Synergist of dsRNA (double-stranded ribonucleic acid) as well as application and method of synergist in improving RNAi (ribonucleic acid interference) efficiency of lepidoptera pests

Abstract

The invention belongs to the technical field of biology, and particularly relates to a dsRNA synergist, and application and a method of the dsRNA synergist in improving the RNAi efficiency of lepidoptera pests. By analyzing factors influencing the RNAi efficiency of the ostrinia furnacalis, a gene which can be induced to be up-regulated by dsRNA and is named as REX4 gene is found, REX4 protein coded by the gene is found to be capable of directly degrading the dsRNA under in-vitro conditions, the degradation efficiency is relatively high, aspartic acid at a first catalytic site is mutated into glycine, and the gene can be used for degrading the dsRNA at a second catalytic site. And the nucleic acid degradation capability of the REX4 protein is obviously reduced. Insect RNAi efficiency can be improved by inhibiting expression of the REX4 gene, and the gene generally exists in lepidoptera insects. The REX4 protein provided by the invention can be used as a core target for improving the RNAi efficiency of the lepidoptera pests, the problem of low RNAi efficiency of the lepidoptera pests is solved, and the application of the RNAi technology in agricultural pest control is promoted.

Description

A dsRNA enhancer and its application and method in improving RNAi efficiency in lepidopteran pests Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a dsRNA enhancer and its application. Background Technology

[0002] The discovery of RNAi technology has greatly advanced research on insect gene function and regulation, particularly in non-model insects. RNAi technology can easily and conveniently reduce the expression of target genes without completely eliminating their messenger RNA, making it easier to analyze the function of target genes based on insect phenotypic changes. Besides being an important technique for insect gene function research, RNAi is also a new technology already applied to pest control. By inhibiting the expression of important genes during insect growth and development, it can cause growth and developmental disorders or even death in insects, thus achieving pest control.

[0003] However, research over the past 20 years has shown that RNAi efficiency is easily affected by a variety of factors, making it a major concern. Some studies indicate that RNAi efficiency is relatively high in Coleoptera, while its effectiveness is less than ideal in Lepidoptera such as moths and butterflies. Researchers have conducted extensive studies on the differences in RNAi efficiency between Coleoptera and Lepidoptera. Patent 201611244499.6 discloses a negative regulatory gene, REase, that affects dsRNA function. This gene can reduce RNAi efficiency by inhibiting the types and number of effective siRNAs cleaved from dsRNA. Therefore, downregulating the expression of the REase gene or its homologous peptides in insects can improve RNAi efficiency in Lepidoptera. Currently, the possible reasons affecting RNAi efficiency in Lepidoptera include: low efficiency of dsRNA uptake by cells, dsRNA degradation, low expression levels of key RNAi proteins, and the presence of specific RNAi pathway-related genes. Degradation of dsRNA by nucleases in the insect midgut or hemolymph is the primary reason for low RNAi efficiency. Therefore, finding nucleases that can degrade dsRNA is crucial for studying RNAi efficiency. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes a dsRNA enhancer and its application and method in improving RNAi efficiency in lepidopteran pests.

[0005] The technical solution of this invention is implemented as follows:

[0006] On one hand, the present invention provides a dsRNA enhancer, wherein the enhancer is dsREX4 or a mutant of the REX4 protein.

[0007] Preferably, the nucleotide sequence of the above-mentioned dsREX4 is shown in SEQ ID No. 3.

[0008] Preferably, the mutant of the REX4 protein is a mutant that loses the function of the REX4 protein.

[0009] Preferably, the amino acid sequence of the REX4 protein is shown in SEQ ID No. 2.

[0010] Preferably, the mutant is obtained by mutating the 7th amino acid of the REX4 protein from aspartic acid to glycine.

[0011] Secondly, this application provides the application of the aforementioned synergist in enhancing the dsRNA of Lepidoptera genes.

[0012] Preferably, the dosage of dsREX4 is 2 μg per insect. The appropriate injection dose should be selected based on the insect size to ensure effective suppression of the REX4 gene.

[0013] Preferably, the concentration of the mutant REX4 protein used is 1 mg / mL, and the volume is 2 μL; the temperature at which the mutant is used is 25-37℃.

[0014] Thirdly, this application provides a method for improving the efficiency of RNAi in lepidopteran pests, characterized by the following steps: simultaneously injecting dsREX4 into the lepidopteran pests during RNAi treatment. The dosage of dsREX4 is 2 μg per pest.

