Citrus fruit fly forked head transcription factor FOXO and coding gene and application thereof

Abstract

The invention provides a bactrocera minax forked head transcription factor FOXO as well as a coding gene and application thereof, and belongs to the technical field of biological prevention and control of crop pests. A nucleic acid molecule of the citrus fruit fly forked head transcription factor FOXO is cloned, dsRNA for interfering the nucleic acid molecule of the citrus fruit fly coded forked head transcription factor FOXO is synthesized, and the dsRNA is introduced into the citrus fruit fly by using an injection method and a feeding method to carry out RNAi on the citrus fruit fly forked head transcription factor gene FOXO. After the dsRNA is injected or fed to the bactrocera minax, the survival rate, the growth rate, the weight and the overall expressive force of the bactrocera minax are remarkably reduced, and it is indicated that the fork head transcription factor FOXO of the bactrocera minax plays an important role in the growth and development process of insects. The invention provides a new method for molecular regulation and control of pests, and also provides a theoretical basis for deeply understanding the growth and development mechanism of insects and creating a new biopesticide preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological control of crop pests, and in particular relates to a forkhead transcription factor FOXO of the citrus fruit fly, a coding gene thereof, and an application thereof. Background Art

[0002] Amidst the booming global citrus industry, the citrus fruit fly, Bactrocera minax Enderlein, a devastating oligophagous pest of citrus, has become a key bottleneck hindering the healthy and sustainable development of the industry. Its unique feeding habits—attacking only immature citrus fruits—cause the fruit to rot internally, causing premature fruit drop and widespread yield reductions. In recent years, frequent outbreaks of the citrus fruit fly have plagued several major citrus-producing areas, impacting numerous orchards. This has severely damaged farmers' economic returns, disrupted the stable supply of citrus fruits, and caused incalculable economic losses to the entire citrus industry.

[0003] Currently, control strategies for the citrus fruit fly primarily focus on chemical and physical control. Chemical control, with its high efficiency and rapid insecticide rates, has long dominated pest control. However, the negative effects of over-reliance on chemical pesticides are becoming increasingly prominent. On the one hand, the extensive use of chemical pesticides has severely damaged the ecological environment, polluting soil, water, and air, while also leading to a sharp decline in the populations of non-target organisms such as bees and birds, undermining biodiversity. On the other hand, pesticide residues have become a significant threat to food safety, sparking significant consumer concern about the quality and safety of citrus products and severely impacting the brand image and market competitiveness of the citrus industry. While physical control measures, such as traps, can reduce chemical pesticide use to a certain extent, they suffer from limitations such as low efficiency and limited coverage, making them inadequate for large-scale pest control. These issues highlight the current shortcomings of crop pest control, which are relatively backward and technologically limited, and urgently require innovation and breakthroughs.

[0004] In the field of insect biology research, the forkhead transcription factor FOXO, as a key regulatory factor, plays a vital role in the detoxification, nutrient absorption and development of insects, 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 cause oxidative stress responses in herbivorous insects, thereby activating the expression of FOXO, a gene downstream 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, peroxidase and other protective enzymes) and detoxification enzymes (such as cytochrome P450 enzyme system, glutathione S-transferase, etc.), and ultimately achieves 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 new targets for pest control, provide a theoretical basis and technical support for the development of efficient, green and safe new pest control agents, and have important practical significance and broad application prospects for breaking through the bottleneck of existing prevention and control technologies and promoting the green and high-quality development of the citrus industry. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the forkhead transcription factor FOXO of the citrus fruit fly, its encoding gene and application. The present invention found that the forkhead transcription factor FOXO of the citrus fruit fly plays an important role in the growth and development of insects. 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 prevention and control.

[0006] The present invention provides a forkhead transcription factor FOXO of the citrus fruit fly, wherein the forkhead transcription factor FOXO is a protein of the following a) or b):

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

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

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

[0010] The present invention provides a biological material expressing the forkhead transcription factor FOXO, comprising any one of the following A1) to A8):

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

[0012] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0013] A3) a recombinant vector containing the nucleic acid molecule described in A1);

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

[0015] A5) a recombinant microorganism containing the nucleic acid molecule described in A1);

[0016] A6) a recombinant microorganism containing the expression cassette described in A2);

[0017] A7) a recombinant microorganism containing the recombinant vector described in A3);

[0018] A8) A recombinant microorganism containing the recombinant vector described in A4).

