Bactrocera minax forkhead transcription factor foxo, gene encoding the same and application

Abstract

The present application provides a Bactrocera minax forkhead transcription factor FOXO, an encoding gene and application thereof, and belongs to the technical field of biological control of crop pests. The present application clones a nucleic acid molecule of the Bactrocera minax forkhead transcription factor FOXO, synthesizes a dsRNA for interfering with the nucleic acid molecule of the Bactrocera minax forkhead transcription factor FOXO, and respectively uses injection and feeding methods to introduce the dsRNA into the Bactrocera minax to perform RNAi on the forkhead transcription factor gene FOXO of the Bactrocera minax. After the dsRNA is injected or fed to the Bactrocera minax, the survival rate, growth rate, body weight and overall performance of the Bactrocera minax are significantly reduced, indicating that the Bactrocera minax forkhead transcription factor FOXO plays an important role in the growth and development of insects. The present application provides a new method for molecular regulation of pests, and provides a theoretical basis for in-depth understanding of the growth and development mechanism of insects and creation of new biological pesticide preparations.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for crop pests, and particularly relates to the transcription factor FOXO of the forkhead of the citrus fruit fly, its encoding gene, and its applications. Background Technology

[0002] In the current booming global citrus industry, the citrus fruit fly (Bactrocera minax Enderlein), a highly destructive oligophagous pest of the citrus genus, has become a key bottleneck restricting the healthy and sustainable development of the industry. Its unique feeding habit—attacking only unripe citrus fruits—causes the affected fruit to rot internally, causing premature fruit drop and resulting in large-scale yield reductions. In recent years, frequent outbreaks of citrus fruit fly infestations have occurred in several major citrus-producing areas, affecting numerous orchards, severely damaging the economic income of fruit farmers, disrupting the stable supply of citrus in the market, and causing incalculable economic losses to the entire citrus industry.

[0003] Currently, the control strategies for the citrus fruit fly mainly focus on two areas: chemical control and physical control. Chemical control, with its high efficiency and rapid insecticidal effect, has long dominated pest control. However, the negative effects of over-reliance on chemical pesticides are becoming increasingly prominent. On the one hand, the large-scale use of chemical pesticides has caused serious damage to the ecological environment, polluting not only soil, water, and air, but also leading to a sharp decline in the populations of non-target organisms such as bees and birds, thus destroying biodiversity. On the other hand, pesticide residues have become a significant threat to food safety, causing high consumer concern about the quality and safety of citrus products, seriously affecting the brand image and market competitiveness of the citrus industry. Although physical control methods, such as trapping, can reduce the use of chemical pesticides to some extent, they have limitations such as low control efficiency and limited coverage, making it difficult to meet the needs of large-scale pest control. These problems fully expose the shortcomings of current crop pest control methods, which are relatively outdated and technologically simplistic, urgently requiring innovative breakthroughs.

[0004] In the field of insect biology research, forkhead transcription factor FOXO, as a key regulatory factor, plays a crucial role in insect detoxification, nutrient absorption, and development, and has become a hot topic in current insect physiology and pest control research. Studies have found that plant secondary metabolites such as flavonoids can induce oxidative stress in herbivorous insects, thereby activating the expression of FOXO, a downstream gene of the insulin signaling pathway (ILP). FOXO helps insects resist oxidative damage and exogenous toxic substances by regulating the activity of antioxidant enzymes (such as superoxide dismutase and peroxidase) and detoxification enzymes (such as cytochrome P450 enzymes and glutathione S-transferase), ultimately achieving precise regulation of insect development and immune processes. In-depth exploration of the mechanism of action of FOXO in the citrus fruit fly is expected to uncover novel pest control targets, providing theoretical basis and technical support for the development of efficient, green, and safe new control agents. This has significant practical implications and broad application prospects for overcoming existing control technology bottlenecks and promoting the green and high-quality development of the citrus industry. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the forkhead transcription factor FOXO of the citrus fruit fly, its encoding gene, and its applications. This invention discovers that the forkhead transcription factor FOXO of the citrus fruit fly plays an important role in the growth and development of the insect. By inhibiting the forkhead transcription factor FOXO, the survival rate, growth rate, weight, and overall performance of the citrus fruit fly can be reduced, which has a very broad application prospect in the field of citrus fruit fly control.

[0006] This invention provides a forkhead transcription factor FOXO for the citrus fruit fly, wherein the forkhead transcription factor FOXO is a protein as follows a) or b):

[0007] a) A protein with the amino acid sequence shown in SEQ ID No. 2;

[0008] b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 2.

[0009] Preferably, the label in b) is Poly-Arg, Poly-His, FLAG, Strep-tag II, or c-myc.

[0010] This invention provides biological materials expressing the forkhead transcription factor FOXO, comprising any one of the following A1) to A8):

[0011] A1) The nucleic acid molecule encoding the forkhead transcription factor FOXO;

[0012] A2) An expression cassette containing the nucleic acid molecules described in A1);

[0013] A3) A recombinant vector containing the nucleic acid molecules described in A1);

[0014] A4) A recombinant vector containing the expression cassette described in A2);

[0015] A5) Recombinant microorganisms containing the nucleic acid molecules described in A1);

[0016] A6) Recombinant microorganisms containing the expression cassette described in A2);

[0017] A7) Recombinant microorganisms containing the recombinant vector described in A3);

[0018] A8) Recombinant microorganisms containing the recombinant vector described in A4).

