Horizontal transfer gene Zadp of tomato leaf miner and application of horizontal transfer gene Zadp in regulation and control of mating rhythm and spawning of tomato leaf miner imago

Abstract

The invention relates to the technical field of biology, in particular to a tomato leaf miner horizontal transfer gene Zadp and application of the tomato leaf miner horizontal transfer gene Zadp to regulation and control of mating rhythm and spawning of tomato leaf miner imagoes. The tomato leaf miner horizontal transfer gene zinc binding ethanol dehydrogenase family gene Zadp encodes a protein with an amino acid sequence as shown in SEQ ID No: 2. The CRISPR / Cas9 is utilized to induce mutation of the Zadp gene of the tomato leaf miner, and the result shows that a Zadp mutation group delays mating, the mating time and the generation length are prolonged, and the egg laying rate within 7 days is remarkably reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the horizontal transfer of genes of tomato leafminers. Zadp And its application in regulating the mating rhythm and oviposition of tomato leafminer adults. Background Art

[0002] Tomato leafminer Tuta absoluta The tomato leafminer (Meyrick) is one of the most destructive quarantine pests worldwide. Its larvae primarily feed on Solanaceae crops, severely damaging many varieties.

[0003] The use of RNAi technology can achieve agricultural pest and disease control, which is in line with the green and safe development strategy. RNAi is a gene silencing phenomenon caused by dsRNA. Obtaining lethal and effective functional target genes is the key core point of using RNAi technology.

[0004] CRISPR-Cas9 technology is a reliable, efficient, and rapid new method for gene knockout. Using CRISPR / Cas for gene editing can achieve long-lasting and stable results, making it an effective genetic tool for studying gene function. CRISPR-Cas9 technology achieves precise genome editing through targeted DNA cleavage by the Cas9 protein guided by gRNA.

[0005] Horizontal gene transfer refers to the transfer of genetic material between distantly related species that are reproductively isolated from each other. The transmission and integration of horizontally transferred genes can provide organisms with some beneficial characteristics and play an important role in their evolution. Horizontal gene transfer in insects is one of the main driving forces for insects to adapt quickly to the environment. Many horizontal gene transfer events have been identified in insects. Different horizontally transferred genes provide different insects with a variety of functions including nutrient synthesis and symbiosis, absorption and digestion, toxin production and detoxification, growth and development, and changes in body color. According to genetic evaluation, the horizontally transferred gene zinc-binding alcohol dehydrogenase family gene Zadp Originated from Listeria monocytogenes, almost all moths and butterflies, except Chilo suppressalis and Pieris striata, contain orthologous genes of this gene. Zadp The number of times male diamondback moths courted females was significantly reduced, showing low desire courtship behavior. Zadp It has the function of enhancing male courtship behavior. Summary of the Invention

[0006] The present invention aims to provide a tomato leafminer horizontal transfer gene zinc binding alcohol dehydrogenase family gene Zadp .

[0007] Another object of the present invention is to provide the role of the above-mentioned coding gene in the mating rhythm and oviposition of tomato leafminer adults.

[0008] According to a specific embodiment of the present invention, the horizontally transferred gene zinc-binding alcohol dehydrogenase family gene of the tomato leafminer was cloned for the first time. Zadp The full-length nucleotide sequence of the cDNA is shown in SEQ ID No: 1:

[0009] Tomato leafminer Zadp The gene encodes a protein with an amino acid sequence as shown in SEQ ID NO: 2: MFLRTVLYIAASIAKANPLVQVAKASTLSNLNMTRTLPSTMKAVGFYKHLDITDPKSLQDITDIALPTVQKNEVLIEVAAVSVNPIDTKIRSPKGKTSDTLPTARILGWDGAGIIAAKGPNVDSLEVGDEVIFTGNIAKSGSNAQYVALNQIMVAPKPKNLTFEQAAAMPLTAVTAYEAIYDRMGLSEKD KRKSLLIINCAGGVGSVAAQLAKNLGMTVIGTASREESKAFALKFGANQVLNHSRNLVSELEHLGLKEVDYIMVNYDPYPYWDTLMHIIKPQGKICLVVDCSQPVDIRALKDKSITLVSEMMATRIKYSTPDVNRHNVILREVSRMLETGELKTTTLTRVMSPINAHNLKDAHKLLESQKLIGKVVLKDF.

