Target gene capable of adapting to hunger stress of harmonia axyridis and application of target gene

Abstract

The invention discloses a target gene capable of adapting to hunger stress of axyridis polyaxyridis, which is an HvarAKR1B1 gene for coding aldose reductase, and is used for detecting the hunger stress and comparing the gene expression and metabolite change of the axyridis polyaxyridis through transcriptomics and metabonomics technologies. The key gene for responding to hunger stress of the heterodylaea axyridis is screened by difference analysis and is verified by an RNAi (Ribonucleic Acid Interference) technology. After the gene is silenced, the survival rate, the eclosion rate and the survival rate from the 4-instar larva to the adult larva of the heterodylaea polyalaea are obviously reduced, and the RNAi-mediated gene reduces the hunger tolerance of the heterodylaea polyalaea. Therefore, the silence or overexpression of the gene can obtain the effect of effectively regulating and controlling the population quantity of the axyridis polyaxyridis, and the gene has relatively good popularization and application values.

Description

Technical Field

[0001] This invention belongs to the field of gene control technology, specifically relating to the technical field of target genes for the adaptation of ladybugs to starvation stress. Background Technology

[0002] Ladybugs, belonging to the family Coleoptera and the family Coccinellidae, are important predatory natural enemies originating from the Palearctic realm and gradually spreading globally. They hold a dominant position in Xinjiang's agricultural ecosystems, serving as primary predatory enemies of aphids and some lepidopteran larvae on various crops. In natural ecosystems, aphids are a major food source for ladybugs, but their availability is significantly unstable, exhibiting dramatic fluctuations in both time and space. Aphid populations can rapidly peak within a short period, followed by a sharp decline due to predation by natural enemies, changes in host plant quality, and environmental factors such as temperature and rainfall. Furthermore, in early spring, aphid populations in agricultural ecosystems are typically low, frequently subjecting ladybugs to starvation stress. This nutritional stress significantly affects the physiological state and behavioral characteristics of ladybugs, thereby altering many of their life activities and developmental status. Simultaneously, releasing natural enemies at low pest densities to prevent delayed release from effectively controlling pests also leads to starvation stress for the natural enemies.

[0003] Insects capable of resisting hunger stress possess a significant survival advantage. Their physiological and behavioral adaptations to hunger, a common stressor, have been refined through long-term evolution. For example, accumulating higher reserves, lowering body energy levels to the minimum permissible for survival, and reducing metabolic rate can significantly increase an insect's resistance to hunger stress. Understanding how natural enemies respond to hunger stress is beneficial for enhancing their potential as biological control services in ecosystems by regulating their resistance to hunger stress. Previous studies have found that life parameters such as developmental duration, egg production, and survival rate of *Heterocarya stenoptera* (a type of ladybug) significantly decreased under hunger stress. Combined transcriptomic and metabolomic analyses revealed changes in metabolic pathways involving carbohydrates, amino acids, and lipids in *Heterocarya stenoptera* under hunger stress. In the carbohydrate metabolism pathway, pathways involving fructose and mannose, galactose, starch and sucrose, as well as pentose and glucuronic acid were significantly enriched. Under starvation stress, the expression level of α-glucosidase gene and the content of substances such as trehalose-6-phosphate in *Heterocystis pilosa* significantly increased, while the content of substances such as trehalose and sucrose significantly decreased. In the amino acid metabolism pathway, pathways involving phenylalanine metabolism, tryptophan, and glycine were relatively enriched. Under starvation stress, the content of various amino acids such as serine, leucine, asparagine, glutamic acid, and phenylalanine significantly decreased. In the lipid metabolism pathway, pathways involving the biosynthesis of unsaturated fatty acids, fatty acid elongation, and glycerophospholipid metabolism were significantly enriched. Under starvation stress, the expression level of glycerol-3-phosphate dehydrogenase gene and the content of substances such as sn-glycerol-3-phosphate in *Heterocystis pilosa* significantly increased. Summary of the Invention