[0019] Preferably, the sequence of the nucleic acid molecule encoding the forkhead transcription factor FOXO is shown in SEQ ID No. 1.

[0020] This invention provides biological materials for inhibiting the expression of the forkhead transcription factor FOXO.

[0021] Preferably, the biological material comprises dsRNA that inhibits the expression of nucleic acid molecules encoding the forkhead transcription factor FOXO.

[0022] Preferably, the sequence of the dsRNA is shown in SEQ ID No. 3.

[0023] The present invention also provides the application of the biomaterial in any one of the following a1) to a12):

[0024] a1) Inhibits insect growth and development;

[0025] a2) Prepare products that inhibit insect growth and development;

[0026] a3) Controlling pests;

[0027] a4) Prepare products for controlling pests;

[0028] a5) Reduces insect survival rate;

[0029] a6) Prepare products that reduce insect survival rates;

[0030] a7) Reduce the amount of weight gain in insects;

[0031] a8) Prepare products that reduce the weight gain of insects;

[0032] a9) Reduce insect growth rate;

[0033] a10) Products for preparing insect growth rates;

[0034] a11) Reduces the overall expressiveness of insects;

[0035] a12) Prepare products that reduce the overall performance of insects.

[0036] This invention provides a method for controlling the citrus fruit fly by applying the biological material to the citrus fruit fly.

[0037] Preferably, the biomaterial is administered via feeding and / or injection.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] This invention first cloned the nucleic acid molecule of the forkhead transcription factor FOXO from the citrus fruit fly (Bactrocera dorsalis), and designed primers to synthesize dsRNA for interfering with the encoding of FOXO. The dsRNA was then introduced into the citrus fruit fly via injection and feeding methods to perform RNAi on the FOXO gene. The results showed that after dsRNA injection or feeding, the survival rate, growth rate, body weight, and overall performance of the citrus fruit fly were significantly reduced, indicating that the forkhead transcription factor FOXO plays an important role in the insect's growth and development. This invention provides a new method for the molecular regulation of pests and also provides a theoretical basis for a deeper understanding of insect growth and development mechanisms and the creation of new biological pesticide formulations. Attached Figure Description

[0040] Figure 1 Statistical analysis of FOXO gene interference efficiency in the citrus fruit fly.

[0041] Figure 2 This study presents the statistical results of the effects of FOXO gene interference in the citrus fruit fly on its survival rate, weight gain, growth rate, and overall performance. Detailed Implementation

[0042] This invention provides a forkhead transcription factor FOXO from the citrus fruit fly, wherein the forkhead transcription factor FOXO is a protein of the following a), b), c), or d): a) a protein with the amino acid sequence shown in SEQ ID No. 2; b) a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 2; c) a protein with the same function obtained by substituting, deleting, and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2; d) a protein with more than 75% homology to the amino acid sequence shown in SEQ ID No. 2 and with the same function.

[0043] In this invention, the tag mentioned in b) is preferably Poly-Arg, Poly-His, FLAG, Strep-tag II, or c-myc. The purpose of the tag in b) is to facilitate the purification of the protein in a), and the specific sequence of the tag is shown in Table 1. This invention does not impose any particular limitation on the preparation method of the forkhead transcription factor FOXO; either artificial synthesis or a method of first synthesizing the encoding gene and then biologically expressing it is acceptable.

[0044] Table 1: Label Sequence

[0045] Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tagII 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0046] This invention provides biological materials expressing the forkhead transcription factor FOXO, including any one of A1) to A8); in this invention, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

[0047] In this invention, the sequence of the nucleic acid molecule encoding the forkhead transcription factor FOXO is preferably as shown in SEQ ID No. 1, consisting of 3612 nucleotides.

[0048] The present invention also provides biomaterials for inhibiting the expression of the forkhead transcription factor FOXO.

[0049] In this invention, the biological material includes dsRNA that inhibits the expression of nucleic acid molecules encoding the forkhead transcription factor FOXO, and the sequence of the dsRNA is preferably as shown in SEQ ID No. 3.

[0050] The present invention also provides a method for controlling the citrus fruit fly, wherein the biological material is applied to the citrus fruit fly.

[0051] In this invention, the biological material is preferably administered via feeding and / or injection, more preferably via feeding. In this invention, when the biological material is dsRNA, the dsRNA is preferably used in solution form, preferably with nuclease-free water as the solvent. The concentration of the dsRNA used is preferably 0.5–2 ng / μL, more preferably 0.8–1.5 ng / μL, and most preferably 1 ng / μL.

[0052] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0055] In the quantitative experiments described below, five replicate experiments were performed, and the average value of the results was taken.