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

[0020] The present invention provides a biological material for inhibiting the expression of the forkhead transcription factor FOXO.

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

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

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

[0024] a1) Inhibit the growth and development of insects;

[0025] a2) preparing products that inhibit the growth and development of insects;

[0026] a3) Pest control;

[0027] a4) Preparation of products for controlling pests;

[0028] a5) Reduce insect survival rate;

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

[0030] a7) reducing the weight gain of insects;

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

[0032] a9) reducing insect growth rate;

[0033] a10) preparing products for increasing insect growth rate;

[0034] a11) reduce the overall performance of insects;

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

[0036] The invention provides a method for preventing and controlling citrus fruit fly, and the biological material is acted on the citrus fruit fly.

[0037] Preferably, the biological material is administered by feeding and / or injection.

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

[0039] The present invention first cloned the nucleic acid molecule encoding the forkhead transcription factor FOXO from the citrus fruit fly, designed primers, and synthesized dsRNA to interfere with the nucleic acid molecule encoding the forkhead transcription factor FOXO in the citrus fruit fly. The dsRNA was then introduced into the citrus fruit fly by injection and feeding, respectively, to perform RNAi on the citrus fruit fly forkhead transcription factor FOXO. 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 citrus fruit fly forkhead transcription factor FOXO plays an important role in the growth and development of insects. This invention provides a new method for the molecular regulation of pests and also provides a theoretical basis for a deeper understanding of the mechanisms of insect growth and development and the creation of new biopesticide formulations. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0041] Figure 2 These are the statistical results of the effects of FOXO gene interference on the survival rate, weight gain, growth rate and overall performance of the citrus fruit fly. DETAILED DESCRIPTION

[0042] The present invention provides a forkhead transcription factor FOXO of the citrus fruit fly. The forkhead transcription factor FOXO is a protein as follows a), b), c) or d): a) a protein with an amino acid sequence as shown in SEQ ID No. 2; b) a fusion protein obtained by connecting 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 replacing, deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2; d) a protein with a homology of more than 75% with the amino acid sequence shown in SEQ ID No. 2 and having the same function.

[0043] In the present invention, the tag described in b) is preferably Poly-Arg, Poly-His, FLAG, Strep-tag II, or c-myc. The tag described in b) facilitates purification of the protein described in a). The specific sequence of the tag is shown in Table 1. The present invention does not specifically limit the preparation method of the forkhead transcription factor FOXO; either artificial synthesis or synthesis of the encoding gene followed by biological expression is acceptable.

[0044] Table 1 Tag sequences

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

[0046] The present invention provides a biological material expressing the forkhead transcription factor FOXO, including any one of A1) to A8); in the present invention, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0047] In the present 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 a biological material for inhibiting the expression of the forkhead transcription factor FOXO.

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

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

[0051] In the present invention, the biomaterial is preferably administered by feeding and / or injection, more preferably by feeding. In the present invention, when the biomaterial is dsRNA, the dsRNA is preferably administered in the form of a solution, preferably using nuclease-free water as the solvent. The dsRNA is preferably administered at a concentration of 0.5 to 2 ng / μL, more preferably 0.8 to 1.5 ng / μL, and most preferably 1 ng / μL.

[0052] The technical solutions provided by the present invention are 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, the materials and reagents used in the following examples can be obtained from commercial sources.

[0055] The quantitative tests in the following examples were all repeated five times, and the results were averaged.

[0056] The test insect source in the following examples: 2-year-old citrus fruit flies (developmentally uniform) were collected from Youyang County, Chongqing City, and raised in an intelligent artificial climate chamber. The breeding conditions were as follows: temperature 26°C, humidity 70%, light-dark ratio of 16h:8h, and uniformly fed with feed (feed formula - 500mL distilled water: sucrose 60g, brewer's yeast 20g, peptone 5g, agarose 5g, sorbic acid 0.5g, ascorbic acid 3.3g, parahydroxybenzoic acid 0.6g).

[0057] The main reagents in the following examples are: RNA isolation reagent (original Invitrogen), RNA spincolumn (Quanshi Gold), gel recovery kit (Axygen), EX Taq DNA polymerase (Takara), T4 DNA ligase (Takara), pGEM-T Easy Vector Systems (Promega), and reagents such as anhydrous ethanol, isopropanol, and glycerol were all domestic analytical alcohols.