[0004] The problem of starvation stress is a common challenge faced by ladybugs in natural ecosystems. This invention aims to solve the technical problem of screening key genes in ladybugs to cope with starvation stress and verifying their functions in this response using RNAi technology. Based on the common survival and reproductive strategies of ladybugs, such as slowing larval development and reducing oviposition, to cope with long-term starvation stress, this invention uses combined metabolomics and transcriptomics analysis to screen key genes in ladybugs' response to starvation stress and verifies their roles using RNAi technology. Silencing of this target gene significantly reduces the starvation tolerance of ladybugs. This not only contributes to a deeper understanding of the ecological adaptability of ladybugs but also provides new theoretical basis for their efficient use in biological control.

[0005] To solve the above technical problems, the present invention provides the following technical solution: This application provides a target gene of *Heteromorpha heteromorpha* for resistance to starvation stress, wherein the target gene is *Heteromorpha heteromorpha*. HvarAKR1B1 The primer sequences used to amplify gene coding region-specific interference fragments include or consist of the following sequences: 1) The nucleotide sequences shown in SEQ ID No. 1-SEQ ID No. 2; or 2) The nucleotide sequences shown in SEQ ID No.3-SEQ ID No.4.

[0006] Furthermore, this application also provides the aforementioned target gene. HvarAKR1B1 Application in at least one of the following: 1) Improve the survival rate of 4th instar larvae of *Heteromorpha chinensis* under starvation stress; 2) Improve the emergence rate of ladybugs under starvation stress; 3) Improve the survival rate of fourth instar larvae to adults of *Heteromorpha maculatus* under starvation stress; The application described uses target genes. HvarAKR1B1 ds were artificially synthesized using the T7RiboMAX™ Express RNAi System synthesis kit. HvarAKR1B1 ds HvarAKR1B1 The solution, 1000 ng, was injected into the body of *Heteromorpha heteromorpha* through the abdominal segments.

[0007] Furthermore, this application also provides a method for reducing the resistance of ladybugs to starvation stress, wherein the method involves targeting genes in ladybugs... HvarAKR1B1 Knock out or silence.

[0008] The method described uses RNAi technology, CRISPR-Cas technology, and T-DNA and promoter insertion to insert target genes from the aforementioned ladybug.HvarAKR1B1 Knock out or silence.

[0009] Furthermore, this application also provides a method for enhancing the resistance of ladybugs to starvation stress, wherein the method involves introducing target genes from ladybugs... HvarAKR1B1 Overexpression.

[0010] The method described uses CRISPR-Cas technology and T-DNA and promoter insertion to insert target genes from the aforementioned ladybug. HvarAKR1B1 Overexpression.

[0011] Through the above technical solutions, this application achieves the following technical effects: This invention utilizes RNAi research technology to discover the effects of injecting 1000 ng ds HvarAKR1B1 The interference efficiencies after 24h, 48h, and 72h were 79.55%, 98.03%, and 70.44%, respectively, indicating that microinjection of 1000 ng ds HvarAKR1B1 It can effectively interfere with the expression of this gene. Interference HvarAKR1B1 Following gene injection, the developmental duration of 4th instar larvae, pupae, and adults in *Heterodactylus spp.* was significantly shortened. Furthermore, compared to the uninjected group, the survival rate of 4th instar larvae, emergence rate, and survival rate from 4th instar larvae to adults were significantly reduced by 6.56%, 2.38%, and 8.04%, respectively. These findings indicate that RNAi-mediated... HvarAKR1B1 This reduced the starvation tolerance of ladybugs. Based on this, HvarAKR1B1 Genes are important for ladybugs to cope with hunger stress. Attached Figure Description

[0012] Figure 1 The image shows the co-expression map of structural genes and metabolite abundance in the glucose metabolism pathway.

[0013] Figure 2 The image shows ladybugs after interference. HvarAKR1B1 The relative expression level of genes.