[0056] The test insect source in the following examples was: second-instar citrus fruit flies (uniformly developed) collected from Youyang County, Chongqing City, and reared in an intelligent artificial climate chamber under the following conditions: temperature 26℃, humidity 70%, light-dark ratio of 16h:8h, and fed uniformly with feed (feed formula - 500mL distilled water: 60g sucrose, 20g brewer's yeast, 5g peptone, 5g agarose, 0.5g sorbic acid, 3.3g ascorbic acid, and 0.6g p-hydroxybenzoic acid).

[0057] The main reagents in the following examples: RNA isolation reagent (original Invitrogen), RNA spincolumn (AllGold), gel extraction kit (Axygen), EX Taq DNA polymerase (Takara), T4 DNA ligase (Takara), pGEM-T Easy Vector Systems (Promega), and anhydrous ethanol, isopropanol, glycerol, etc. are all domestically produced analytical alcohols.

[0058] The main instruments used in the following examples are: a clean bench (Shanghai Boxun Industrial Co., Ltd.), an ETC-811 PCR instrument (Beijing Dongsheng Innovation Biotechnology Co., Ltd.), a Sigma 3K15 refrigerated centrifuge (Sigma Centrifuge GmbH, Germany), a NanoPhotometer micro spectrophotometer (IMPLEN GmbH, Germany), an HPX-9052MBE digital display electric heating incubator (Shanghai Boxun Industrial Co., Ltd.), a THZ-D benchtop thermostatic shaker (Huamei Biochemical Instrument Factory), a QL-901 vortex shaker (Haimen Qilinbei Instrument Manufacturing Co., Ltd.), and a YXQ-LS-50SII autoclave (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory).

[0059] Example 1

[0060] Obtaining the FOXO transcription factor forkhead of the citrus fruit fly and its encoding gene.

[0061] 1. Extraction of total RNA from *Bactrocera dorsalis*

[0062] use RNA was extracted from tissue samples of *Bactrocera dorsalis* using RNA separation reagents. The specific steps are as follows:

[0063] 1) Place a 2mL homogenizer in an oven and sterilize at 160℃ for 3 hours. Cool to room temperature before use.

[0064] 2) Place the homogenizer on ice and add 1 mL RNA isolation reagent and 200 mg of citrus fruit fly tissue were ground together.

[0065] 3) Transfer the homogenate to a 1.5 mL centrifuge tube and let it stand at room temperature for 5 min. Centrifuge at 13000 rpm for 5 min at 4℃.

[0066] 4) Transfer the supernatant to a clean 1.5 mL centrifuge tube, add 200 μL of chloroform, and vortex for 15 seconds. Incubate at room temperature for 5 minutes. Centrifuge at 13,000 rpm for 10 minutes at 4 °C.

[0067] 5) Transfer 400 μL of supernatant to a new 1.5 mL centrifuge tube, add 200 μL of chloroform, and vortex for 30 seconds. Incubate at room temperature for 5 minutes. Centrifuge at 13,000 rpm for 10 minutes at 4 °C.

[0068] 6) Take 300 μL of supernatant, add 300 μL of isopropanol, vortex for 30 seconds, transfer to RNA spin column, and incubate on ice for 10 minutes.

[0069] 7) Centrifuge at 4℃ and 13000rpm for 2 minutes, then discard the filtrate.

[0070] 8) Add 600 μL of RNAwash solution, pipette and wash the sediment, centrifuge at 13000 rpm for 2 min at 4°C, and discard the filtrate. Add another 600 μL of RNAwash solution, pipette and wash the sediment, centrifuge at 13000 rpm for 2 min at 4°C, and discard the filtrate. Centrifuge at 13000 rpm for 3 min at 4°C to remove excess ethanol, and air dry for 3 min.

[0071] 9) Replace with a new collection tube, add 50 μL of RNase-free water preheated at 65℃ to the RNA spin column, incubate under residual heat for 5 min, and centrifuge at 13000 rpm for 3 min at 4℃.

[0072] 10) Collect the filtrate and use a NanoPhotometer micro-spectrophotometer to detect the RNA concentration and OD260 / 280 value to confirm RNA quality. Simultaneously, take 2 μL of the extracted RNA for agarose gel electrophoresis. Store the remaining RNA at -20℃ for later use.

[0073] 2. Reverse transcription

[0074] Using PrimeScript TMThe 1st strand cDNA Synthesis Kit is used to obtain cDNA through reverse transcription. The specific steps are as follows:

[0075] 1) Prepare a 10μL system: Oligo dT Primer (50μM) 1μL, dNTP Mixture (10mM each) 1μL, total RNA <5μL, and RNase Free dH2O to make up to 10μL.

[0076] 2) After holding at 65℃ for 5 minutes, cool rapidly on ice.

[0077] 3) Prepare a 20 μL reaction solution: 4 μL of 5×PrimeScript Buffer, 0.5 μL (20 units) of RNase Inhibitor (400 U / μL), 1 μL (200 units) of PrimeScript RTase (200 U / μL), and RNase Free dH2O to make up to 20 μL.

[0078] 4) Mix slowly.

[0079] 5) Keep warm at 42℃ for 30-60 minutes.

[0080] 6) Incubate at 95℃ for 5 minutes to inactivate the enzyme, then place on ice and store the cDNA at -20℃.

