Tomato leaf miner beta-fructofuranosidase gene Suc and application thereof

Abstract

The invention relates to the technical field of agricultural biology, in particular to a tomato leaf miner beta-fructofuranosidase gene Suc and application thereof. The tomato leaf miner beta-fructofuranosidase gene Suc is obtained through cloning, CRISPR / Cas9 is used for inducing mutation of the tomato leaf miner Suc gene, and the result shows that the survival rate of F0 generation imagoes in a Suc mutation group within 7 days is remarkably reduced by 64% compared with that of a control group, and eggs generated by survival F1 generation cannot be normally hatched.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and in particular to a beta-fructofuranosidase gene Suc of a tomato leafminer and an application thereof. Background Art

[0002] The tomato leaf miner Tuta absoluta (Meyrick), native to Peru in South America, is a worldwide quarantine pest.

[0003] Horizontal gene transfer, also known as lateral gene transfer, refers to the transfer of genetic material between distantly related species across reproductive isolation. Horizontal gene transfer is considered a common phenomenon observed in the genomes of bacteria, fungi, plants or animals. The transmission and integration of horizontally transferred genes can provide some beneficial characteristics and play an important role in the evolution of organisms. The horizontally transferred gene β-furanosidase Suc gene is widely found in Lepidoptera and sporadically appears in Coleoptera and Hymenoptera species. Mutant individuals lacking this gene weigh less than controls, and the food intake of mutants is generally reduced.

[0004] RNAi is a gene silencing phenomenon caused by dsRNA. The key to using RNAi technology is to obtain a functional target gene that is effective and lethal. CRISPR-Cas9 technology is a new method for reliable, efficient and rapid gene knockout. Using CRISPR / Cas for gene editing can achieve a long-lasting and stable effect, and is an effective genetic tool for studying gene function.

[0005] Studies have reported that the β-fructofuranosidase Suc gene can promote the metabolic homeostasis of the midgut and silk gland of the lepidopteran insect, Bombyx mori, indicating that it has the effect of enhancing the digestion and metabolic adaptation of the silkworm. The body weight of mutant individuals lacking this gene is lower than that of the control, and the food intake of mutants is generally lower than that of the control group.

[0006] The functional study of the horizontally transferred gene β-fructofuranosidase Suc gene of tomato leafminer will help to gain a deeper understanding of its regulatory role in the growth and development of tomato leafminer, provide a research basis and target genes for the prevention and control of tomato leafminer, and is of great significance for the use of RNAi to prevent and control the damage of tomato leafminer. Summary of the invention

[0007] The purpose of the present invention is to provide a β-fructofuranosidase gene Suc of tomato leafminer.

[0008] Another object of the present invention is to provide the role of the above gene in the adult development and egg development of tomato leafminer.

[0009] According to a specific embodiment of the present invention, the tomato leafminer β-fructofuranosidase gene Suc was cloned for the first time, and its cDNA full-length nucleotide sequence is shown in SEQ ID No: 1:

[0010]

[0011] The amino acid sequence encoded by the Suc gene of the tomato leafminer is shown in SEQ ID NO: 2:

[0012] MALKKFLFLCFLGLVGTEKVRQNNAVEELEKYIEDKKTEINQRYRLHYHVAGPVGWINDPNGFSFYKGEYHLFYQFYPYDSVWGPMHWGHVKSKDLVTWEQLPTALIPEEENCFSGSAVVD GNNLTLIYTAHIETPNEVANQSQYLAFSSDGVNFEKYEGNPVIAAAPNGSPDFRDPKAWKYESHWYVVIGSKTDDNRGRVLLYRSVDMINWEFLTVLGESDGSLGYMWECPDFFELDGKFVL LMSPQGMEPEGDRYKNVHQTGYIIGSFNYDTFEFVPEVDFQEMDYGHDFYAAQTTENKGRRYVVGWFNMWDNPHPEDVDGWAGAMTIVRELSISNNRIIMTPVEDMVTLRENIVHLEAMKT NAVTVLGKAVELNLEPDLNHDVVLQLEGVDGGEVAWIRWNTTSSIVAVDRGDDDVRQVEWKPLGSRSWRLFLDASSLELFCGDGEVVFSSRVYPEGGWKITNLSPQQVPATAYALRRSVPV.

[0013] The invention provides the application of the tomato leafminer Suc.

[0014] The present invention cloned the β-fructofuranosidase gene Suc of the tomato leafminer, and used CRISPR / Cas9 to induce mutation in the Suc gene of the tomato leafminer. The results showed that the survival rate of F0 generation adults in the Suc mutation group was significantly reduced by 64% within 7 days compared with the control group, and the eggs produced by the surviving F1 generation could not hatch normally. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shows the expression analysis of Suc at different developmental stages in tomato leafminer;

[0016] Figure 2 Shows the effect of the Suc gene of the tomato leafminer on the survival rate of F0 generation adults;

[0017] Figure 3 Shows the effect of the Suc gene of the tomato leafminer on the hatching rate of F1 generation eggs. DETAILED DESCRIPTION

[0018] Example 1: Cloning of the full-length cDNA sequence of the Suc gene of the tomato leafminer

[0019] Place the adult sample of the tomato leafminer in a 1.5 mL centrifuge tube, freeze it in liquid nitrogen for 5 minutes, then grind it with a grinding rod, extract RNA, and store it at -80°C for later use. Use a reverse transcription kit to reverse transcribe the extracted RNA into cDNA. Design primers and use cDNA as a template for PCR amplification. The primers used are as follows:

[0020] Upstream primer: 5'-CGCCTTCTGGACTAAA-3',

[0021] Downstream primer: 5′-AACATCTTCTGGGTGA-3′;

[0022] Upstream primer: 5'-GACAATCCTCACCCAG-3',

[0023] Downstream primer: 5′-CGCCTTCTGGACTAAA-3′.