[0015] Annotating dsREX4 while injecting target dsRNA (dsCTP8, dsKTI) can effectively increase the inhibition efficiency of the target gene and improve the RNAi effect, indicating that the expression level of the REX4 gene can affect the RNAi efficiency of lepidopteran pests.

[0016] The REX4 gene is mainly expressed in the midgut of the corn borer larvae and can only be upregulated by dsRNA. The REX4 gene participates in the regulation of RNAi efficiency in the Asian corn borer and has a strong response to dsRNA. The REX4 protein does not have specific selectivity for nucleic acid degradation and can directly degrade nucleic acids such as dsRNA and DNA, with better degradation effect on dsRNA.

[0017] Preferably, the nucleotide sequence of the above-mentioned dsREX4 is shown in SEQ ID No. 3.

[0018] The present invention has the following beneficial effects:

[0019] This invention analyzes factors affecting the efficiency of RNAi in the Asian corn borer and identifies a REX4 gene that can be upregulated by dsRNA. The REX4 gene is mainly expressed in the midgut of the corn borer larvae and can only be specifically upregulated by dsRNA. The REX4 protein can directly degrade dsRNA and DNA in vitro, exhibiting high efficiency in dsRNA degradation. Mutating the first catalytic site of the REX4 protein, aspartic acid "D", to glycine "G", significantly reduces the protein's ability to degrade nucleic acids. Simultaneously inhibiting the target gene and injecting dsREX4 to suppress REX4 gene expression effectively increases the inhibition efficiency of the target gene and improves RNAi efficacy. The expression level of the REX4 gene affects the efficiency of insect RNAi, and this gene is widely present in lepidopteran insects. The REX4 protein provided by this invention can serve as a core target for improving the efficiency of RNAi in lepidopteran pests, addressing the problem of low RNAi efficiency in lepidopteran pests and promoting the application of RNAi technology in agricultural pest control. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 shows the analysis of conserved amino acid sites in the REX4 sequence.

[0022] Figure 2 shows the expression pattern analysis of the REX4 gene; where A represents the expression level of REX4 in different tissues of corn borer without any treatment, B represents the expression level of REX4 at different developmental stages, C represents the response of REX4 to different immune stimuli, and D represents the expression level of REX4 at different time points after dsRNA treatment. "*" indicates significant difference (p<0.05), and "**" indicates extremely significant difference (p<0.01).

[0023] Figure 3 shows the prokaryotic expression and protein purification of REX4-pET44b; where A represents the solubility detection of REX4-pET44b fusion protein, "1" represents the total lysate of uninduced REX4-pET44b cells, "2" represents the total lysate of induced REX4-pET44b cells, "3" represents the supernatant after ultrasonic disruption, "4" represents the precipitate resuspension after ultrasonic disruption, M: pre-stained protein marker, and B represents the quantitative analysis of purified REX4-pET44b fusion protein, where "1" represents 1 µL of purified protein solution, "2" represents 2 µL of purified protein solution, "3" represents 1 µg of BSA standard, "4" represents 2 µg of BSA standard, and "5" represents 4 µg of BSA standard.

[0024] Figure 4 shows that the REX4 protein has a good degradation effect on dsRNA under in vitro conditions; where A indicates that the REX4 protein can degrade different nucleic acids under in vitro conditions, and B indicates that the REX4 protein has a better degradation effect on dsRNA.

[0025] Figure 5 shows the efficiency of REX4 protein in degrading nucleic acids.

[0026] Figure 6 shows the REX4 homologous sequence alignment and phylogenetic tree analysis; where A is the REX4 amino acid sequence alignment of different insects, B is the REX4 gene phylogenetic tree analysis, and the red text marks the short REX4 homologous sequences.

[0027] Figure 7 shows the response of the REX4 homolog of the cabbage white butterfly to dsRNA and its prokaryotic expression sequence; where A shows that the long REX4 fragment of the cabbage white butterfly does not respond to dsRNA stimulation, B shows that the short REX4 fragment can be significantly induced and upregulated by dsRNA, C shows the amino acid sequence alignment of REX4 from the corn borer, cotton bollworm and cabbage white butterfly, and D shows different prokaryotic expression sequences designed for the REX4 genes of cotton bollworm and cabbage white butterfly. "H.REX4-0" represents the full-length sequence (1-1035 bp) of the exonuclease 4-like RNA gene of cotton bollworm, "H.REX4-1" represents the partial sequence (1-528 bp) of the 5′ end of the REX4 gene of cotton bollworm, and "H.REX4-2" represents the partial sequence (496-1035 bp) of the 3′ end of the REX4 gene of cotton bollworm. “P.REX4-332” represents the full-length sequence (1-999 bp) of the long REX4 gene in the Chinese cabbage white butterfly, and “P.REX4-154” represents the full-length sequence (1-465 bp) of the short REX4 gene in the Chinese cabbage white butterfly. Mean+SD, n=3, “**” indicates extremely significant difference (p<0.01).