[0010] The present invention is based on CRISPR / Cas technology to horizontally transfer genes of zinc-binding alcohol dehydrogenase family genes in tomato leafminer Zadp The function of genes was explored and the Zadp The functional role of genes in tomato leafminer. Zadp Gene-induced mutations, the results showed that Zadp The mutant group had delayed mating, prolonged mating time and generation length, and a significant decrease in egg-laying rate within 7 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Tomato leafminer Zadp Expression analysis at different developmental stages; Figure 2 Tomato leafminer Zadp The effect of genes on the egg-laying rate of F0 generation adults; Figure 3 Tomato leafminer Zadp Genetic influences on mating rhythms; Figure 4 Tomato leafminer Zadp Genetic influences on mating duration; Figure 5 Tomato leafminer Zadp Genetic influences on generation length. DETAILED DESCRIPTION Example 1: Cloning of the full-length cDNA sequence of the Zadp gene of the tomato leafminer

[0012] Place a sample of adult tomato leafminer in a 1.5 mL centrifuge tube and freeze in liquid nitrogen for 5 minutes. Grind the sample with a grinding rod, extract RNA, and store at -80°C until needed. Reverse transcribe the extracted RNA into cDNA using a reverse transcription kit. Design primers and perform PCR amplification using the cDNA as a template. The primers used are as follows: Upstream primer: 5′-AAACGGTGGTGAGGTA-3′ (SEQ ID NO: 3); Downstream primer: 5'-CAGGGATAATAGATGTGATA-3' (SEQ ID NO: 4).

[0013] PCR amplification, obtained Zadp The cDNA sequence of the gene encodes 379 amino acids as shown in SEQ ID No: 2. Example 2: Expression analysis of Zadp gene at different developmental stages in tomato leafminer

[0014] The eggs, 1st to 4th instar larvae, pupae, female adults and male adults were collected. RNA extraction and cDNA synthesis were the same as in Example 1.

[0015] Fluorescence quantitative PCR analysis was performed using an ABI 7500 fluorescence quantitative PCR instrument. The reaction conditions were as follows: cycling phase (40 cycles): 95°C for 5 min, 95°C for 10 s, and 60°C for 30 s; melting curve phase: 95°C for 15 s, 60°C for 1 min, 95°C for 30 s, and 60°C for 15 s.

[0016] Fluorescence quantitative PCR results were obtained using 2 -△△Ct The relative expression of genes was calculated by SPSS software, and the significant differences were expressed by letters ( P <0.05). The primers for real-time fluorescence quantitative PCR are shown below. The results show that Zadp The gene mRNA is expressed at all stages, with the highest expression in male adults ( Figure 1 ).

[0017] TaZadp: q Zadp -F: 5'TCCGTTAGTTCAAGTC3' (SEQ ID NO: 5); q Zadp-R: 5'TCCATCCCAACCCAGA3' (SEQ ID NO: 6); Rpl5: qRpl5-F: 5'CAGTCGTCGAGCCAGCAACA3' (SEQ ID NO:7); qRpl5-R: 5'TCCCGCATTGAAGGAGACCA3' (SEQ ID NO:8). Example 3: Identification of the Suc gene function in the tomato leafminer

[0018] sgRNAs were designed in the CDS region, and editing efficiency and potential off-target effects were evaluated simultaneously. Finally, three sgRNAs were selected, and the sgRNA primer sequences are shown below: sgRNA-TaZadp-1: 5'ATGCACATAATCAAGCCCCAGGG3' (SEQ ID NO: 9); sgRNA-TaZadp-2: 5'CCTAGTGGTGGATTGTAGTCAAC3' (SEQ ID NO: 10); sgRNA-TaZadp-3: 5'CCTTGTATCTGAGTTAGAACATC3' (SEQ ID NO: 11).

