Peroxide reductase coding gene dsRNA for preventing and treating cedar macrophaga longibrachii and application of peroxide reductase coding gene dsRNA

Abstract

The invention discloses a peroxide reductase coding gene dsRNA for preventing and treating cedar macrotropis longipes and application, and belongs to the field of reductase coding, the peroxide reductase coding gene comprises a dsRNA sequence of a targeted cedar macrotropis longipes peroxide reductase Peroxiredoxin gene, and the dsRNA sequence is a dsRNA sequence of the targeted cedar macrotropis longipes peroxide reductase Peroxiredoxin gene. The sequence has a good silencing effect on the Peroxiredoxin gene of the cedar macrophis longibrachii, and the survival rate of the cedar macrophis longibrachii is remarkably reduced.

Description

Technical Field

[0001] This invention relates to the field of reductase encoding, and more particularly to a peroxidase encoding gene dsRNA for controlling the cedar aphid and its application. Background Technology

[0002] The cedar aphid is a major agricultural and forestry pest that infests coniferous trees such as cedar. It weakens trees and causes yellowing leaves by piercing and sucking the sap of its host plants, severely impacting their ornamental and economic value. Currently, control of the cedar aphid mainly relies on chemical pesticides. However, the long-term and excessive use of chemical pesticides leads to serious environmental pollution problems. Furthermore, pesticide residues harm soil, water sources, and non-target organisms. Long-term use of pesticides with a single mechanism of action also leads to aphid resistance, resulting in a gradual decline in control effectiveness. Therefore, developing a novel, environmentally friendly, highly targeted, and less resistant green pest control technology has become an urgent need in this field.

[0003] RNA interference (RNAi) is a highly efficient and specific gene silencing mechanism induced by double-stranded RNA (dsRNA). By designing dsRNAs homologous to the target gene sequence, the messenger RNA (mRNA) of that gene can be specifically degraded, thereby inhibiting the synthesis of the corresponding protein and leading to phenotypes such as developmental arrest and death in the target organism. Due to its high sequence specificity, RNAi technology is considered a highly promising next-generation pest control technology, which can be used to develop novel biopesticides or cultivate insect-resistant transgenic plants. Peroxidases are important antioxidant enzymes in organisms, playing a crucial role in scavenging reactive oxygen species (ROS) and maintaining cellular redox homeostasis. For herbivorous insects, they face stress from defensive secondary metabolites such as ROS produced by host plants during feeding; therefore, peroxidases are essential for their survival.

[0004] However, as of the date of this application, no research has reported cloning of the peroxidase gene of the cedar aphid, nor has any research revealed that this gene can serve as an effective target for RNAi, and no specific dsRNA sequence that can efficiently silence this gene and cause significant mortality in the cedar aphid has been disclosed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a peroxidase encoding gene for the control of the cedar aphid, thereby addressing the problem that the control of the cedar aphid mainly relies on chemical insecticides in the existing technology.

[0006] The present invention is achieved through the following technical solution: a peroxidase encoding gene for controlling the cedar aphid, the amino acid sequence of the protein of the peroxidase of this gene is shown in SEQ ID NO: 2.

[0007] The present invention also provides an isolated nucleic acid molecule that encodes the protein as described above, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0008] The present invention also provides a dsRNA for controlling the peroxidase encoding gene of the cedar aphid, which can specifically downregulate the expression of the nucleic acid molecules described above in the cedar aphid, and the dsRNA contains a nucleotide sequence that is SEQ ID NO: 3 or has at least 95% sequence identity with SEQ ID NO: 3.

[0009] The present invention also provides a DNA molecule that can be transcribed into dsRNA as described above.

[0010] The present invention also provides an application of the peroxidase encoding gene for controlling the cedar aphid, the protein as described above, the dsRNA as described above, and the DNA molecule as described above in the preparation of an aphid-killing composition for controlling the cedar aphid.

[0011] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0012] 1. This invention is the first to identify the peroxidase (CcPeroxiredoxin) and its encoding gene in the cedar aphid, and confirms that it is a key gene for maintaining the survival of aphids. Experiments show that this gene is highly expressed in multiple developmental stages and key tissues (such as the head and midgut) of aphids, revealing its important function in the life activities of aphids and providing a scientific basis for targeted prevention and control.