[0058] The main instruments in the following embodiments are: clean bench (Shanghai Boxun Industrial Co., Ltd.), Dongshenglong ETC-811 PCR instrument (Beijing Dongsheng Innovation Biotechnology Co., Ltd.), German Sigma 3K15 refrigerated centrifuge (German Sigma Centrifuge Co., Ltd.), NanoPhotometer microspectrophotometer (Germany IMPLEN Company), HPX-9052MBE digital display electric incubator (Shanghai Boxun Industrial Co., Ltd.), THZ-D desktop constant temperature oscillator (Huamei Biochemical Instrument Factory), vortex oscillator QL-901 (Haimen Qilin Bell Instrument Manufacturing), and high pressure sterilizer YXQ-LS-50SII (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory).

[0059] Example 1

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

[0061] 1. Extraction of total RNA from Bactrocera citri

[0062] use RNA isolation reagent was used to extract RNA from citrus fruit fly tissue samples. The specific steps are as follows:

[0063] 1) Sterilize a 2 mL homogenizer in an oven at 160°C for 3 hours, then cool to room temperature for later use.

[0064] 2) Place the homogenizer on ice and add 1 mL RNA isolation reagent and 200 mg of B. citri tissue, ground.

[0065] 3) Transfer the homogenate to a 1.5 mL centrifuge tube and let it stand at room temperature for 5 minutes. Centrifuge at 13,000 rpm at 4°C for 5 minutes.

[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 at 4°C for 10 minutes.

[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 at 4°C for 10 minutes.

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

[0069] 7) Centrifuge at 13,000 rpm for 2 min at 4°C and discard the filtrate.

[0070] 8) Add 600 μL RNAwash solution, pipette to wash the pellet, centrifuge at 13,000 rpm at 4°C for 2 min, and discard the filtrate. Add 600 μL RNAwash solution again, pipette to wash the pellet, centrifuge at 13,000 rpm at 4°C for 2 min, and discard the filtrate. Centrifuge at 13,000 rpm at 4°C for 3 min to remove excess ethanol, and air dry for 3 min.

[0071] 9) Replace a new collection tube and add 50 μL of 65°C preheated RNase-free water to the RNA spin column. Heat the tube for 5 minutes under residual heat and centrifuge at 13,000 rpm at 4°C for 3 minutes.

[0072] 10) Collect the filtrate and measure RNA concentration and OD260 / 280 using a NanoPhotometer to confirm RNA quality. Simultaneously, remove 2 μL of the extracted RNA and analyze by agarose gel electrophoresis. Store the remaining RNA at -20°C until further use.

[0073] 2. Reverse transcription

[0074] Using PrimeScript TMcDNA was obtained by reverse transcription using the 1st strand cDNA Synthesis Kit. The specific steps are as follows:

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

[0076] 2) Keep at 65℃ for 5 min and then 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 add RNase-Free dH2O to a volume of 20 μL.

[0078] 4) Mix slowly.

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

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

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

[0082] 1) Primer design

[0083] Based on the previously obtained transcriptome of the fruit fly, the FOXO gene sequence was obtained. DNAMAN8 was used to design full-length primers or fragment primers for the FOXO gene. 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] The PCR product was obtained by using the cDNA of Bactrocera melanogaster as template and primers FOXO-F / FOXO-R for PCR amplification.

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

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

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

[0091] 3-1) Run the PCR product on a 1% agarose gel in TAE. When the target band is well separated, cut the gel block containing the target band with a razor blade and place it in a sterile centrifuge tube. Then, use an Axygen gel extraction kit to recover and purify the target band. Follow the kit instructions for recovery and purification.

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

[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, place on ice for 15 minutes, incubate at 42°C in a water bath for 90 seconds, and then place on ice for 10 minutes. Add 500 μL of liquid LB medium to each 1.5 mL centrifuge tube and shake at 37°C at 200 rpm for 2 hours. After shaking, transfer 100 μL of the culture to 1‰ AMP LB solid medium and incubate at 37°C overnight. Select a single colony and place it in a 2 mL centrifuge tube containing 1 mL of 1‰ AMP LB liquid medium. Shake at 37°C at 200 rpm for 6 hours and observe growth.