[0081] 3. Obtaining the transcription factor FOXO and its encoding gene from the citrus fruit fly forkhead.

[0082] 1) Primer design

[0083] Based on the previously obtained transcriptome of *Bactrocera dorsalis*, the FOXO gene sequence was obtained. Primers for the full-length FOXO gene or fragment primers were designed using DNAMAN8. The designed primers are as follows:

[0084] FOXO-F (SEQ ID No. 4): 5'-ATGAATTCGAAATGCGCAAAATG-3';

[0085] FOXO-R (SEQ ID No. 5): 5'-TAAATACGGATCCCCATCCAGG-3'.

[0086] 2) PCR reaction

[0087] Using citrus fruit fly cDNA as a template, PCR amplification was performed using primers FOXO-F / FOXO-R to obtain the PCR product.

[0088] The PCR reaction system is as follows (total volume 50 μL): 1 μL cDNA template, 4 μL dNTP, 5 μL 10×Buffer, 1 μL front primer, 1 μL back primer, 0.25 μL Taq enzyme, and 37.75 μL ddH2O.

[0089] The PCR reaction conditions were as follows: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min and 30 s, 35 cycles; 72℃ for 10 min; stored at 4℃.

[0090] 3) PCR product recovery, cloning, and sequencing

[0091] 3-1) Perform electrophoresis on a 1% agarose gel prepared with TAE. When the target band is well separated, cut off the gel block containing the target band with a blade and place it in a sterile centrifuge tube. Then, use a gel extraction kit (Axygen) to recover and purify the target band, following the kit instructions.

[0092] 3-2) After PCR product recovery, ligation was performed using the pGEM-T Easy vector to obtain the recombinant vector. The ligation system was as follows: T4 DNA ligase 1 μL, 2× buffer 5 μL, pGEM-T Easy 1 μL, and PCR product recovery 3 μL. Ligation conditions: ligation at room temperature for 6 h.

[0093] 3-3) Preparation and transformation of competent cells

[0094] Add 33.3 μL of Trans1-t1 competent cells to a 1.5 mL centrifuge tube, incubate on ice for 15 min, then incubate at 42°C for 90 s, and then on ice for 10 min. Add 500 μL of liquid LB medium to each 1.5 mL centrifuge tube, and incubate at 37°C and 200 rpm for 2 h. After incubation, transfer 100 μL of the bacterial culture to 1‰ AMP LB solid medium and incubate overnight at 37°C. Select a single colony and place it in a 2 mL centrifuge tube containing 1 mL of 1‰ AMP LB liquid medium. Incubate at 37°C and 200 rpm for 6 h, and observe the growth.

[0095] 3-4) Bacterial PCR

[0096] The bacterial culture in 3-3) was verified by PCR. The bacterial culture PCR reaction system (total volume 50 μL) was as follows: 1 μL bacterial culture, 4 μL dNTP, 5 μL 10× Buffer, 1 μL front primer, 1 μL back primer, 0.25 μL Taq enzyme, and 37.75 μL ddH2O.

[0097] The reaction conditions were as follows: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min and 30 s, 35 cycles; 72℃ for 10 min; stored at 4℃.

[0098] Positive clone strains were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing and analysis of the sequencing results.

[0099] Sequencing results showed that a DNA fragment of 3612 bp was obtained by PCR amplification, and its nucleotide sequence is shown in SEQ ID No. 1. The gene shown in SEQ ID No. 1 was named Citrus fruit fly forkhead transcription factor FOXO-1, and the amino acid sequence of the forkhead transcription factor it encodes is shown in SEQ ID No. 2. The amino acid sequence shown in SEQ ID No. 2 was named Citrus fruit fly forkhead transcription factor FOXO.

[0100] SEQ ID No. 1:

