An HcKynU gene, dsRNA targeting this gene, and their applications

Abstract

This invention relates to a HcKynU Genes, dsRNAs targeting these genes, and their applications. This invention focuses on horizontal gene transfer. HcKynU Designing dsRNAs specifically for the control of the fall webworm (Russian white moth) not only allows for precise pest control but also avoids threatening non-target species. Injecting this dsRNA into the fall webworm effectively silences its target gene, leading to increased mortality and impaired normal growth and development, ultimately achieving control. This invention provides a new and effective approach for controlling the fall webworm.

Description

Technical Field

[0001] This invention relates to the field of forestry genetic engineering technology, and more specifically, to a... HcKynU Genes, dsRNAs targeting these genes, and their applications. Background Technology

[0002] The American white moth (Hyphantria cunea) is a typical invasive leaf-eating pest, native to North America, and now spread to most parts of the world. It has a wide range of host plants and a large feeding capacity, consuming more than 600 host plant species, causing serious economic losses.

[0003] The development of molecular biology techniques has provided more possibilities for the green control of the fall webworm. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) refers to the phenomenon in which organisms induce the degradation of target RNA through endogenous or exogenous small RNAs (Neumeier and Meister, 2021). RNA interference technology is highly specific, harmless to non-target species, and has great application potential. This technology has become an important option for developing new forest pest management methods, and genes that increase mortality and affect normal growth and development can serve as good candidate target genes for RNA interference in the larval stage of the fall webworm.

[0004] Horizontal gene transfer (HGT) refers to the non-genetic exchange or lateral dissemination of genetic information between distantly related organisms. With the deepening research on insect biological adaptation, HGT events are considered a significant driving force for adaptive evolution in eukaryotes. Although horizontally transferred genes constitute a very small proportion of the genome of a single species, studies have shown that they possess positive adaptive evolutionary functions. Therefore, using horizontally transferred genes for pest control has advantages such as high specificity, low off-target effects, and good efficacy. Exploring RNA interference targeting horizontally transferred genes will provide a new method for green, efficient, and precise molecular regulation of the fall webworm. Summary of the Invention

[0005] This invention provides HcKynU Genes, the ones mentioned HcKynU The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] The present invention also provides a dsRNA, wherein the dsRNA is a target for the fall webworm. HcKynU dsRNA of genes.

[0007] Furthermore, the dsRNA is a double-stranded RNA composed of the nucleotide sequence shown in SEQ ID NO.6 and its inverse complementary sequence.

[0008] The present invention also provides a primer pair for amplifying the aforementioned dsRNA, the primer pair comprising a forward primer and a reverse primer, the forward primer being shown in SEQ ID NO.4 and the reverse primer being shown in SEQ ID NO.5.

[0009] The present invention also provides the application of the aforementioned dsRNA in the preparation of pesticides for controlling the fall webworm.

[0010] The present invention also provides a biological pesticide formulation for controlling the fall webworm, wherein the biological pesticide formulation includes the aforementioned dsRNA.

[0011] The present invention also provides a method for controlling the fall webworm, the method comprising interfering with the normal growth and development of the fall webworm or causing its death by injecting or feeding it the aforementioned dsRNA.

[0012] The beneficial effects of this invention include: this invention uses horizontal gene transfer HcKynU A gene-targeted dsRNA was designed for the control of the fall webworm, enabling precise pest control without threatening non-target species. Injecting this dsRNA into the fall webworm effectively silences its target gene, leading to increased mortality and impaired normal growth and development, ultimately achieving control. This invention provides a new and effective approach for controlling the fall webworm. Attached Figure Description

[0013] Figure 1 American white moth HcKynU Phylogenetic analysis of genes; Figure 2 To amplify using American white moth DNA and cDNA as templates, respectively HcKynU Electrophoresis diagram of genes; Figure 3 This is an electrophoresis diagram of the dsRNA amplification products in Example 2; Figure 4 In Example 3 HcKynU RNA interference effects on genes; Figure 5 The survival rate of the American white moth after injection of dsRNA in Example 4; Figure 6 The relative growth rate of the American white moth after injection of dsRNA in Example 5; Figure 7 The amount of feces from the American white moth after injection of dsRNA in Example 5. Detailed Implementation

[0014] The present invention will be further described and illustrated below with reference to embodiments. However, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the present invention and the embodiments, all other inventions and embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0016] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0017] Example 1: Horizontal gene transfer in the fall webworm - HcKynU Gene A search of the genome data of the American white moth (GenBank: GCA_003709505.1) yielded a gene with the nucleotide sequence shown in SEQ ID NO. 1. Blastp alignment of this gene in the GeneBank database and a search for homologous protein sequences in the NCBI RefSeq database revealed a high degree of similarity to the bacterial kynurenase gene, suggesting it may be a horizontal transfer gene with potential specific functions. Therefore, this gene was named... HcKynU Genes. The matching results were sorted by sequence consistency, and species with broad taxonomic representativeness were selected. The processed data included 16 bacterial sequences, 6 fungal sequences, and 5 animal sequences. The selected sequences were imported into MEGA 6.0 software, and protein sequence alignment was performed using Clustal W, with missing or incomplete regions pruned. A neighbor-joining phylogenetic tree was constructed using the alignment results, and the confidence of each branch node of the phylogenetic tree was evaluated using 1000 replicates of Bootstrap.