[0095] 3-4) Bacterial liquid PCR

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

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

[0098] The positive clone strains were sent to Shanghai Sangon Bioengineering Technology Service Co., Ltd. for sequence determination and analysis of the sequencing results.

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

[0110] GGCGTTCTTTATGACAATTATGTATACTGAATTATGGGCACTGTCAGAGAGCATATCG

[0111] AACGATAAGCAGTCCAAAGTAGAGGAGCATTTAAAATCCAGAATTTCAGGAAGT

[0112] AGCAATAAAGGCTTATAAATCCATTGGTTGTCATGATGCCATATCTGGGATTCTCTCA

[0113] CCTTTACAATCTCGTCTTGAATTTCTGAATTTAAACAACGATTGGTCCGAAATGTTAC

[0114] TTCAAAACTCTTTTAAGAATACATCGTCGAATACACTTTGTACTTCAGCATTAAAACG

[0115] GAACGGTATTTTATGCCTCGCAAATCTGGACAATAAAGACAATTTAGTTGACTATGA

[0116] AGTATGTTGGAGATTATGTCAATGGGATACGCCCGTAGAAGGGCATTTGAAAGTGAA

[0117] TATTGAAAATGATCCCGAATTGGAATTTAAGAAGCATCATTTCAATGCTTTAAAATGC

[0118] CTATATAATCGCGAACAACAAAATTGTTTAGCGGCCATCGCAAACGCACGGCTATGC

[0119] GTAATTTACACTTTGACGGGAATAAGCACCGATTGCCTTCAAAGTATATACAAATATC

[0120] TAACATGGCTGCACATACTTCAACAAACGGAGGATTTTTGTCAAATCCAGTTTACTT

[0121] CTGAAGTAGATATTAAATCGATCTTTTCAAAGTGGCAGTCGGAAAATAAACTAAAAT

[0122] ACGGTAGCTTTTATTGCAAAGAACTAATAATGTCGCATCAGATGACTCTATTTAACAC

[0123] TGCTGGAGTCCGTGGTCAAAGAAGGATAATAGATTTTTACAAATTTAGTCCGACCGA

[0124] AACTGGTTTGTTAAATATCATTAGAGAATGCCAAAAATCAGGGGAAATTAACTTGGC

[0125] GAAACGTAATATATTAGCTCTTAGAGAAGCAGAAATTACAAACGAACATGTCAAGCT

[0126] TAATTTGTTACTTGAAGATGCAGAAATTTCTTTTCGATGCGGAAATATTGAAATAACT

[0127] GAAGCATTGTTAAAACACACGTTGACACATAAAGATCTAACTGCTTGCCCTCAACA

[0128] AGCACGTGCTTCTCCGAATGTATGGAGAATTTCTCTTGGAGACTAATTCGCAAAGTTT

[0129] TGAATACGTTCTAGAAAAATGTTTTAATAGATCTATGCTTTACCTGGAGAAGATATTA

[0130] AAATCTCAAAAACAGTGTGATAATGACGAATTAAGTTTTTTGTATTTAGAAGGCCTA

[0131] AAACCAGCCGAATTTGAAAAGGAAAACAAAAAAGAAGCATATCAATTAATAGCGA

[0132] AATATGCCGATCGTGAATATGCGCAATCGAATGTGTACATCAATTCTGAGGAATTTAA

[0133] ACTGAAGTGTCAAATCATACAACACAACAGACAAACAGCGGATTCAATTGGTCGAC

[0134] AACACAAAGATCGTGATATTAACCACGGTGTAATCATAATGAAAAAGTATGCGAATT

[0135] TGGATGAAACGGAAATAAAGTTTATTGAGGAGAAACGCACAAATAACTTGTGTATT

[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] AATATATTGATTGATGAGAAGACTGCTGAAGTAATTCATATCGATTTTGGAATTGCTT

[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 the forkhead transcription factor FOXO of the citrus fruit fly and its application in pest control

[0189] 1. Synthesis of dsRNA

[0190] Using T7RiboMAX TM The Express RNAi System kit synthesizes 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 of about 500 bp. The T7 promoter was introduced at the 5' end of the primer. The primer sequence is 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 liquid plasmid was extracted using a kit, and the plasmid containing the gene fragment (the recombinant vector in 3-2 in Example 1) was used as a template to perform PCR amplification using FOXO-2F and FOXO-2R to obtain the target fragment containing the T7 promoter sequence.