[0101] ATGAATTCGAAATGCGCAAAATGTATTGTAGAACTGTACCCGTATTTGACTATTTTTC

[0102] AAACCAATAAGAAGGCGAATCAAGAGTGGGATCCAAGTTTTGGTATCGAACCTATT

[0103] GATTGGCTATGTATTTTAAATTCAACGGAGGCACTAAGGTGGGATAAATGGATTTGTA

[0104] TGTTTGTTAGTAAAGTGCTTGAAAGTTGCGGTTGGATAGCACTCTGCAACTTGGCTT

[0105] CTCGAGATATGTATGTGGCAAATGAAATACTTTTGCCTTTTATTACGCTCTTAATGTCA

[0106] AATAAAAGGCGACATATAGATAGTATTATGTCAATGCTCATTCATTATTTCAAAATGTT

[0107] GGATATGGTTCTTGGAAAGAGTGAAACCGATCAGATGAAAGCTCAGGAGATTTATA

[0108] AAGACAAGCGAATAATAAAAATATTTCTAAAAATATGTGAAAGTATTCGTTTGAATAA

[0109] TAAGGTGTTCTGTGCCAATGAATTTGCTTTATGTTGCTAAAGCCAGTAACCATTGCCA

[0110] GGCGTTCTTTATGACAATTATGTATACTGAATTATGGGCACTGTCAGAGAGCATATCG

[0111] AACGATAAGCAGTCCAAAGTAGAGGAGCATTTAAAATCCAGAATTTCAGGAAGT

[0112] AGCAATAAAGGCTTATAAATCCATTGGTTGTCATGATGCCATATCTGGGATTCTCTCA

[0113] CCTTTACAATCTCGTCTTGAATTTCTGAATTTAAACAACGATTGGTCCGAAATGTTAC

[0114] TTCAAAACTCTTTTAAGAATACATCGTCGAATACACTTTGTACTTCAGCATTAAAACG

[0115] GAACGGTATTTTATGCCTCGCAAATCTGGACAATAAAGACAATTTAGTTGACTATGA

[0116] AGTATGTTGGAGATTATGTCAATGGGATACGCCCGTAGAAGGGCATTTGAAAGTGAA

[0117] TATTGAAAATGATCCCGAATTGAATTTAAGAAGCATCATTTCAATGCTTTAAAATGC

[0118] CTATATAATCGCGAACAACAAAATTGTTTAGCGGCCATCGCAAACGCACGGCTATGC

[0119] GTAATTTACACTTTGACGGGAATAAGCACCGATTGCCTTCAAAGTATATACAAATATC

[0120] TAACATGGCTGCACATACTTCAACAAACGGAGGATTTTTGTCAAATCCAGTTTACTT

[0121] CTGAAGTAGATATTAAATCGATCTTTTCAAAGTGGCAGTCGGAAAATAAACTAAAAT

[0122] ACGGTAGCTTTTATTGCAAAGAACTAATAATGTGCGCATCAGATGACTCTATTTAACAC

[0123] TGCTGGAGTCCGTGGTCAAAGAAGGATAATAGATTTTTACAAATTTAGTCCGACCGA

[0124] AACTGGTTTGTTAAAATATCATTAGAGAATGCCAAAAATCAGGGGAAATTAACTTGGC

[0125] GAAACGTAATATATTAGCTCTTAGAGAAGCAGAAATTACAAACGAACATGTCAAGCT

[0126] TAATTTGTTACTTGAAGATGCAGAAATTTCTTTTCGATGCGGAAATATTGAAATAACT

[0127] GAAGCATTGTTAAAACACACGTTGACACATAAAGATCTAACTGCTTGCCCTCAACA

[0128] AGCACGTGCTCTCCGAATGTATGGAGAATTTCTCTTGGAGACTAATTCGCAAAGTTT

[0129] TGAATACGTTCTAGAAAAAATGTTTAATAGATCTATGCTTTACCTGGAGAAGATATTA

[0130] AAATCTCAAAAACAGTGTGATAATGACGAATTAAGTTTTTTGTATTTAGAAGGCCTA

[0131] AAACCAGCCGAATTTGAAAAGGAAAACAAAAAAGAAGCATATCAATTAATAGCGA

[0132] AATATGCCGATCGTGAATATGCGCAATCGAATGTGTACATCAATTCTGAGGAATTTAA

[0133] ACTGAAGTGTCAAATCATACAACAACAACAGACAAACAGCGGATTCAATTGGTCGAC

[0134] AACACAAAGATCGTTAATTAAACCACGGTGTAATCATAATGAAAAAGTATGCGAATT

[0135] TGGATGAAACGGAAATAAAGTTTATTGAGGAGAAACGCACAAAATAACTTGTGTATT

[0136] GCCGTTAAGAATTATATGAAATTTTGTCAAATTGATTCTGGATTTTCTAATGCCGCAAT

[0137] ATATAGGATAATAGCATTGTGGTTCGCCAATAAACAAGATCAAGTGTTGTGTAAAGA

[0138] AATAAAAGATAATATTGAAATAATTCCTTCCTATAAGTTTATTTGCACTTTAAATCAGA

[0139] TCACCGCTAGATTAAATACGAAACACGTAGATTTTATTACCATTATCAAAGAAATATT

[0140] AGTTAAGTGCTTACAAGACCATCCACATCACACACTTTACCAGTTGTATCCGCTAAT

[0141] TTTTGATGATACAGGAGGTAAAACAAATAAGTCGTTCAACCATTGCAGCAGAAA

[0142] TTATATCAAAGGGGCGAAACTCGTCAAATGCACAGTCCGCAAAACAGTTGGCAGTA

[0143] GTGTTTCCTGCTTTAATTCAGTTCGCAAATGCTGATTGTGGAAGGGGCCACACTATG