[0018] Furthermore, genomic analysis revealed HcKynUThe gene is located on scaffold-3411 and consists of a single exon without introns. Based on this, primers HcKynU-F: 5'-ATGGAATCAGATTATCAAGAG-3' (SEQ ID NO. 2) and HcKynU-R: 5'-TTAAACAACCAAATTTCTTTTC-3' (SEQ ID NO. 3) were designed to identify the presence of this gene in the genome of the fall webworm at both the genomic and transcriptional levels, and to confirm whether the gene is expressed at the transcriptional level. This experiment used 4th instar fall webworm larvae. Total genomic DNA was extracted from the fall webworm genome using the TIANamp Genomic DNA Kit (TIANGEN), and total RNA was extracted using the RNeasy Mini Kit (QIAGEN). Then, 1 μg of cDNA was synthesized using the GoScript™ Reverse Transcription System kit (Promega), and the cDNA product was diluted to 200 ng / μL. The PCR reaction mixture consisted of: 12.5 μL of 2' Easy Taq PCR SuperMix, 1 μL each of forward and reverse primers, 1.5 μL of template, and 9 μL of ddH2O. The PCR program was: 94℃ for 3 min; 94℃ for 30 s; 50℃ for 30 s; 72℃ for 1 min; 35 cycles; 72℃ for 10 min. The amplified products were detected by electrophoresis.

[0019] Phylogenetic analysis results as follows Figure 1 As shown, the selected gene sequences form two stable branches, with the fungal and animal sequences stably clustering into one branch, while... HcKynU Gene sequences and bacterial sequences stably cluster together, which can be inferred from high-confidence topological relationships. HcKynU The gene was horizontally transferred from bacteria of the genus Staphylococcus.

[0020] Electrophoresis results as follows Figure 2 As shown, a 1281 bp fragment was amplified using both the DNA and cDNA of the American white moth as templates, indicating that fragments could be identified at both the genomic and transcriptional levels. HcKynU The gene indicates that it exists in the genome and is expressed during the larval stage.

[0021] In conclusion, the American white moth HcKynU The gene is a horizontally transferred gene derived from bacteria, which exists in the genome of the fall webworm and is expressed at the transcriptional level. Further research can be conducted to explore its potential as a molecular target for the control of the fall webworm.

[0022] Example 2: Targeting the American White Moth HcKynU dsRNA of genes Targeting the American white moth HcKynU Specific dsRNA primer pairs were designed for the gene, specifically HcKynURi-F: 5'-GAAATATCATTATTGGGTGG-3' (SEQ ID NO. 4) and HcKynURi-R: 5'-TGTATCCTTCTTCGTGTTCTA-3' (SEQ ID NO. 5), and the synthesized dsRNA was 483 bp in length (as shown in SEQ ID NO. 6).

[0023] The T7 promoter sequence (TAATACGACTCACTATAGG, SEQ ID NO. 7) was added to the 5' end of two specific primers, HcKynURi-F and HcKynURi-R, respectively. Target bands with T7 at both the 5' and 3' ends were amplified by PCR. The PCR reaction system consisted of: 12.5 μL of 2´Easy Taq PCR SuperMix, 1 μL each of forward and reverse primers, 1.5 μL of dsRNA template, and 9 μL of ddH2O. The PCR program was: 94℃ for 3 min; 94℃ for 30 s; 52℃ for 30 s; 72℃ for 1 min; 35 cycles; 72℃ for 10 min. The amplified products, confirmed by electrophoresis, were used as templates for dsRNA synthesis using the MEGAscript RNAi Kit.

[0024] dsRNA was synthesized according to the MEGAscript RNAi Kit instructions. The concentration of dsRNA was detected using a micro-spectrophotometer, and 1 μL of dsRNA was analyzed by 1% agarose gel electrophoresis (see [link to kit]). Figure 3 The concentration of dsRNA was confirmed and uniformly adjusted to 5 μg / μl, and stored at -80℃ for later use. Green fluorescent protein (GFP) dsRNA of the same concentration was prepared as a control.

[0025] like Figure 3 As shown, this embodiment yielded a target moth of approximately 483 bp in size. HcKynU dsRNA of genes.

[0026] Example 3: Fall webworm HcKynU Gene RNA interference effect detection Take 2 μl of the synthesized product from Example 2 HcKynUdsRNA of the gene and GFP was microinjected into the abdomen of healthy, uniformly growing fourth-instar fall webworms, with a blank control (CK) set up. Each group had three replicates. Whole webworms were collected as samples 24 hours later, flash-frozen in liquid nitrogen, and stored at -80°C. Total RNA was extracted from the fall webworm samples using the RNeasy Mini Kit (QIAGEN). Then, 1 μg of cDNA was synthesized using the GoScript™ Reverse Transcription System (Promega) (PCR reaction system and procedure were the same as in Example 1). The cDNA product was diluted to 200 ng / μL and used as a template for quantitative real-time RT-PCR to detect the effects of dsRNA injection. HcKynU Gene expression levels.