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

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

[0199] Recover the PCR product and detect the target DNA concentration using a NanoPhotometer micro-spectrophotometer. The recovery concentration must be greater than 150 ng / μL.

[0200] 3) Synthesis of dsRNA

[0201] Using T7RiboMAX TM The Express RNAi System kit uses in vitro transcription of recovered DNA to synthesize dsRNA targeting the forkhead transcription factor gene FOXO of the citrus fruit fly. The dsRNA concentration is measured using a NanoPhotometer microspectrophotometer, which requires it to be greater than 1000 ng / μL. The dsRNA concentration is then adjusted to 1 ng / μL to create a dsRNA solution (solvent: Nucleaue-free water).

[0202] The dsRNA of the forkhead transcription factor gene FOXO of the citrus fruit fly obtained in the present 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 complement of SEQ ID No. 3. The dsRNA of the forkhead transcription factor gene FOXO of the citrus fruit fly can also be obtained by artificial synthesis. The dsRNA of the forkhead transcription factor gene FOXO of the citrus fruit fly is named dsFOXO.

[0203] SEQ ID No.3:

[0204] TGGGACGAAAAAAGTGTTGCTGGTAGGGGGTATAAACGCTCCAAAAAAACTCCAATGTTTATGTTCAGATGGAAAATCACGACCTCAACTCTTAAAAGGTCGAGATGATTTACGCCAAGACGCCGTAATGGAGCAATATTTTTCACTCATGAACACACTATTGTGTTGCGATCCGAAAACAAGTGAACGAAAAATTAATATTCGCACCTACAAAGTTGTACCGCTCTCAATGAGAAGTGGAATATTAGAG TGGTGCGAAAATACCGTACCGATTGGTGTATATTTGGGTAGTGGAAGTGACAAAACTGGCGCACATCGGAAATATCGACCTGCTGATATCTCACCCCATAAGTGCCGTCAAATATCCATGCAACATCTTAAATCCGACATACAAAAACGACTTTTAATTTACGAGCAAATTTGCGCACAAATAAAGCCTGTTTTCCATTATTTTCTGCTGGAAAATTTTGTAGTTCCAGGTATTTGGTTTGAACGACGCC

[0205] 4) Control GADPH dsRNA

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

[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 by injection. The specific steps were as follows: 5 μL of a 1 ng / μL dsGADPH solution (experimental group) or a dsGADPH solution (dsGADPH control group) was injected into the intersegmental membrane between the second and third abdominal segments of the abdomen of third-instar larvae, respectively, using a 10 μL microsyringe. The needle of the syringe was parallel to the abdomen to avoid damaging the fruit fly's internal organs and tissues. Each treatment was repeated five times, with 20 third-instar citrus fruit flies (1:1 male:female ratio) treated per replicate. After injection, the flies were reared in an intelligent artificial climate chamber under the following conditions: temperature of 26°C, humidity of 70%, and a light:dark ratio of 16 hours:8 hours. The growth and development of the citrus fruit flies were observed daily. After entering the adult stage, samples were collected to analyze survival rate, weight gain, growth rate, and overall performance. The number of statistical days was D, the number of survivors in each treatment group was S, and the survival rate was S / 20; the average body length of each treatment group before the experiment was A1, and the average body length of each treatment group after entering the adult stage was A2, and the increase in body length was A2-A1; the average weight of each treatment group before the experiment was B1, and the average weight of each treatment group after entering the adult stage was B2, and the increase in weight was B2-B1; growth rate was (B2-B1) / D; overall performance was (S / 20)×[(B2-B1) / D].

[0214] dsRNA was introduced into the citrus fruit fly (B. citri) by feeding. The specific steps were as follows: Third-instar larvae of the fruit fly were selected for feeding and divided into two groups: the experimental group was fed an artificial diet containing 0.3 μg / μL dsFOXO, and the control group (CK) was fed an artificial diet containing 0.3 μg / μL dsGADPH. The artificial diet was placed in insect feeding boxes (disposable boxes with a diameter of 10 cm and a height of 5 cm, with the lid punctured with multiple holes using a 3 mm insect needle). Thirty third-instar larvae were released from each box, with five replicates per group. Fresh artificial diet was replaced daily, and the growth, development, and survival of the fruit fly were observed and recorded. After reaching adulthood, samples were collected to analyze survival rate, weight gain, growth rate, and overall performance. Statistical methods were the same as for the dsFOXO-injected treatment. The experiment was conducted in an artificial climate chamber at a temperature of (26±1)°C, a relative humidity of (75±5)%, and a 16h:8h light:dark cycle.

