Interference sequence of ddc gene of oedaleus asiaticus and its application in locust control

Abstract

The application discloses an interference sequence of a DDC gene of Asian migratory locust and application of the interference sequence in locust control. The DDC gene is a key gene for regulating growth and development of the Asian migratory locust. The specific double-stranded RNA is designed by using the gene, and is injected into the Asian migratory locust, so that the Asian migratory locust is caused to die, and growth and development of the Asian migratory locust are significantly inhibited. The application of the DDC gene in locust control includes the following aspects: the DDC gene is applied to regulate growth and development of the Asian migratory locust; the DDC gene is applied to increase the mortality of the Asian migratory locust due to the lack of the DDC gene; the proportion of the Asian migratory locust with morphological changes is reduced due to the lack of the DDC gene; and the DDC gene is applied to regulate body color of the Asian migratory locust. The DDC gene can be used to regulate the growth and development process of the Asian migratory locust, a grassland pest. By means of gene regulation, the growth and development of the Asian migratory locust are inhibited, the Asian migratory locust is prevented from gathering and causing damage, the mortality of the Asian migratory locust is increased, and the harm of the pest to the grassland ecological environment is reduced.

Description

Technical Field

[0001] This invention belongs to the field of grassland pest control technology, specifically relating to an interference sequence of the DDC gene of the Asian grasshopper and its application in locust control. Background Technology

[0002] Dopa decarboxylase is an important enzyme in organisms, participating in various biochemical reactions. It is a key enzyme in dopamine biosynthesis, catalyzing the decarboxylation of L-dopa to form dopamine. Dopamine, as a neurotransmitter, regulates the growth, development, reproduction, behavior, and body color of insects. Numerous studies have found that dopa decarboxylase is a key enzyme in the melanization process of insect epidermis, playing an important regulatory role in insect embryonic development, molting, and exoskeleton development.

[0003] The Asian grasshopper is a significant agricultural pest in the northern grasslands and agro-pastoral transition zones. Outbreaks severely impact the healthy development of agriculture, animal husbandry, and the socio-economic system. A key characteristic of these outbreaks is the locust's transformation into two life forms and its swarming migration. In areas experiencing grassland locust outbreaks, the Asian grasshopper becomes the dominant species, typically requiring chemical control methods. However, these methods place significant pressure on the environment and livestock, hindering sustainable development. Developing green and safe control methods is urgently needed. Controlling pest occurrence and damage at the genetic level is an important sustainable approach to pest management. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides an interference sequence for the DDC gene of the Asian grasshopper and the application of its effective interference sequence. The double-stranded RNA fragment of this gene can effectively interfere with the transcriptional expression of the DDC gene. Another objective of this invention is to provide a method for effectively inhibiting the dimorphic transformation of the Asian grasshopper.

[0005] This invention is achieved through the following technical solution: This invention discloses an interference sequence for the DDC gene of the Asian grasshopper, the nucleotide sequence of which is shown below:

[0006]

[0007]

[0008] The DDC gene is a key gene regulating the growth and development of the Asian carp. Injecting specific double-stranded RNA designed using this gene into the Asian carp can cause death, thereby significantly inhibiting the growth and development of the Asian carp.

[0009] The applications of the interference sequence of the DDC gene in locust control include: the use of the DDC gene to regulate the growth and development of the Asian grasshopper; the application of DDC gene deficiency leading to increased mortality in the Asian grasshopper; the application of DDC gene deficiency leading to a reduced proportion of morphological changes in the Asian grasshopper, thus inhibiting the two-type transition of locusts; the application of DDC gene transcription regulation of the phenotype transition of the Asian grasshopper from solitary to gregarious; and the application of DDC gene participation in regulating the body color of the Asian grasshopper.

[0010] The main problem solved by this invention is the discovery of a key gene, DDC, that regulates the growth and development of the Asian carp. By using this gene to design specific double-stranded RNA and injecting it into the Asian carp, the growth and development of the Asian carp can be significantly inhibited, leading to its death.