[0013] 2. The specific dsRNA sequence provided by this invention can efficiently silence the expression of the CcPeroxiredoxin gene (mRNA level downregulated by 40.17%) after delivery via feeding. More importantly, this efficient gene silencing can be converted into a significant aphid-killing effect. The mortality rate of aphids in the experimental group was significantly higher than that in the control group, proving that the dsRNA of this invention has great potential to be developed into a biological insecticide.

[0014] 3. Based on the high sequence specificity of RNAi, the dsRNA provided by this invention is effective only against the cedar aphid containing the homologous gene sequence. It is safe for non-target organisms (such as natural enemies and pollinating insects) and the environment, making it a green pest management method. Furthermore, the provided dsRNA can be developed into a biological aphid that can be sprayed directly, or it can be integrated into the host plant (such as cedar) through transgenic technology to make it persistently express dsRNA, thereby obtaining endogenous and stable aphid resistance, which has broad application prospects. Attached Figure Description

[0015] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0016] Figure 1 Aphids of different ages provided in Example 2 of the present invention Peroxiredoxin A diagram illustrating the representation of 1.

[0017] Figure 2 Different tissues of adult aphids provided in Example 2 of the present invention Peroxiredoxin Expression pattern analysis diagram.

[0018] Figure 3 This is a feeding statistics chart provided in Embodiment 4 of the present invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated herein by reference to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. The terms “comprising,” “including,” “having,” “containing,” etc., as used herein are open-ended terms, meaning that they include but are not limited to. Unless the context clearly indicates otherwise, the expressions “a” and “an” as used herein include plural references. It should be noted that “first,” “second,” etc., are used merely for convenience of description and distinction and should not be construed as indicating or implying relative importance. The term “about” as used herein indicates a range of ±20% of the following value. In some embodiments, the term “about” indicates a range of ±10% of the following value. In some embodiments, the term “about” indicates a range of ±5% of the following value. Unless otherwise specified, the reagents and materials used in the examples are commercially available.

[0021] Example 1

[0022] Cloning and sequence analysis of the CcPeroxiredoxin gene from the cedar aphid.

[0023] 1) Extraction of total RNA from aphids:

[0024] Healthy mixed-stage samples of cedar aphids raised in the laboratory were collected, rapidly frozen in liquid nitrogen, and ground into powder.

[0025] Add 1 mL of TRIzol reagent to every 100 mg of sample to extract total RNA. The concentration and purity of RNA were determined using a NanoDrop 2000 spectrophotometer (A260 / A280 ratio should be between 1.8 and 2.1), and its integrity was assessed by 1.2% agarose gel electrophoresis.

[0026] 2) Synthesis of the first strand of cDNA:

[0027] Take 2 μg of high-quality total RNA and reverse transcribe it using the PrimeScript™ RT Reagent Kit with gDNA Eraser. The reaction system is as follows:

[0028] First, 2 μg RNA, 2 μL 5×gDNA Eraser Buffer, 1 μL gDNA Eraser, add ddH2O to 10 μL, and react at 42℃ for 2 min to remove genomic DNA.

[0029] Subsequently, 4 μL of 5×PrimeScript Buffer 2, 1 μL of RT Primer Mix, 1 μL of PrimeScript RT Enzyme Mix I, and 4 μL of RNase-free H2O were added to form a 20 μL reverse transcription system.

[0030] The reaction procedure is as follows:

[0031] Incubate at 37°C for 15 min, then at 85°C for 5 s. The product is the first strand of cDNA, which should be stored at -20°C for later use.

[0032] 3) Cloning of the full-length CDS: Based on the known conserved sequence information of Peroxiredoxin genes in other aphids, degenerate primers (not shown) were designed, and the core fragment was obtained by PCR amplification. Then, specific primers were designed based on the core fragment sequence, and the 5' and 3' end sequences of the gene were amplified using RACE (Rapid Amplification of cDNA Ends) technology. The obtained sequences were then spliced ​​together to obtain the complete coding region (CDS) sequence of the CcPeroxiredoxin gene.

[0033] 4) Sequence analysis:

[0034] The assembled cDNA sequence was sequenced for verification. Sequencing results showed that a complete CDS sequence of 681 bp was obtained, and its nucleotide sequence is shown in SEQ ID NO: 1. This CDS sequence encodes a protein composed of 226 amino acids, named CcPeroxiredoxin, and its amino acid sequence is shown in SEQ ID NO: 2.