[0028] Figure 8 shows that inhibiting REX4 gene expression can increase host RNAi efficiency. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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.

[0030] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0031] This application analyzed factors affecting the efficiency of RNAi in the Asian corn borer and discovered a gene that can be upregulated by dsRNA. This gene contains a conserved DEDDh domain, possesses 3′-5′ exonuclease activity, and belongs to the RNA exonuclease 4-like family. Based on its structural characteristics, it was named the REX4 gene. We found that the REX4 protein can degrade various nucleic acids under in vitro conditions, with high efficiency in degrading dsRNA. When one of the catalytic sites was mutated, the nucleic acid degradation ability of the REX4 protein was significantly reduced; and this gene is widely distributed in lepidopteran insects.

[0032] Table 1 Primer information used in this application

[0033]

[0034] The primer information used in this application is shown in Table 1.

[0035] Example 1: Analysis of the amino acid sequence encoded by REX4

[0036] Primers were designed based on sequence information from transcriptome data to amplify the full-length sequence of the REX4 gene. The gene is 477 bp in length, and its sequence is shown in SEQ ID No. 1. It encodes 158 amino acids, the amino acid sequence of which is shown in SEQ ID No. 2. Prediction of the signal peptide sequence revealed that the gene does not encode a signal peptide, suggesting it is a non-secretory protein. A search of the gene sequence on the NCBI website revealed that the sequence it encodes belongs to the Asian corn borer RNA exonuclease 4-like protein family, hence we named it the REX4 gene (GeneBank accession no. XM_028309538.1). This gene contains a conserved DEDDh 3′-5′ exonuclease domain. Gene families containing this domain include REX4 (Rex4p), XPMC2, ISG20, and other similar proteins. Genes in this family play an important role in the processing of rRNA precursors.

[0037] We downloaded and analyzed sequences from the NCBI website that showed high amino acid similarity to the REX4 gene. We found that only a few lepidopteran insects and *Ctenopharynx* species had protein sequences of similar length to the REX4 gene from the Asian corn borer. Other protein sequences annotated as RNA exonuclease 4-like were relatively long fragments. We first performed multiple alignments on the short RNA exonuclease 4-like sequences, revealing that they all possessed the five conserved DEDDH catalytic sites (Figure 1). The black highlighted areas indicate conserved sites between different sequences, and the * indicates the locations of several conserved DEDDH catalytic sites. Therefore, we hypothesize that the REX4 gene from the corn borer possesses the typical 3′-5′ exonuclease function of the RNA exonuclease 4-like family. Based on the data we have found so far, most of the homologous genes of REX4 are distributed in Lepidoptera insects. Sequences with high similarity to REX4 have been found in Lepidoptera species such as Pieridae, Papilionidae, and Nymphalidae, as well as in Paphiopedilum fruticosa.

[0038] Example 2: Tissue Expression Analysis of REX4 Gene

[0039] The expression pattern of the REX4 gene was analyzed using quantitative real-time fluorescence detection. The expression levels of the REX4 gene in the head, brain, midgut, hemolymph, and fat body of the fifth instar larvae of the corn borer were detected. As shown in Figure 2A, the results indicate that the gene is highly expressed only in the midgut, and almost not expressed in the other sites. Analysis of expression levels at different developmental stages showed that the gene is highly expressed throughout the larval and pupal stages (Figure 2B).

[0040] The expression of this gene was detected in corn borers after different immune stimuli. Samples were taken after treatment with dsRNA (dsEGFP, sequence as shown in SEQ ID No.4), bacteria (Escherichia coli), fungi (Saccharomyces cerevisiae), and virus (NPV virus) for 6 h. The results showed that only dsEGFP treatment could induce the expression of the REX4 gene in corn borers (Figure 2C).

[0041] Further sampling at different time points after dsRNA treatment revealed that the REX4 gene showed upregulation 1 h after dsRNA treatment, and the upregulation trend could continue for 8 h or even longer (Figure 2D). Therefore, we believe that the REX4 gene is mainly expressed in the midgut of the corn borer larvae and can only be upregulated by dsRNA.

[0042] Based on the above results, we speculate that the REX4 gene may be involved in the regulation of RNAi efficiency in Asian corn borers and has a strong response to dsRNA.