[0011] The beneficial effects of this invention are as follows: This invention mainly relates to the field of grassland pest control technology, and provides an effective interference sequence for the DDC gene of the Asian grasshopper for locust control. By injecting a double-stranded DDCRNA fragment, the growth and development of the Asian grasshopper are inhibited, its mortality is promoted, and the outbreak rate of the Asian grasshopper is reduced, thereby achieving the purpose of effectively controlling locust outbreaks and damage. This invention synthesizes a specific double-stranded RNA of the DDC gene and injects it into the Asian grasshopper. By observing its growth and development process, the ratio of two-type transitions, and the mortality rate of pests after injection, the function of the DDC gene and the interference efficiency of the dsDDC fragment are explored. This fragment can be applied to regulate the growth, development, and aggregation outbreaks of the Asian grasshopper, which is beneficial for the effective control of the Asian grasshopper.

[0012] This invention belongs to the field of grassland plant protection and relates to the regulatory role of the DOP decarboxylase (DDC) gene in the growth and development of the Asian grasshopper. Normal expression of the DDC gene maintains the growth and development of the Asian grasshopper; high expression promotes melanization; and silent expression inhibits growth and development, increases mortality, and causes albinism. The technical solution in this invention is of great significance for the control of the Asian grasshopper, a grassland pest. This invention controls the occurrence and damage of the pest at the gene level, representing an important and sustainable approach to pest management. This invention interferes with the expression level of the DDC gene by injecting specific double-stranded RNA into individual Asian grasshoppers, thereby regulating the growth, development, and metamorphosis of the Asian grasshopper at the molecular level through gene intervention, reducing the aggregation and outbreak rate of the Asian grasshopper, and achieving effective locust control. This invention utilizes the DDC gene to regulate the growth and development of the Asian grasshopper, inhibiting its growth and development, preventing aggregation and damage, increasing the mortality rate of the Asian grasshopper, and reducing the harm of the pest to the grassland ecological environment through gene regulation. Attached Figure Description

[0013] Figure 1This is a bar chart showing the mRNA expression level of the DDC gene in Asian carp injected with dsRNA 48 hours prior.

[0014] Figure 2 This is a bar chart showing the mortality rates of the Asian carrion locust 7 days after injection of dsRNA into the treatment group and each control group.

[0015] Figure 3 This is a bar chart showing the proportion of individuals in the Asian carp that transitioned from solitary to gregarious behavior 7 days after injection of dsRNA into the treatment group and each control group.

[0016] Figure 4 This is a comparative chart showing the differences in phenotypes between the solitary (left) and gregarious (right) forms of the Asian caricature locust.

[0017] Figure 5 This is a bar chart showing the proportion of individuals in the Asian caricature locust treatment group and each control group who developed albino body color 7 days after injection of dsRNA.

[0018] Figure 6 This is a comparative analysis of the differences in body color among albino individuals of the Asian caricature locust after injection of dsDDC. Detailed Implementation

[0019] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following non-limiting examples are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the scope of protection of the present invention. All reagents used in the present invention are commercially available.

[0020] The main reagents and their manufacturers used in this invention are as follows: RNA extraction kit, reverse transcription kit (Promega, USA); 4.2×Taq PCR Master Mix, DNA recovery reagent (Tiangen Biotech, China); pGEM-T Easy Vector (Takara, Japan); plasmid DNA extraction kit (Tiangen Biotech, China); E. coli DH5α competent cells (Tiangen Biotech, China); T7 RiboMAX. TM Express RNAi System, qPCR Master Mix (Promega, USA).

[0021] The primer sequences used in the embodiments of this invention were all completed by Beijing Housheng Biotech Co., Ltd. Experimental methods not specifying specific conditions in the embodiments are generally performed under conventional conditions or according to the conditions recommended by the manufacturer.

[0022] Based on the early transcriptome results of *Heliotropium indicum* in our laboratory, the DDC gene sequence of *Heliotropium indicum* was obtained. The CDS region of this gene was predicted using NCB-BIAST. The primer sequences (DDC-3F, DDC-3R) for double-stranded RNA fragment and expression level determination were designed as follows:

[0023]

[0024] Total RNA was extracted from *Heliotropium indicum* using an RNA extraction kit, and then reverse transcribed into cDNA using a reverse transcription kit. The *Heliotropium indicum* DDC gene was amplified using the cDNA as a template.

[0025] The PCR amplification conditions for the DDC gene were as follows: 94℃ pre-denaturation for 4 min; followed by 94℃ denaturation for 30 s, 56℃ annealing for 30 s, and 72℃ extension for 1 min, repeated 34 times; and a final extension at 72℃ for 10 min. The reaction mixture consisted of 25 μl: 9.5 μl ddH2O; 1 μl template cDNA; 1 μl each of forward and reverse primers; and 12.51 μl 2×Taq PCR Master Mix.