[0144] TTGTGTGATAAATTGAAAAGAATTAAATGCTTAGACGCTGTTCATTGCCCGACAATT

[0145] GAACTTCCGGTATTACCTAGTAAGGATTACACAATAATAAGTATCGTACGGTGGGAC

[0146] GAAAAAGTGTTGCTGGTAGGGGGTATAAACGCTCCAAAAAAACTCCAATGTTTATG

[0147] TTCAGATGGAAAATCACGACCTCAACTCTTAAAAGGTCGAGATGATTTACGCCAAG

[0148] ACGCCGTAATGGAGCAATATTTTTCACTCATGAACACACTATTGTGTTGCGATCCGA

[0149] AAACAAGTGAACGAAAAATTAATATTCGCACCTACAAAGTTGTACCGCTCTCAATG

[0150] AGAAGTGGAATATTAGAGTGGTGCGAAAATACCGTACCGATTGGTGTATATTTGGGT

[0151] AGTGGAAGTGACAAAACTGGCGCACATCGGAAATATCGACCTGCTGATATCTCACC

[0152] CCATAAGTGCCGTCAAATATCCATGCAACATCTTAAATCCGACATACAAAAACGACT

[0153] TTTAATTTACGAGCAAATTTGCGCACAAATAAAGCCTGTTTTCCATTATTTTCTGCTG

[0154] GAAAATTTTGTAGTTCCAGGTATTTGGTTTGAACGACGCCTGGCCTACACCAACAGC

[0155] TTGGCTGTGAATTCCATGGTTGGCTTTGTAGTGGGCTTAGGCGATCGACATACTCAA

[0156] AATATATTGATTGATGAGAAGACTGTGAAGTAATTCATATCGATTTTGGAATTGCTT

[0157] TTGAGCAAGGAAAAATAATGCCAACCCCAGAAACCGTACCTTTCCGTCTAACCCGA

[0158] GATATGATTGCGCCAATGGGCATTTGTGAAACAGGTGGGGTATTTAAGAAAGCATGC

[0159] CAATCAACACTAGAAGTTTTGCGAAAAAATCATTCCGTAATAATTACAATTCTTGAA

[0160] GTTCTGCTATATGATTCCGCTATATATTTGGAATGTAGTGCCAACTCCCGCTGGATCTAA

[0161] AGATGATGAAAAAAATCTAACTGCACAACGTGCGCTTTTATGCGTGCAGCATAAGTT

[0162] GGAAGGAAGGTTAAGTAACATAACAGGTACCGCCAATACTGACGTTCAAGTTCACA

[0163] GATTAATAAATGACGCTGTTTCTAAACAAAATCTATGTCGTCTATACCCTGGATGGGA

[0164] TCCGTATTTA

[0165] SEQ ID No.2:

[0166] MNSKCAKCIVELYPYLTIFQTNKKANQEWDPSFGIEPIDWLCILNSTEALRWDKWICM

[0167] FVSKVLESCGWIALCNLASRDMYVANEILLPFITLLMSNKRRHIDSIMSMLIHYFKMLD

[0168] MVLGKSETDQMKAQEIYKDKRIIKIFLKICESIRLNNKCSVPMNLLYVAKASNHCQAFF

[0169] MTIMYTELWALSESISNDKQSKVEEHLKNPEFQEVAIKAYKSIGCHDAISGILSPLQSRL

[0170] EFLNLNNDWSEMLLQNSFKNTSSNTLCTSALKRNGILCLANLDNKDNLVDYEVCWRL

[0171] CQWDTPVEGHLKVNIENDPELEFKKHHFNALKCLYNREQQNCLAAIANARLCVIYTL

[0172] TGISTDCLQSIYKYLTWLHILQQTEDFCQIQFTSEVDIKSIFSKWQSENKLKYGSFYCKE

[0173] LIMSHQMTLFNTAGVRGQRRIIDFYKFSPTETGLLNIIRECQKSGEINLAKRNILALREA

[0174] EITNEHVKLNLLLEDAEISFRCGNIEITEALLKHTLTHKDLTACPQQARALRMYGEFLL

[0175] ETNSQSFEYVLEKMFNRSMLYLEKILKSQKQCDNDELSFLYLEGLKPAEFEKENKKEA

[0176] YQLIAKYADREYAQSNVYINSEEFKLKCQIIQQNRQTADSIGRQHKDRDINHGVIIMKK

[0177] YANLDETEIKFIEEKRTNNLCIAVKNYMKFCQIDSGFSNAAIYRIIALWFANKQDQVLC

[0178] KEIKDNIEIIPSYKFICTLNQITARLNTKHVDFITIIKEILVKCLQDHPHHTLYQLYPLIFDD

[0179] TGGKTNKSRSTIAAEIISKGRNSSNAQSAKQLAVVFPALIQFANADCGRGHTMLCDKL

[0180] KRIKCLDAVHCPTIELPVLPSKDYTIISIVRWDEKVLLVGGINAPKKLQCLCSDGKSRPQ

[0181] LLKGRDDLRQDAVMEQYFSLMNTLLCCDPKTSERKINIRTYKVVPLSMRSGILEWCEN

[0182] TVPIGVYLGSGSDKTGAHRKYRPADISPHKCRQISMQHLKSDIQKRLLIYEQICAQIKPV

[0183] FHYFLLENFVVPGIWFERRLAYTNSLAVNSMVGFVVGLGDRHTQNILIDEKTAEVIHID

[0184] FGIAFEQGKIMPTPETVPFRLTRDMIAPMGICETGGVFKKACQSTLEVLRKNHSVIITILE

[0185] VLLYDPLYIWNVVPTPAGSKDDEKNLTAQRALLCVQHKLEGRLSNITGTANTDVQVH

[0186] RLINDAVSKQNLCRLYPGWDPYL

[0187] Example 2

[0188] dsRNA of FOXO, the forkhead transcription factor of citrus fruit fly, and its application in pest control.