[0014] Figure 3 The image shows silence. HvarAKR1B1 The influence of genes on the developmental stage of the ladybug. Detailed Implementation

[0015] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] The reagent used in this invention is: T7 RiboMAXTM The Express RNAi System was purchased from Promega. E.Z.N.A. ® Gel Extraction Kit purchased from OMEGA; pET1 expression vector, *E. coli* Escherichiacoli competent cells, and EasyPure Plasmid MiniPerp Kit. TransZol Up Plus RNA Kit TransScript All-in-One First-Strand cDNA Synthesis was purchased from TransGen Biotech Ltd., and SuperMix for qPCR and PerfectStart Green qPCR SuperMix Kit were purchased from Tiangen Biotech Ltd.

[0017] Unless otherwise specified, the technical means used in the following implementation examples are conventional means well known to those skilled in the art.

[0018] Example: Functional identification of a gene related to starvation stress in a ladybug. This embodiment provides a method for functional identification of genes related to starvation stress adaptation in ladybugs, specifically including the following steps: (1) Rearing of ladybugs The tested insects were a stable population of *Heteromorpha heterophylla* reared indoors by cotton aphids at the Korla Experimental Base of the Institute of Plant Protection, Xinjiang Academy of Agricultural Sciences (Korla Village, Heshilik Township, Korla City, Bayingolin Mongol Autonomous Prefecture, Xinjiang, 41.75ºN, 85.81ºE).

[0019] Based on the maximum predation capacity of *Heterodon spp.* at different instars, two treatments were set up: starvation stress and normal rearing (control). In the control group, approximately 40, 60, 120, 180, and 240 cotton aphids were provided for 1st, 2nd, 3rd, and 4th instar larvae and adults, respectively. In the starvation stress group, 25% of the aphid quantity of the control group was provided for each instar, i.e., approximately 10, 15, 30, 45, and 60 cotton aphids for 1st, 2nd, 3rd, and 4th instar larvae and adults, respectively. Active, newly emerged adult *Heterodon spp.* were placed in transparent plastic rearing boxes, with 10 boxes each for the starvation stress group and the control group. Each box was paired with males and females at a 1:1 ratio, for a total of 15 pairs (F0 generation). The rearing was conducted in an incubator at 25℃, 65% RH, and 14L:10D. During the peak oviposition period (5-8 days after F0 generation pairing), egg masses were collected daily and placed in the same incubator for incubation (F1 generation). Subsequently, each treatment was repeated in triplicate, with 140 newly hatched larvae selected from each replicate. Each larva was individually reared in numbered glass beakers (sealed with a 120-mesh nylon sieve and secured with rubber bands) in the aforementioned incubator until the F1 generation completed its pupal stage and emerged as adults. During rearing, the survival status of the ladybugs was observed daily at 10:00 AM. After sexing the F1 generation adults using a stereomicroscope, they were paired at a 1:1 female-to-male ratio (each pair placed in an independent glass beaker), with the same aphid supply as described above.

[0020] (2) Sampling of ladybugs Two treatments were set up: starvation stress and normal feeding (control). Each treatment had three replicates, and each replicate included all developmental stages of *Heterocarya heterophylla* (egg, 1st-4th instar larvae, pupa, and adult females). For each replicate, approximately 32 mg of *Heterocarya heterophylla* was collected from each developmental stage, with approximately 16 mg collected from each adult female, and the samples were mixed thoroughly. The collected samples were quickly washed with distilled water to remove surface impurities, blotted dry on absorbent paper, and then placed in 1.5 mL RNase-free centrifuge tubes. The centrifuge tubes were then flash-frozen in liquid nitrogen for 2 min and stored at -80°C for transcriptomics and metabolomics analysis.