[0035] SEQ ID NO: 1 is:

[0036] .

[0037] SEQ ID NO: 2 is:

[0038] MRLNSVVPDFSGPSTKGPIDFYNWLGDSWCVLFAHPADFTPVCTTELGKMAVLVDEFTKRNTKVLGLSCDKLESHVNWINDIKSYCVDIEGEFPFPIISDSSRELAIKLD MLAEEDKNNPDTAMTIRSLYIIGPDKKVKLMMVYPTSTGRNIQEVLRCIDSLQLCDKKKIVATPVNWVPGEKVMILPSVDDKDLNALFPNGHEKCSMPSSINYLRMTSDY.

[0039] Example 2:

[0040] Expression pattern analysis of the CcPeroxiredoxin gene.

[0041] 1) Sample collection:

[0042] Different developmental stages: Synchronized cedar aphids were reared on cedar branches, and 1st, 2nd, 3rd, and 4th instar nymphs, as well as adults 3-5 days after emergence, were collected. Four biological replicates were set up for each stage, with each replicate containing 4 aphids.

[0043] Different tissues: Dissect adult aphids 3-5 days after emergence and separate their head, thorax, abdomen, epidermis, midgut, ovaries, and other tissues. Each tissue sample requires the dissection of 30-50 adult aphids, and four biological replicates are set up.

[0044] 2) Real-time quantitative PCR (qRT-PCR):

[0045] The extraction of total RNA and the synthesis of cDNA for each sample were the same as in Example 1.

[0046] Design qRT-PCR primers:

[0047] qCcPrxF: 5'-GTCTGAACTCTGTCGTCCG-3';

[0048] qCcPrxR: 5'-AAAACGCACCAAGAATCGCC-3'.

[0049] The CcActin primers (designed based on the NCBI published sequence) were used in the NovoStart® SYBR qPCR SuperMix Plus kit on an ABI 7500 real-time quantitative PCR instrument. The reaction mixture (20 μL) consisted of 10 μL 2×SuperMix, 0.4 μL forward and reverse primers (10 μM), 1 μL cDNA template, and ddH2O added to a final volume of 20 μL.

[0050] Reaction program: 95℃ for 1 min; 95℃ for 20 s, 60℃ for 20 s, 72℃ for 20 s, for a total of 40 cycles. Using 2... - The ΔΔCt method is used to calculate the relative expression level of genes.

[0051] 3) Results Analysis:

[0052] Figure 1 The expression patterns of Peroxiredoxin1 in aphids at different instars are shown. As can be seen from the figure, the expression level of the peroxidase Peroxiredoxin in the cedar aphid gradually increases from the first instar, with the lowest expression level observed in the first instar aphids. The expression level reaches its highest point from the third instar nymphs to adults. It is speculated that Peroxiredoxin expression is related to the aphid's feeding amount and growth and development. The first instar nymphs consume less food, causing the least damage to pine trees, resulting in low gene expression. With increasing instar, the gene expression level increases significantly, suggesting that Peroxiredoxin is a key factor in the aphid's resistance to plant defense during its growth and development from nymph to adult.

[0053] Figure 2 The figure shows an analysis of Peroxiredoxin expression patterns in different tissues of adult aphids. It can be seen that the expression levels of Peroxiredoxin are high in the head, abdomen, and midgut of 3-5 day old adults of *Aphis cedarina*, while the expression levels are low in the thorax, ovary, and epidermis. It is speculated that the high expression levels in the midgut and abdomen may be due to the need to maintain the stability of the digestive system and the detoxification metabolism of plant secondary metabolites during feeding. The highest expression level is found in the head, indicating that it plays an important role in aphid feeding on host plants, possibly related to salivary proteins. Aphids use this gene to reduce the impact of plant defenses during feeding, and the role differs in different tissues, hence the significant differences in expression patterns among different tissues of *Aphis cedarina*.

[0054] Example 3

[0055] Synthesis of dsRNA targeting the CcPeroxiredoxin gene.