[0189] 1. Synthesis of dsRNA

[0190] Uses T7RiboMAX TM The Express RNAi System kit is used to synthesize dsRNA. The specific steps are as follows:

[0191] 1) Synthesis of dsRNA primers

[0192] Primers were designed based on the cloned gene fragment to amplify the target fragment, which was approximately 500 bp. A T7 promoter was introduced at the 5' end of the primers. The primer sequences are as follows: FOXO-2F (SEQ ID No. 6): 5'-TAATACGACTCACTATAGGGTGGGACGAAAAAGTGTTGCT

[0193] -3'; FOXO-2R (SEQ ID No.7): 5'-TAATACGACTCACTATAGGGGGCGTCGTTCAAACCAAATAC

[0194] -3'.

[0195] 2) Preparation of DNA template

[0196] The bacterial culture plasmid was extracted using a kit. Using the plasmid containing the gene fragment (the recombinant vector in Example 1, 3-2) as a template, PCR amplification was performed using FOXO-2F and FOXO-2R to obtain the target fragment containing the T7 promoter sequence.

[0197] The PCR reaction system is as follows (total volume 50 μL): plasmid 1 μL, dNTP 4 μL, 10×Buffer 5 μL, front primer 1 μL, back primer 1 μL, Taq enzyme 0.25 μL, ddH2O 37.75 μL.

[0198] The PCR reaction conditions were as follows: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min; stored at 4℃.

[0199] The PCR products were recovered, and the concentration of the target DNA was detected using a NanoPhotometer micro-spectrophotometer. The recovered concentration must be greater than 150 ng / μL.

[0200] 3) Synthesis of dsRNA

[0201] Uses T7RiboMAX TM The Express RNAi System kit transcribed the recovered DNA in vitro to synthesize dsRNA of the FOXO gene, a transcription factor in the citrus fruit fly forkhead. The dsRNA concentration was then measured using a NanoPhotometer micro-spectrophotometer, and the concentration needed to be greater than 1000 ng / μL. The dsRNA concentration was then adjusted to 1 ng / μL to obtain the dsRNA solution (using Nuclease-free water as the solvent).

[0202] The dsRNA of the citrus fruit fly forkhead transcription factor gene FOXO obtained in this invention is a double-stranded RNA, consisting of a sense strand and an antisense strand. The nucleotide sequence of the sense strand is shown in SEQ ID No. 3, and the nucleotide sequence of the antisense strand is the reverse complementary sequence of SEQ ID No. 3. The aforementioned dsRNA of the citrus fruit fly forkhead transcription factor gene FOXO can also be obtained through artificial synthesis. The dsRNA of the citrus fruit fly forkhead transcription factor gene FOXO is named dsFOXO.

[0203] SEQ ID No. 3:

[0204] TGGGACGAAAAAAGTGTTGCTGGTAGGGGGTATAAACGCTCCAAAAAAACTCCAATGTTTATGTTCAGATGGAAAATCACGACCTCAACTCTTAAAAGGTCGAGATGATTTACGCCAAGACGCCGTAATGGAGCAATATTTTTCACTCATGAACACACTATTGTGTTGCGATCCGAAAACAAGTGAACGAAAAATTAATATTCGCACCTACAAAGTTGTACCGCTCTCAATGAGAAGTGGAATATTAGAG TGGTGCGAAAATACCGTACCGATTGGTGTATATTTGGGTAGTGGAAGTGACAAAACTGGCGCACATCGGAAATATCGACCTGCTGATATCTCACCCCATAAGTGCCGTCAAATATCCATGCAACATCTTAAATCCGACATACAAAAACGACTTTTAATTTACGAGCAAATTTGCGCACAAATAAAGCCTGTTTTCCATTATTTTCTGCTGGAAAATTTTGTAGTTCCAGGTATTTGGTTTGAACGACGCC

[0205] 4) dsRNA of control GADPH

[0206] The dsRNA of control GADPH was synthesized according to the above method, and named dsRNA of control GADPH, yielding a dsGADPH solution with a concentration of 1 ng / μL. The primer sequences for synthesizing dsGADPH are as follows:

[0207] dsGADPH-T7F (SEQ ID No. 8):

[0208] TAATACGACTCACTATAGGGGTTTCACCTTGATGCCGTTC;

[0209] dsGADPH-T7R (SEQ ID No. 9):

[0210] TAATACGACTCACTATAGGGCACAAGTTCAAGCGTGTCCG.