[0021] (3) Gene screening This invention utilizes transcriptomics and metabolomics techniques to analyze gene expression and metabolite changes in *Heteromorpha heterophylla* under starvation stress and normal treatment. Differential analysis revealed that differentially expressed genes and metabolites were mainly enriched in sugar, amino acid, and lipid metabolism pathways. Subsequently, from the top 30 enriched differential metabolic pathways, the pathway with the most enriched pathways and six pathways related to carbohydrate biosynthesis and degradation were selected, and a gene-metabolite co-expression network was constructed. Under starvation stress, the expression levels of three genes encoding aldose reductase, glucosidase, and glucuronyl transferase were significantly upregulated in *Heteromorpha heterophylla*, while the expression levels of two genes encoding hydroxymethylglutaryl-CoA synthase and hexokinase were significantly downregulated. Specifically, under starvation stress, the expression level of genes encoding aldose reductase... HvarAKR1B1 The gene exhibited the highest log2FC value; see appendix. Figure 1 As shown.

[0022] (4) Synthesis dsGFP and ds HvarAKR1B1 Combined transcriptomic and metabolomic analysis was used to screen for strains related to starvation stress in *Heteromorpha multifida*. HvarAKR1B1 and control genes GFP In vitro dsRNA synthesis was performed separately. First, siRNA target sites were predicted using Eurofins Genomics, siDirect2, and Invitrogen siRNA Wizard. Primers were designed based on 400-500 bp sequences containing the most predicted sites (see Table 1). Using *Heliotropium indicum* cDNA as a template, the target fragment was amplified by PCR, purified via gel extraction, ligated into the pEASY-T1 vector, transformed into Trans1-T2 competent cells, and positive clones were screened and sequenced for verification. After extracting the recombinant plasmid, it was used as a template to amplify the dsRNA template. Finally, dsRNA was synthesized using the T7RiboMAX™ Express RNAi System. GFP The gene was used as a negative control and processed using the same procedure.

[0023] Table 1: Primer sequences for synthesized dsRNA.

[0024]

[0025] Note: The underlined sequences in the table represent the T7 promoter sequences introduced by primer 5'. (5) dsHvarAKR1B1 Optimal Interference Concentration Detection The experiment used newly molted (<12 h) 4th instar larvae of the ladybug, and employed the Nanoject III microinjection system to inject purified ds HvarAKR1B1 The solution (1000 ng) was injected into the *Heterodactylus spp.* through the abdominal segments. dsGFP*Heliotropium indicum* was used as a negative control, and those not injected with dsRNA served as a blank control. Each treatment was repeated three times, with 30 *Heliotropium indicum* per replicate. After injection, each *Heliotropium indicum* was placed individually in a glass beaker with ample food and reared in the aforementioned incubator. At 24 h, 48 h, and 72 h post-injection, 10 *Heliotropium indicum* were collected from each replicate to determine the dsRNA interference efficiency. The collected *Heliotropium indicum* were washed with distilled water, blotted dry with absorbent paper, placed in 1.5 mL centrifuge tubes, rapidly treated with liquid nitrogen, and then stored at -80°C for later use. qRT-PCR was used to detect the different treatment groups (uninjected, injected with dsRNA, and uninjected)... GFP and injection ds HvarAKR1B1 The expression level of the target gene in the qPCR was determined (qPCR program: pre-denaturation: 95℃, 5 min; denaturation: 95℃, 15 s; annealing: 58℃, 30 s; extension: 72℃, 30 s). Data were normalized using the *Dipladenia multifiliis* internal reference gene EF1α as an internal standard. The qPCR primers are shown in Table 2.

[0026] Table 2: Internal reference gene (EF1α) and HvarAKR1B1 qPCR primer sequences for the gene (6) Silence HvarAKR1B1 Effects of genes on the growth and development of *Heteromorpha maculatus* under starvation stress Uninjected dsRNA, Injected dsRNA GFP and injection ds HvarAKR1B1 The test insects were continued to be fed under starvation stress. Each treatment was repeated three times, with 30 ladybugs per replicate. The survival and development of the ladybugs were observed and recorded every 12 hours.