[0019] Total RNA extraction and cDNA synthesis were performed as in Example 1. sgRNA template synthesis, PCR product purification, in vitro sgRNA synthesis, and sgRNA purification were performed using a kit according to the kit instructions.

[0020] Purchase Cas9 protein and prepare injection solution. The total system is 10 μL, including 0.75 μL of synthetic 4 mg / mL Cas9 protein (final concentration 300 ng / μL) and Suc 1.5 μL of sgRNA (final concentration 300 ng / μL, 150 ng / μL of each sgRNA), 1.5 μL each of sgRNA-Pamasc-1 and sgRNA-Pamasc-2, and the rest was made up with ddH2O.

[0021] Collect eggs from tomato leaves within two hours of germination and sequentially tape them onto coverslips with thin strips of double-sided tape, arranging them in a vertical line. Place the coverslips with eggs on a glass slide, and inject the injection solution into the embryos using a microinjection platform. Mark the coverslip where the eggs were injected, transfer them to a 150mm disposable Petri dish, place a damp cotton ball in the dish to keep them moist, and incubate them in an incubator.

[0022] After injection of target gene, compared with the control, Zadp The egg-laying rate of the mutant group decreased significantly within 7 days ( Figure 2 ), Zadp The mutant group delayed mating ( Figure 3 ), prolonged mating time ( Figure 4 ), generation length extension ( Figure 5 ).

[0023] The above embodiments are only used to understand the technical solutions of the present application and do not limit the scope of protection of the present application.

Claims

1. Horizontally transferred genes of the zinc-binding alcohol dehydrogenase family in tomato leafminer Zadp, It is characterized by ， The tomato leafminer horizontally transferred gene zinc-binding alcohol dehydrogenase family gene Zadp The encoded amino acid sequence is shown in SEQ ID No:

2.

2. The tomato leafminer horizontal transfer gene zinc-binding alcohol dehydrogenase family gene according to claim 1 Zadp, It is characterized by ， The tomato leafminer horizontally transferred gene zinc-binding alcohol dehydrogenase family gene Zadp The nucleotide sequence is shown in SEQ ID No:

1.

3. A zinc-binding alcohol dehydrogenase family gene comprising the tomato leafminer horizontal transfer gene according to claim 1 or 2. Zadp Recombinant expression vector 。 4. The tomato leafminer horizontally transferred gene zinc-binding alcohol dehydrogenase family gene according to claim 1 Zadp Application for regulating the reproductive characteristics of tomato leafminer.

5. The use according to claim 4, characterized in that Silencing of horizontally transferred genes by the tomato leafminer zinc-binding alcohol dehydrogenase family genes Zadp The expression of regulates the reproductive characteristics of tomato leafminer.

6. The use according to claim 4, characterized in that The tomato leafminer horizontally transferred gene zinc-binding alcohol dehydrogenase family gene Zadp Used to control the following growth characteristics of tomato leafminer: Delaying mating, increasing mating time and generation length, or reducing egg production.

7. A method for regulating the reproductive characteristics of tomato leafminer, characterized in that: The method comprises silencing the horizontally transferred gene zinc-binding alcohol dehydrogenase family gene of the tomato leafminer Zadp The step of expressing the zinc-binding alcohol dehydrogenase family gene Zadp The encoded amino acid sequence is shown in SEQ ID No:

2.

8. The method for regulating the growth characteristics of tomato leafminer according to claim 7, characterized in that: Silencing of horizontally transferred zinc-binding alcohol dehydrogenase family genes in the tomato leafminer by CRISPR-Cas9 Zadp expression.

9. The method for regulating the growth characteristics of tomato leafminer according to claim 8, characterized in that: The following sgRNAs were used in the CRISPR-Cas9 method: sgRNA-TaZadp-1: 5´ATGCACATAATCAAGCCCCAGGG3´; sgRNA-TaZadp-2:5´CCTAGTGGGTGGATTGTAGTCAAC3´; sgRNA-TaZadp-3: 5´CCTTGTATCTGAGTTAGAACATC3´.

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