[0043] Example 3: Expression of REX4 fusion protein

[0044] The correctly sequenced fusion expression vector was transformed into expression strain BL21(DE3), and positive expression strains were selected to explore the protein induction conditions. It was ultimately determined that the highest expression level of the fusion protein was achieved at 16℃ and a final IPTG concentration of 0.5 mM; therefore, this induction condition was used for subsequent protein expression. Solubility analysis showed that almost all of the REX4-pET44b fusion protein was present in the supernatant of the lysis buffer (Figure 3A), indicating that the fusion protein could be expressed in a soluble form in expression strain BL21(DE3), which facilitated subsequent purification. The prokaryotic expression protein was purified using Ni-NTA column affinity chromatography. The quality of the purified protein was assessed by SDS-PAGE gel electrophoresis, and the concentration of the purified protein was determined by BCA method and serial dilution with BSA standards. The final results showed that the concentration of the purified fusion protein was approximately 1 mg / mL (Figure 3B). The protein was then aliquoted and stored at -80℃ for later use.

[0045] Example 4: REX4 protein functional analysis

[0046] The REX4 gene possesses a typical DEDDh 3′-5′ exonuclease domain, leading us to hypothesize that it may have a function in degrading nucleic acids. We incubated the purified REX4 protein with different types of nucleic acids, and analyzed the degree of degradation by the protein using agarose gel electrophoresis. Complete degradation of all nucleic acids was detected after 8 h of incubation at 37°C, indicating that the REX4 protein does not exhibit specific selectivity for nucleic acid degradation (Figure 4A). We then further analyzed the degradation rate of nucleic acids by the REX4 protein. We used equal amounts of dsRNA and dsDNA (500 ng / μL each), totaling 10 μL, for the experiment. The dsDNA consisted of the DNA fragment used to synthesize dsREX4, or any dsRNA or dsDNA.

[0047] Samples were taken at five time points—2 h, 4 h, 6 h, 8 h, and 10 h—after co-incubation of REX4 protein and nucleic acids. The results showed that dsRNA samples were almost completely degraded within 2–4 h, while dsDNA was only completely degraded around 8 h (Figure 4B). This result further demonstrates that REX4 protein has a better degradation effect on dsRNA.

[0048] We then further verified the nuclease function of REX4 as a member of the DEDDh gene family by mutating the key active site of REX4. We mutated the first catalytic site of REX4, aspartic acid "D", to glycine "G" (Figure 5A) through site-directed mutagenesis, and incubated the protein and nucleic acid before and after the catalytic site mutation to detect the changes in its nuclease activity.

[0049] We analyzed the degradation rates of dsRNA and dsDNA by the REX4 protein at two temperature conditions: 25℃ and 37℃. At 25℃, dsRNA began to degrade after 2 hours of incubation with the REX4 protein, and was almost completely degraded after about 4 hours. However, the protein with the REX4 site mutation could not effectively degrade dsRNA (Figure 5B). dsDNA, on the other hand, did not begin to degrade after 4 hours of incubation, and undegraded DNA fragments could still be detected after about 8 hours. There was no difference in degradation efficiency before and after the site mutation (Figure 5C). At 37℃, the REX4 protein was more effective at degrading dsRNA. The protein with the site mutation could completely degrade dsRNA after 8 hours (Figure 5D). dsDNA showed significant degradation after about 4 hours and was completely degraded after about 8 hours (Figure 5E).

[0050] The degradation rate of nucleic acids by the REX4 protein in vitro is temperature-dependent, with higher efficiency observed at 37°C. REX4 can degrade various nucleic acids, but its degradation effect on dsRNA is stronger. Mutating one of the catalytic sites in REX4 significantly reduced its nucleic acid degradation efficiency, but did not completely eliminate it. This result further confirms that the REX4 gene belongs to the DEDDh exonuclease family, cleaving nucleic acids through a relatively conserved catalytic site.