[0056] 1) Interference primer design:

[0057] Interference primers were designed using the primer design website https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=BlastHome. Off-target effects on non-target organisms were evaluated using the off-target assessment website https: / / dsrna-engineer.cn / based on the designed dsRNA fragment. Results showed no off-target effects on species such as ladybugs, multicolored ladybugs, bear beetles, and *Smallseii barkei*, indicating that the dsRNA has very high species specificity and is only effective against *Aphidius cedarina*. A T7 promoter sequence (TAATACGACTCACTATAGGG) was added to the 5' end of the primers to design primers for in vitro transcription.

[0058] dsCcPrxF:

[0059] 5'-TAATACGACTCACTATAGGGACCGAATTGGGCAAAATGGC-3';

[0060] dsCcPrxR:

[0061] 5'-TAATACGACTCACTATAGGGAGTGCGTTGAGGTCCTTGTC-3'.

[0062] 2) Preparation of DNA template:

[0063] Using the primers described above and the cDNA obtained in Example 1 as templates, PCR amplification was performed to obtain the target DNA fragment with T7 promoter sequences at both ends. The PCR product was purified and used for later use.

[0064] 3) In vitro transcription to synthesize dsRNA:

[0065] Using the TranscriptAid T7 High Yield Transcription Kit, the purified DNA fragment was used as a template for in vitro transcription at 37°C for 4 hours to synthesize two complementary single-stranded RNAs. After the reaction, the system was incubated at 95°C for 5 min, and then slowly cooled to room temperature to allow the single-stranded RNA to anneal and form a double-stranded structure (dsRNA). The dsRNA was extracted with phenol-chloroform and purified by isopropanol precipitation. After quantification, it was stored at -80°C.

[0066] 4) Preparation of control dsRNA: Using the same method, dsGFP was synthesized as a negative control using the template and primers of the green fluorescent protein (GFP) gene.

[0067] The final dsRNA fragment is shown in SEQ ID NO: 3. SEQ ID NO: 3 is:

[0068] ACCGAATTGGGCAAAATGTTTTAGTCGACGAATTCACCAAGCGCAACACCAAAGTGTTAGGTTTGTCTTGTGACAAACTCGAAAGCCACGTTAATTGGATAAATGATATTAAATCGTACTGTGTGGATATCGAGGGGGAATTCCCTTTTCCGATTATAAGCGACAGCTCGAGAGAATTAGCTATAAAATTGGACATGCTCGCCGAAGAAGACAAGAACAATCCTG ATACGGCCATGACTATTCGATCCCTTTATATCATCGGTCCTGACAAGAAAGTTAAACTCATGATGGTCTATCCTACAAGCACTGGCCGTAACATACAAGAGGTTTTACGTTGCATCGATTCTCTTCAATTATGCGATAAAAAGAAAATTGTTGCTACACCTGTAAACTGGGTTCCTGGAGAAAAAGTCATGATCCTTCCTTCAGTCGACGACAAGGACCTCAACGCACT.

[0069] Example 4

[0070] RNAi bioassay.

[0071] 1) Artificial feeding: Make artificial feeding devices.

[0072] One end of a 5cm long plastic tube was sealed with a double-stretched Parafilm membrane to form a small feeding capsule. 50 μL of liquid containing dsRNA was injected between the two membranes.

[0073] The experimental group contained 1000 ng / μL of dsCcPeroxiredoxin, while the control group contained 1000 ng / μL of dsGFP. Both solutions contained 1% (w / v) of blue food dye to indicate whether aphids had fed on the food.

[0074] 2) Bioassay: Select healthy, uniformly sized 1-3 day old adult aphids, and put 15 aphids into each treatment group. Let them feed on dsRNA liquid for 24 hours through a paraffin film feeder at 25℃.

[0075] 3) Silencing efficiency test: 24 hours after feeding, a portion of aphids were randomly selected from each treatment, total RNA was extracted and detected by qRT-PCR, the method was the same as in Example 2.

[0076] 4) Survival observation: The remaining aphids (4 biological replicates per treatment group, 15 aphids per replicate) were removed from the feeder and transferred to fresh cedar branches for further culture. The culture conditions were: temperature 25±1℃, relative humidity 60±10%, and photoperiod 14h:10h (light:dark). Observations were conducted for 7 consecutive days, and dead aphids were recorded and removed daily.