[0024] The cDNA sequence of the Suc gene obtained by PCR amplification is 1458 bp in length. The obtained gene has a nucleotide sequence as shown in SEQ ID No: 1. The gene encodes 495 amino acid sequences as shown in SEQ ID No: 2, contains a Glyco_32 protein domain, and belongs to the glycoside hydrolase family.

[0025] Example 2: Expression analysis of Suc gene in different developmental stages of tomato leafminer

[0026] The eggs, 1-4 instar larvae, pupae, female adults and male adults were collected. RNA extraction and cDNA synthesis were the same as in Example 1.

[0027] Fluorescence quantitative PCR results were The relative expression of genes was calculated by the method; the significance analysis of expression was performed using SPSS software, and letters were used to indicate significant differences (P < 0.05). The primers for real-time fluorescence quantitative PCR detection are shown in Table 1. The results showed that the Suc gene mRNA was expressed in all stages, with the highest expression in the male adult stage ( Figure 1 ).

[0028] Table 1. Primers used for real-time quantitative PCRSuc

[0029]

[0030] Example 3: Identification of the function of the Suc gene of the tomato leafminer

[0031] Design sgRNA and evaluate editing efficiency and potential off-target effects at the same time. Finally, 3 sgRNAs were selected, and the sgRNA sequences are as follows:

[0032] Suc-sgRNA1:GTTTGGGGTCCGATGCACTGGGG,

[0033] Suc-sgRNA2:GAGCAAGGATTTAGTCACATGGG,

[0034] Suc-sgRNA3:ATTGCCTACAGCCCTCATCCCGG.

[0035] Use the kit to synthesize and purify sgRNA, and follow the instructions of the kit. Purchase Cas9 protein and prepare injection solution, the total system is 10μL, including 0.75μL of synthesized 4mg / mL Cas9 protein (final concentration 300ng / μL) and Suc sgRNA (final concentration 300ng / μl, each sgRNA 150ng / μL), 1.5μL each of sgRNA-TaSuc-1, sgRNA-TaSuc-2 and sgRNA-TaSuc-3, and the rest is made up with ddH2O.

[0036] Collect the eggs on the tomato leaves within two hours, stick them on the cover glass with thin double-sided tape in sequence, and arrange them in a line vertically. Place the cover glass with eggs on a slide and inject the injection solution into the egg embryo. Mark the cover glass where the eggs are after injection, transfer them to a 150mm disposable culture dish, put in a cotton ball with water for moisture retention, and culture in an incubator.

[0037] After injection of the target gene, the survival rate of F0 generation adults was only 20% within 7 days, which was significantly lower than that of the control group (64%). Figure 2 ), the eggs produced by the F1 generation cannot hatch normally ( Figure 3 ).

[0038] The above embodiments are only used to understand the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. A β-fructofuranosidase gene from the tomato leafminer, characterized in that: The tomato leafminer β-fructofuranosidase gene encodes a protein whose amino acid sequence is shown in SEQ ID NO:

2.

2. The tomato leafminer β-fructofuranosidase gene according to claim 1, characterized in that: The nucleotide sequence of the tomato leafminer β-fructofuranosidase gene is shown in SEQ ID NO:

1.

3. A recombinant expression vector comprising the tomato leafminer β-fructofuranosidase gene according to claim 1.

4. A method for controlling tomato leafminer, characterized in that: The method comprises the step of silencing the β-fructofuranosidase gene of the tomato leafminer, wherein the β-fructofuranosidase gene encodes a protein with an amino acid sequence as shown in SEQ ID NO:

2.

5. The method for controlling tomato leafminer according to claim 4, characterized in that: The method mutates the β-fructofuranosidase gene of the tomato leafminer by the following steps: The sgRNA fragment of the β-fructofuranosidase gene of the tomato leafminer is injected into the eggs of the tomato leafminer.

6. The method for controlling tomato leafminer according to claim 5, characterized in that: The sgRNA fragment is: Suc-sgRNA1: 5'GTTTGGGGTCCGATGCACTGGGG3', Suc-sgRNA2: 5'GAGCAAGGATTTAGTCACATGGG3', or Suc-sgRNA3: 5'ATTGCCTACAGCCCTCATCCCGG3'.

7. Use of the tomato leafminer β-fructofuranosidase gene according to claim 1 for controlling tomato leafminer.

8. The use according to claim 7, characterized in that: The tomato leafminer β-fructofuranosidase gene is used for the following purposes: Reduce the survival rate of adult tomato leafminers; or Make the eggs of the F1 generation of tomato leafminer unable to hatch.

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