NO. 10; S3, taking the DNA fragment containing the T7 promoter sequence obtained in the step S2 as a template, and performing in vitro transcription reaction by using an in vitro transcription kit and purifying to obtain the dsRNA of the targeted DcPDF gene.
4. 4. A preparation for preventing and controlling diaphorina citri is characterized by comprising dsRNA of a target DcPDF gene as an active ingredient, wherein the nucleotide sequence of the DcPDF gene is shown as SEQ ID NO.1, the nucleotide sequence of a target area of the dsRNA of the target DcPDF gene is shown as SEQ ID NO.13, the nucleotide sequence of an upstream primer of a primer pair for preparing the dsRNA of the target DcPDF gene is shown as SEQ ID NO.9, and the nucleotide sequence of a downstream primer of a primer pair for preparing the dsRNA of the target DcPDF gene is shown as SEQ ID NO. 10.
5. 5. Use of the formulation of claim 4 for inhibiting development of the egg nest of a diaphorina citri female or for reducing the oviposition of a diaphorina citri female.
6. 6. The use according to claim 5, comprising the step of feeding diaphorina citri with said formulation.

Description

DcPDF gene and application of dsRNA thereof in prevention and control of diaphorina citri Technical Field The invention belongs to the field of green prevention and control of agricultural pests, and particularly relates to DcPDF genes and application of dsRNA thereof in prevention and control of diaphorina citri. Background Pathogen candidate bacillus phloem (Candidatus Liberibacter asiaticus, CLas) for citrus yellow longdisease (Huanglongbing, HLB) cannot be cultured ex vivo and lacks effective control agents. Diaphorina citri (Diaphorina citri) is a natural transmission medium of the disease, so that effective prevention and control of the Diaphorina citri is a key for inhibiting the spread of yellow dragon disease and guaranteeing the healthy and sustainable development of the citrus industry. At present, the control of diaphorina citri mainly depends on chemical pesticides, and is supplemented with measures such as agricultural management, physical control, biological control and the like. Although the chemical control takes effect quickly, the long-term use of the pesticide composition can easily cause the drug resistance of pests, and simultaneously causes the problems of pesticide residues and environmental pollution. Agricultural and physical control methods (such as unified tip placement, yellow insect attracting plate suspension, etc.) tend to be relatively costly to manage and difficult to eradicate on a large scale. The biological control mode of natural enemies or microbial pesticides is utilized, the effect is greatly influenced by environmental conditions, the effect is relatively slow, and the quick control is difficult when the diaphorina citri insect pests occur on a large scale. In general, the existing control technology has obvious limitations in terms of environmental safety, action sustainability and control efficiency. Researches show that carrying the yellow-shoot germ CLas can obviously enhance the fertility of the female diaphorina citri, so that the spawning quantity of the female diaphorina citri is improved by 30% -50%, and a malignant cycle of 'pathogen promoting medium proliferation' is formed, but the inherent molecular regulation mechanism is not clear. Pigment-dispersing factor (PDF) is an evolutionarily conserved neuropeptide that has been shown to regulate reproductive development (e.g., activate vitellogenin synthesis) through signaling pathways such as cAMP/PKA in Drosophila-like mode insects. However, the role of PDF in the reproductive regulation of vector insects (especially diaphorina citri), especially in its interaction with pathogenic bacteria, has not been reported. In recent years, RNA interference (RNAi) technology has shown great potential in the field of agricultural pest control due to its high target specificity and environmental friendliness. Disclosure of Invention Based on the defects and shortcomings of the prior art, the invention aims to provide DcPDF genes and application of dsRNA thereof in controlling diaphorina citri. The invention provides a PDF gene of diaphorina citri as a novel target for RNAi control for the first time. The gene can deeply participate in the key physiological process of regulating psyllids. The invention not only provides a high-efficiency specific dsRNA sequence aiming at the DcPDF gene and a synthesis method thereof, but also provides an innovative solution for developing a green, accurate and high-efficiency novel control strategy for diaphorina citri, and is hopeful to overcome a plurality of defects of the existing control method. The first object of the invention is to provide an application of inhibiting expression of diaphorina citri DcPDF genes in controlling diaphorina citri, wherein the nucleotide sequence of the DcPDF genes is shown as SEQ ID NO. 1. Preferably, the application is the application of inhibiting the gene expression of the diaphorina citri DcPDF in regulating and controlling the reproduction of the diaphorina citri female insects. Preferably, the application is the application of inhibiting the gene expression of the diaphorina citri DcPDF in inhibiting the development of the egg nest of the female diaphorina citri or reducing the spawning quantity of the female diaphorina citri. Preferably, the inhibition of diaphorina citri DcPDF gene expression is achieved by feeding dsRNA targeting DcPDF gene to reduce DcPDF gene, and the nucleotide sequence of the target region of the dsRNA targeting DcPDF gene is shown as SEQ ID NO. 13. Preferably, the concentration of dsRNA targeting DcPDF genes is 500 ng/. Mu.L. The second object of the invention is to provide a dsRNA targeting DcPDF genes, the nucleotide sequence of the target area of which is shown as SEQ ID NO. 13. Preferably, the preparation method of the dsRNA comprises the following steps: S1, extracting total RNA of diaphorina citri, and reversely transcribing the total RNA into cDNA; S2, designing a specific primer pair with a T7 sequence, and carry