[0077] Figure 3 The feeding statistics in this embodiment are shown in the figure. "dsCcPeroxiredoxin" represents the treatment group fed with CcPeroxiredoxin gene dsRNA; "dsGFP" represents the control group fed with GFP gene dsRNA. Independent t-tests were performed. " indicates p < 0.01, " "" indicates p < 0.001. Survival rate observation sample size: dsGFP, N = 60; dsCcPeroxiredoxin, N = 60. As shown in the figure, compared with the control group fed dsGFP, the relative expression level of CcPeroxiredoxin mRNA in the experimental group fed dsCcPeroxiredoxin was significantly downregulated by 40.17%, indicating that dsRNA successfully entered the aphids and effectively silencing the gene. Statistical analysis (independent samples t-test) showed that the cumulative mortality rate of aphids in the experimental group fed dsCcPeroxiredoxin was significantly higher than that in the control group fed dsGFP (P < 0.05). Table 1 shows the survival rate statistics of *Aphis cedarina* after treatment with dsCcPeroxiredoxin in this example.

[0078] Table 1. Survival rate of the cedar aphid after treatment with dsCcPeroxiredoxin.

[0079]

[0080] The above results indicate that feeding dsRNA targeting the CcPeroxiredoxin gene can significantly inhibit CcPeroxiredoxin gene expression and effectively kill adult cedar aphids.

[0081] Example 5

[0082] The aphid-killing composition was prepared by dissolving the freeze-dried powder of dsCcPeroxiredoxin synthesized and purified in Example 3 in sterile water to prepare a stock solution of 1000 ng / μL. This stock solution was then mixed with 0.1% (v / v) of a nonionic surfactant (such as Tween-20) as a spreading agent to obtain an aphid-killing composition suitable for spray application. This composition can be directly sprayed onto the surface of plants infested by the cedar aphid.

[0083] Example 6

[0084] Construction of aphid-resistant transgenic cedar.

[0085] 1) Construction of the expression vector: The DNA fragment used to amplify the dsRNA template in Example 3 was modified to construct a reverse repeat sequence capable of generating hairpin RNA. This sequence was placed between a plant constitutive strong promoter (such as the CaMV 35S promoter) and a terminator (such as the Nos terminator) to form a gene expression cassette for efficient dsRNA expression. This expression cassette was cloned into a plant expression vector (such as pCAMBIA1301) containing a selection marker (such as a kanamycin resistance gene) to obtain a recombinant expression vector.

[0086] 2) Genetic transformation: The above recombinant expression vector was introduced into the embryogenic callus of cedar using Agrobacterium-mediated transformation or gene gun transformation.

[0087] 3) Screening and regeneration: Transformed callus tissues are screened on a culture medium containing appropriate screening agents (such as kanamycin) and induced to differentiate and regenerate, ultimately obtaining transgenic cedar plants.

[0088] 4) Molecular detection: PCR is used to detect whether the target DNA fragment has been successfully integrated into the genome of the transgenic plant, and qRT-PCR is used to detect whether the target dsRNA is stably expressed in the transgenic plant.

[0089] 5) Resistance identification: The long-legged cedar aphid was inoculated onto transgenic cedar plants and non-transgenic control plants. The survival rate, reproduction rate and other indicators of the aphids were observed to evaluate the aphid resistance effect of the transgenic plants.

[0090] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A gene encoding peroxide reductase for controlling the cedar aphid, characterized in that, The amino acid sequence of the oxidoreductase protein is shown in SEQ ID NO:

2.

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the protein of claim 1, and its nucleotide sequence is shown in SEQ ID NO:

1.

3. A dsRNA encoding a peroxidase gene for controlling the cedar aphid, characterized in that, The dsRNA can specifically downregulate the expression of the nucleic acid molecule of claim 2 or 3 in the cedar aphid, and the dsRNA contains a nucleotide sequence that is SEQ ID NO: 3 or a sequence that has at least 95% sequence identity with SEQ ID NO:

3.

4. A DNA molecule, characterized in that, The DNA molecule can be transcribed into the dsRNA of claim 3.

5. An application of a peroxide reductase encoding gene for controlling the cedar aphid, characterized in that, The protein in claims 1-2, the dsRNA in claim 3, and the DNA molecule in claim 4 are used to prepare an aphid-killing composition for controlling the cedar aphid.

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