[0026] The DDC gene fragment obtained by PCR amplification was ligated into a plasmid, transformed into E. coli, and then sent for sequencing. After obtaining the correct sequence, the conserved region was extracted and primers (DDC-2F, DDC-2R) were redesigned. A T7 promoter was added to the 5' end of the primers for double-stranded RNA synthesis. The primers were then subjected to PCR amplification, ligated into a vector, transformed into E. coli, and the bacterial culture was sequenced. The correct bacterial culture was cultured overnight and the plasmid was extracted.

[0027] The plasmids obtained above were used as templates to obtain double-stranded RNA with a length of 417 bp using the T7 RNA synthesis kit. The concentration was detected by spectrophotometer and then stored at -20℃ for later use.

[0028] 5 μl of 1000 ng / μl double-stranded RNA was injected into the 2nd-3rd abdominal segments of 3rd-4th instar solitary locusts. The experiment was conducted in triplicate, with 60 locusts per replicate. An equal volume of dsGFP was injected into the control group, and an equal volume of no RNA was injected into the blank control group. The growth, development, phenotypic changes, and ratios of the treated and control groups were observed and recorded over one week. Gene expression levels were detected after dsRNA interference. 48 h after dsRNA injection, whole locusts were treated in liquid nitrogen to extract RNA, which was then reverse transcribed to generate template cDNA. Primers (DDC-3F, DDC-3R) were redesigned based on the conserved region of the DDC gene for DDC gene expression determination. β-actin from the Asian grasshopper was used as an internal reference gene. The relative expression levels of the DDC gene in the treated and control groups were detected using real-time quantitative PCR. Each sample was tested in triplicate, and each biological replicate was tested in quadruplicate using RT-qPCR.

[0029] dsDDC segment interference efficiency such as Figure 1 As shown, after 48 hours of injection of dsDDC into the Asian grasshopper, the expression level of the DDC gene was significantly lower than that of the dsGFP-injected and uninjected groups, with a 95.1% reduction compared to the control group. The results indicate that dsDDC has a high silencing efficiency for the DDC gene.

[0030] The mortality rate of *Heliotropium indicum* in the treatment and control groups after dsRNA injection was observed and recorded for 7 days to assess the effect of dsDDC on the growth and development of *Heliotropium indicum*. Results are as follows: Figure 2 As shown, the mortality rate of Asian grasshopper injected with dsDDC reached 81% after 7 days, which was 11.6 times that of the control group. This indicates that the injection of dsDDC can significantly increase the mortality rate of Asian grasshopper and severely inhibit its growth and development.

[0031] The dsDDC-injected treatment group, the dsGFP-injected group, and the uninjected control group were housed at a density of 60 individuals / cage for 7 days. Phenotypic changes and the percentage of individuals exhibiting phenotypic changes were observed. The results are as follows: Figure 3 The blank control and control groups shown had 49.9% and 48.9% of individuals with body color as indicated. Figure 4 The model shows a transformation from a solitary, green form to a gregarious, brown form. Only 5.6% of individuals in the dsDDC-treated group exhibited this bimorphic change, significantly lower than the two control groups. This indicates that DDC participates in the bimorphic transformation process of the Asian grasshopper, and dsDDC can significantly inhibit the transformation from a solitary to a gregarious form. Furthermore, the experiment revealed that 13% of individuals injected with dsDDC experienced albino body coloration (e.g., ...). Figure 5 As shown in the image, the body color of the Asian grasshopper after injection of dsDDC is as follows: Figure 6 As shown, the color changes from green to pale yellow. This albino individual did not appear in individuals injected with dsGFP or those not injected with any substance, indicating that the DDC gene may be involved in the regulation of body color in the Asian grasshopper, and that dsDDC can cause albinoization of the Asian grasshopper's body surface.

[0032] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of the interfering nucleic acid dsDDC of the DDC gene in the control of the Asian carp locust, characterized by: The nucleotide sequence of the dsDDC is shown in SEQ ID NO.2 of the sequence listing; The DDC gene is a key gene regulating the growth and development of the Asian carp. By using this gene to design specific double-stranded RNA and injecting it into the Asian carp, the death of the Asian carp was caused, thereby significantly inhibiting the growth and development of the Asian carp.

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