[0215] 2) Real-time fluorescence quantitative PCR

[0216] 72 hours after injection, total RNA was extracted from the experimental group and the control group (dsGADPH control group) and cDNA was synthesized with the Takara reverse transcription kit to measure FOXO gene expression. 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 GADPH gene primer sequences 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 FOXO gene in citrus fruit fly in the dsFOXO experimental group was significantly reduced, indicating that dsFOXO successfully interfered with the expression of FOXO gene in citrus fruit fly. There was no significant difference in the results of the dsGADPH control group ( Figure 1 ).

[0224] 3) Survival rate, weight gain, growth rate and overall performance test results

[0225] The results are as follows Figure 2 As shown in the results, 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 the feeding effect is better than the injection effect.

[0226] As can be seen from the above examples, the present invention cloned the citrus fruit fly forkhead transcription factor FOXO from the citrus fruit fly, which plays an important role in the growth and development of the citrus fruit fly. The present invention synthesized dsRNA for interfering with the citrus fruit fly FOXO gene, and introduced the dsRNA into the citrus fruit fly by injection and feeding, respectively, to perform RNAi on the citrus fruit fly forkhead transcription factor FOXO gene. After interfering with FOXO expression, the citrus fruit fly's survival rate, growth rate, body weight, and overall performance were significantly reduced. This invention provides a new method for the molecular regulation of pests and a theoretical basis for a deeper understanding of the growth and development mechanisms of insects and the creation of new biopesticide formulations.

[0227] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A forkhead transcription factor FOXO of the citrus fruit fly, characterized in that The forkhead transcription factor FOXO is a protein of a) or b) as follows: a) a protein with an amino acid sequence as shown in SEQ ID No. 2; b) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No.

2.

2. The forkhead transcription factor FOXO according to claim 1, characterized in that b) The tag is Poly-Arg, Poly-His, FLAG, Strep-tag II or c-myc.

3. The biomaterial expressing the forkhead transcription factor FOXO according to claim 1, characterized in that: Including any one of the following A1) to A8): A1) a nucleic acid molecule encoding the forkhead transcription factor FOXO according to claim 1; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1); A4) a recombinant vector containing the expression cassette described in A2); A5) a recombinant microorganism containing the nucleic acid molecule described in A1); A6) a recombinant microorganism containing the expression cassette described in A2); A7) a recombinant microorganism containing the recombinant vector described in A3); A8) A recombinant microorganism containing the recombinant vector described in A4).

4. The biomaterial according to claim 3, characterized in that The sequence of the nucleic acid molecule encoding the forkhead transcription factor FOXO according to claim 1 is shown in SEQ ID No.

1.

5. A biomaterial for inhibiting the expression of the forkhead transcription factor FOXO according to claim 1.

6. The biomaterial according to claim 5, characterized in that The biological material includes dsRNA that inhibits the expression of a nucleic acid molecule encoding the forkhead transcription factor FOXO.

7. The biomaterial according to claim 6, characterized in that The sequence of the dsRNA is shown in SEQ ID No.

3.

8. Use of the biomaterial according to any one of claims 5 to 7 in any one of the following a1) to a12): a1) Inhibit the growth and development of insects; a2) preparing products that inhibit the growth and development of insects; a3) Pest control; a4) Preparation of products for controlling pests; a5) Reduce insect survival rate; a6) preparing products that reduce insect survival rates; a7) reducing the weight gain of insects; a8) preparing products that reduce the weight gain of insects; a9) reducing insect growth rate; a10) preparing products for increasing insect growth rate; a11) reduce the overall performance of insects; a12) Preparation of products that reduce the overall performance of insects.

9. A method for controlling citrus fruit fly, characterized in that: The biological material according to any one of claims 5 to 7 is applied to the citrus fruit fly.

10. The method according to claim 9, characterized in that The biological material may be administered by feeding and / or injection.

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