[0211] 2. Application of dsRNA in pest control

[0212] 1) Experimental methods

[0213] dsRNA was introduced into the citrus fruit fly via injection. The specific steps were as follows: Using a 10 μL microsyringe, 5 μL of 1 ng / μL dsGADPH solution (experimental group) and dsGADPH solution (dsGADPH control group) were drawn and injected into the intersegmental membrane between the second and third abdominal segments of the third instar larvae, respectively. The needle of the syringe was parallel to the abdomen to avoid damaging the fly's internal organs. Each treatment was repeated 5 times, with 20 third instar citrus fruit flies (female to male ratio 1:1) per repeat. After injection, the flies were reared in an intelligent artificial climate chamber under the following conditions: temperature 26℃, humidity 70%, light-dark ratio 16h:8h. The growth and development of the citrus fruit flies were observed daily. Once they entered the adult stage, samples were taken to record survival rate, weight gain, growth rate, and overall performance. The number of days for the experiment is D, the number of survivors in each treatment group is S, and the survival rate is S / 20. The average body length of each treatment group before the experiment is A1, and the average body length of each treatment group after entering the adult stage is A2, with the increase in body length being A2-A1. The average weight of each treatment group before the experiment is B1, and the average weight of each treatment group after entering the adult stage is B2, with the increase in weight being B2-B1. The growth rate is (B2-B1) / D. The overall performance is (S / 20)×[(B2-B1) / D].

[0214] dsRNA was introduced into the citrus fruit fly via feeding. The specific steps were as follows: Third-instar larvae of the fruit fly were selected and fed. The experiment was divided into two groups: the experimental group was fed artificial feed containing 0.3 μg / μL dsFOXO, and the control group (CK) was fed artificial feed containing 0.3 μg / μL dsGADPH. The artificial feed was placed in insect feeding boxes (disposable containers, 10 cm in diameter and 5 cm high, with multiple small holes punched in the lid using a 3 mm insect needle). Thirty third-instar larvae were released into each feeding box, with five replicates per group. The artificial feed was changed daily, and the growth, development, and survival status of the citrus fruit fly were observed and recorded. After entering the adult stage, samples were taken to collect data on survival rate, weight gain, growth rate, and overall performance. The statistical methods were the same as for dsFOXO injection treatment. The experiment was conducted in an artificial climate chamber at a temperature of (26±1)℃, relative humidity of (75±5)%, and a light:dark ratio of 16h:8h.

[0215] 2) Real-time quantitative PCR

[0216] Total RNA was extracted from the citrus fruit fly bodies of the experimental group and the control group (dsGADPH control group) 72 hours after injection. cDNA was synthesized using a Takara reverse transcription kit, and the expression level of the FOXO gene was detected. The GADPH gene was used as an internal reference gene.

[0217] The primer sequences for the FOXO gene are as follows:

[0218] A-FOXO-F(SEQ ID No.10):CCCGAATACCCGTGTGCAA

[0219] A-FOXO-R (SEQ ID No. 11): CAGACTTCCCAACTTGCGGC;

[0220] The primer sequences for the GADPH gene are as follows:

[0221] A-GADPH-F (SEQ ID No. 12): GCAAACTGTGGCGTGATG

[0222] A-GADPH-R (SEQ ID No. 13): GGTGTTGGGACACGGAAT.

[0223] The results showed that compared with the control group (dsGADPH control group), the expression level of the FOXO gene in the citrus fruit fly dsFOXO experimental group was significantly reduced, indicating that dsFOXO successfully interfered with the expression of the FOXO gene in the citrus fruit fly dsFOXO. There was no significant difference in the dsGADPH control group. Figure 1 ).

[0224] 3) Results of survival rate, weight gain, growth rate, and overall performance.

[0225] The results are as follows Figure 2 As shown, the survival rate, weight gain, growth rate, and overall performance of the citrus fruit fly after FOXO gene RNAi were significantly different from those of the control group (dsGADPH control group) (P < 0.05). All indicators of the citrus fruit fly after GADPH gene interference were significantly lower than those of the control group. This indicates that GADPH gene RNAi has an inhibitory effect on the growth and development of the citrus fruit fly, and that feeding is more effective than injection.

[0226] As demonstrated by the above embodiments, this invention clones the forkhead transcription factor FOXO from the citrus fruit fly, which plays a crucial role in the growth and development of the citrus fruit fly. This invention synthesizes dsRNA to interfere with the FOXO gene in the citrus fruit fly, and introduces the dsRNA into the citrus fruit fly via injection and feeding, respectively, to perform RNAi on the FOXO gene. After interfering with FOXO expression, the survival rate, growth rate, body weight, and overall performance of the citrus fruit fly were significantly reduced. This invention provides a new method for the molecular regulation of pests and also provides a theoretical basis for a deeper understanding of insect growth and development mechanisms and the creation of new biological pesticide formulations.

[0227] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for controlling the citrus fruit fly, characterized in that, Biological materials that inhibit the expression of proteins with amino acid sequences as shown in SEQ ID No. 2 were applied to the citrus fruit fly; The biological material is dsRNA that inhibits the expression of nucleic acid molecules with sequences as shown in SEQ ID No. 1; The sequence of the dsRNA is shown in SEQ ID No.

3.

2. The method according to claim 1, characterized in that, The biomaterials are administered via feeding and / or injection.

3. The method according to claim 1, characterized in that, Control of the citrus fruit fly includes: a1) Inhibits the growth and development of the citrus fruit fly; a2) Reduce the survival rate of the citrus fruit fly; a3) Reduce the weight gain of the citrus fruit fly; a4) Reduces the growth rate of the citrus fruit fly; a5) Reduces the overall performance of the citrus fruit fly.

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