[0027] The results are as follows Figure 4 As shown, compared with CK and control (dsGFP, injected with dsRNA of the GFP gene), the injection... HcKynU The American white moth (dsRAN) HcKynU ) inside the body HcKynU Gene expression levels decreased significantly within 24 hours. Figure 4 * indicates p < 0.05, ** indicates p < 0.01, indicating that the dsRNA has a significant effect and can be used for RNA interference.

[0028] Example 4 HcKynU Survival rate of fall white moth after gene silencing 2 μl of the synthesized in Example 2 HcKynU The dsRNA of the gene was microinjected into the abdomen of healthy, uniformly grown larvae at the late 3rd or early 4th instar of the fall webworm. The control group was injected with an equal amount of dsGFP, and a blank control (CK) was also included. Each treatment had 40 replicates. A booster injection was performed on the third day after initial injection to ensure complete remission. HcKynU Gene expression levels continued to decrease. After injection, the patients were fed fresh mulberry leaves, and the survival rate was observed and recorded daily for 12 days.

[0029] The results are as follows Figure 5 As shown, during injection HcKynU Following the addition of dsRNA to the gene, the survival rate of American white moth larvae was significantly lower than that of the control group (dsGFP) and the CK group, indicating that the dsRNA synthesized in Example 2 of this invention... HcKynU The dsRNA of a gene can interfere HcKynU The expression, and HcKynU Low gene expression affects the survival rate of fall webworm larvae. Furthermore, continuous observation has revealed that interference... HcKynU Genetic modification lengthens the time interval between the development of the American white moth to the next instar, affecting its development.

[0030] Example 5 HcKynU Growth and development of the fall webworm after gene silencing Before the experiment, early-stage fourth instar fall webworms were prepared and weighed using an analytical balance to ensure that the size and weight of the test insects remained within a certain range (weight difference less than 0.005 g). The test insects were starved for 6 hours, and their initial fresh weight was measured. Then, 2 μl of the pre-synthesized material from Example 2 was added. HcKynU The dsRNA of the gene was microinjected into the abdomen of fall webworm larvae, while the control group was injected with an equal amount of dsGFP. A blank control (CK) was also included. The tested insects were fed with fresh mulberry leaves. Each treatment contained 15 fall webworm larvae and three replicates. Forty-eight hours after injection, the tested insects were starved for 6 hours to empty their feces. The fresh weight of the larvae and the fresh weight of the feces were then measured two days later. HcKynU Deaths occurred within 2 days after gene dsRNA injection, so the relative growth rate was calculated based on the average value of the insects. No molting occurred during the experiment.

[0031] The formula for calculating the relative growth rate is: relative growth rate

[0032] Where A is the average weight gain of the tested insects (where the weight gain of each tested insect is the fresh weight after 2 days minus the initial fresh weight), B is the average weight of the tested insects (where the weight of each tested insect is the average of the initial fresh weight and the fresh weight after 2 days), and T is the number of experimental days (2 days in this experiment).

[0033] like Figure 6 , 7 As shown, compared to the GFP group (dsGFP) and the CK group, the injection... HcKynU After gene dsRNA (dsHcKynU) was introduced, the relative growth rate and the amount of feces produced by the American white moth both decreased significantly. Figure 6 **** represents p < 0.0001, *** represents p < 0.001. Figure 7 * indicates p < 0.05, ** indicates p < 0.01, indicating interference. HcKynU In addition to increasing the mortality rate of the fall webworm, the genetic modification also produces a sublethal effect, affecting the normal growth and development of the fall webworm.

Claims

1. A dsRNA, characterized in that, The dsRNA is a double-stranded RNA consisting of a nucleotide sequence represented by SEQ ID NO. 6 and a reverse complement thereof, which targets HcKynU a gene, wherein the nucleotide sequence of the gene is represented by SEQ ID NO.

1. HcKynU a gene, wherein the nucleotide sequence of the gene is represented by SEQ ID NO.

1.

2. A primer pair for amplifying the dsRNA of claim 1, characterized in that, The primer pair comprises a forward primer and a reverse primer, the forward primer is shown as SEQ ID NO. 4, and the reverse primer is shown as SEQ ID NO.

5.

3. The use of the dsRNA in claim 1 in the preparation of a Malacosoma americanum prevention and treatment agent.

4. A biopesticide preparation for controlling Malacosoma americanum, characterized by, The biological pesticide preparation comprises the dsRNA in claim 1.

5. A method of controlling Malacosoma americanum, characterized by, The normal growth and development of Malacosoma americanum is interfered or the Malacosoma americanum is killed by injecting the dsRNA in claim 1.

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