[0051] Example 5: Functional Conservatism Analysis of REX4

[0052] During sequence analysis, we found that among the homologous genes of REX4 found in other species, the sequences annotated as RNA exonuclease 4-like included both short fragments with few amino acids and long fragments with many amino acids. We performed multiple alignments on 37 amino acid sequences from 31 insect species (species and sequence information are shown in Appendix Table 1). The results showed that the long fragments had an additional sequence with an unknown function at the 5′ end. Currently, RNA exonuclease 4-like family genes have been found in Lepidoptera, Diptera, Hymenoptera, Blattodea, and Siphonaptera insects. Furthermore, both long and short fragment genes annotated as RNA exonuclease 4-like proteins exist in seven Lepidoptera species: Asian corn borer, silkworm (Bombyx mori), cabbage white butterfly (Pieris rapae), golden swallowtail butterfly (Papilio machaon), white-banded swallowtail butterfly (Papilio polytes), citrus swallowtail butterfly (Papilio xuthus), and variegated eye butterfly (Bicyckus anynana) (Figure 6A). Based on these sequences, we constructed a phylogenetic tree (Figure 6B). We found that the REX4 sequence of the corn borer is most closely related to the homologous gene in the cabbage white butterfly. Short fragment sequences of several lepidopteran insects clustered together, while the long and short fragments in the same species were relatively distantly related. We speculate that RNA exonuclease 4-like sequences of different lengths may have different biological functions.

[0053] Taking the cabbage white butterfly as an example, we first analyzed the response of two different genes annotated as RNA exonuclease 4-like in the cabbage white butterfly to dsRNA (Figure 7A, B). Four hours after injection of dsEGFP, the expression level of the short REX gene was upregulated, while the long REX4 gene could not be induced.

[0054] Table 1. Homologous sequence information of REX4 in insects

[0055]

[0056] To investigate the functional differences among RNA exonuclease 4-like sequences of different sizes, we selected three genes annotated as RNA exonuclease 4-like from cotton bollworm and cabbage white butterfly (Figure 7C, D, Table 1), and constructed corresponding expression vectors with gene fragments of different lengths. We plan to obtain several different proteins through a prokaryotic expression system and further analyze their nucleic acid degradation function through in vitro experiments.

[0057] Example of implementation results: The effect of REX4 on the efficiency of insect RNAi

[0058] The CTP8 (Chymotrypsins 8) and KTI (Kunitz trypsin inhibitor) genes in the corn borer were selected as targets, and the corresponding dsCTP8 (sequence shown in SEQ ID No. 5) and dsKTI (sequence shown in SEQ ID No. 6) genes were injected into the fourth-instar corn borer. Simultaneously, an additional treatment group was established, which, while inhibiting the target gene, was injected with dsREX4 at a dosage of 2 μg per worm. The injection doses per worm were as follows: dsEGFP (1000 ng / μL × 2 μL), dsREX4 (1000 ng / μL × 2 μL), dsKTI (1000 ng / μL × 2 μL), dsCTP8 (1000 ng / μL × 2 μL), dsREX4+dsCTP8 (2000 ng / μL × 1 μL + 2000 ng / μL × 1 μL), and dsREX4+dsKTI (2000 ng / μL × 1 μL + 2000 ng / μL × 1 μL); this inhibited the expression of the REX4 gene.

[0059] Twenty-four hours later, samples were taken, RNA was extracted, and the expression of the corresponding target genes was detected. It was found that injecting dsREX4 simultaneously with the injection of target dsRNA (dsCTP8, dsKTI) could effectively increase the inhibition efficiency of the target genes and improve the RNAi effect (Figure 8). This indicates that the expression level of the REX4 gene can affect the efficiency of insect RNAi.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A dsRNA enhancer, characterized in that: The synergist is dsREX4 or a mutant of the REX4 protein.

2. The dsRNA enhancer according to claim 1, characterized in that: The nucleotide sequence of the dsREX4 is shown in SEQ ID No.

3.

3. The dsRNA enhancer according to claim 1, characterized in that: The mutant of the REX4 protein is a mutant that loses the function of the REX4 protein.

4. The dsRNA enhancer according to claim 1, characterized in that: The amino acid sequence of the REX4 protein is shown in SEQ ID No.

2.

5. The dsRNA enhancer according to claim 4, characterized in that: The mutant is obtained by mutating the 7th amino acid of the REX4 protein from aspartic acid to glycine.

6. The use of the synergist according to any one of claims 1-5 in enhancing dsRNA in Lepidoptera insect genes.

7. The application according to claim 6, characterized in that: The dosage of dsREX4 was 2 μg per insect.

8. The application according to claim 6, characterized in that: The concentration of the mutant REX4 protein used is 1 mg / mL; the temperature for using the mutant is 25-37℃.

9. A method for improving the efficiency of RNAi in lepidopteran pests, characterized in that, The procedure is as follows: dsREX4 is injected into lepidopteran pests while they are being treated with RNAi.

10. The method according to claim 9, characterized in that: The nucleotide sequence of the dsREX4 is shown in SEQ ID No. 3; the dosage of dsREX4 is 2 μg / insect.

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