[0027] The results showed that during the synthesis of dsRNA, samples were taken before and after RNase and DNase digestion, and the purity was determined by 1% agarose gel electrophoresis. The dsRNA obtained after digestion... HvarAKR1B1 and ds GFP The lane appears as a single band, indicating that ds HvarAKR1B1 and ds GFP Purity meets standards. Inject DS. HvarAKR1B1 Upon entering the body of 4th instar larvae of *Heterodactylus simonii*, the gene was successfully interfered with within 24 hours, 48 ​​hours, and 72 hours. After interference for 24 hours, 48 ​​hours, and 72 hours... HvarAKR1B1 Gene expression levels compared to the blank control group injected with dsRNA and the group injected with dsRNA GFP Compared with the negative control, all were significantly reduced. The interference efficiency was highest at 48h, showing an overall trend of first increasing and then decreasing. Specifically, the interference efficiencies at 24h, 48h, and 72h were 79.55%, 98.03%, and 70.44%, respectively. (See Appendix) Figure 2 As shown.

[0028] interference HvarAKR1B1 The gene significantly shortened the developmental time of 4th instar larvae, pupae, and the period from 4th instar larvae to adults in *Heterodactylus spp.* Compared to the uninjected group, the developmental time of 4th instar larvae, pupae, and the period from 4th instar larvae to adults was only significantly reduced by 0.09 days, 0.15 days, and 0.24 days, respectively. [The text then abruptly shifts to a discussion of dsRNA injection and its effects on developmental time.] GFP The study had no significant impact on the developmental duration of the above-mentioned developmental stages of *Heteromorpha multifida*. (See attached table for results.) Figure 3 As shown in Table 3, the life table parameter results indicate that... HvarAKR1B1 Genes are involved in the ability of ladybugs to resist hunger stress.

[0029] Table 3: Silent Ladybug HvarAKR1B1 The Influence of Genes on Survival Rate and Initial Emergence Weight

[0030] The above embodiments are only for illustrating the technical concept and features of the present invention in a specific scenario. Their purpose is to enable those who need this technology to understand the content of the present invention and implement it. They do not limit the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A target gene of *Heteromorpha heterocerca* for adapting to starvation stress, characterized in that: The target gene is *Heterophyllum oxypetalum*. HvarAKR1B1 Primer sequences used to amplify gene coding region-specific interference fragments include or consist of the following sequences: 1) The nucleotide sequences shown in SEQ ID No. 1-SEQ ID No. 2; or 2) The nucleotide sequences shown in SEQ ID No.3-SEQ ID No.

4.

2. The use of the target gene as described in claim 1 in at least one of the following: 1) Improve the survival rate of 4th instar larvae of *Heteromorpha chinensis* under starvation stress; 2) Improve the emergence rate of ladybugs under starvation stress; 3) Improve the survival rate of fourth instar larvae to adults of ladybug under starvation stress.

3. The application as described in claim 2, characterized in that: Using the target gene of claim 1 and artificially synthesizing ds using the T7RiboMAX™ Express RNAi System synthesis kit HvarAKR1B1 ds HvarAKR1B1 The solution, 1000 ng, was injected into the body of *Heteromorpha heteromorpha* through the abdominal segments.

4. A method for reducing the resistance of ladybugs to starvation stress, characterized in that: Knockout or silence of the target gene as described in claim 1 in ladybugs.

5. The method as described in claim 4, characterized in that: The target gene in the aforementioned ladybug was inserted using RNAi technology, CRISPR-Cas technology, and T-DNA and promoter insertion. HvarAKR1B1 Knock out or silence.

6. A method for enhancing the resistance of ladybugs to starvation stress, characterized in that: Overexpression in *Heteromorpha spp.* as described in claim 1.

7. The method as described in claim 6, characterized in that: The target gene in the aforementioned ladybug was inserted using CRISPR-Cas technology, T-DNA, and promoter insertion. HvarAKR1B1 Overexpression.

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