Solanum lycopersicum leaf miner sex determining gene Tra-2 and application thereof in prevention and control of Solanum lycopersicum leaf miner

Abstract

The invention relates to the technical field of agricultural biology, in particular to a tomato leaf miner sex determining gene Tra-2 and application of the tomato leaf miner sex determining gene Tra-2 in prevention and control of tomato leaf miners. The tomato leaf miner sex determining gene Tra-2 encodes a protein with an amino acid sequence as shown in SEQ ID NO: 1. The CRISPR / Cas9 is utilized to induce mutation of the tomato leaf miner Tra-2 gene, and the result shows that the pupation time is remarkably prolonged, and the pupation rate and the adult eclosion rate are remarkably reduced, which indicates that knockout of the Tra-2 gene plays a key role in growth and development of the tomato leaf miner.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and particularly relates to the sex determination gene Tra-2 of the tomato leafminer and its application in controlling the tomato leafminer. Background Art

[0002] Tomato leafminer Tuta absoluta (Meyrick) is a devastating pest of tomatoes worldwide, with a fast reproduction rate, a wide range of host plants, and strong survival ability. It mainly damages solanaceous crops by the larvae mining.

[0003] Currently, pests can be controlled by spraying RNAi biopesticides. RNAi is a gene silencing phenomenon caused by dsRNA. Obtaining a lethal and effective functional target gene is the key core point of using RNAi technology. The CRISPR-Cas9 technology is a reliable, efficient, and rapid new method for gene knockout, and multiple mutations can be introduced simultaneously at different sites. Using CRISPR / Cas for gene editing can achieve a persistent and stable effect, and it is an effective genetic tool for studying gene functions.

[0004] Sex determination is the result of natural selection of species and is also the basic guarantee for the life activities of organisms. The reproductive mode of Lepidoptera is mainly bisexual reproduction, mostly with the ZW / ZZ system. Females are chromosomal heterogametic, i.e., ZW type, and males are ZZ type. The unique W chromosome of Lepidoptera insects makes their sex determination mechanism different from that of other insects.

[0005] In the ZW / ZZ sex regulation system of Lepidoptera insects, masculinization key genes such as Masc can regulate downstream genes such as doublesex gene Dsx to form male-specific spliceosomes and achieve male sex differentiation; if the Masc gene is silenced, Dsx the gene is specifically spliced by females, thereby regulating the expression of female-biased genes. Research reports on the Lepidoptera insect Bombyx mori show that the sex determination gene Transformer 2 (Tra-2) can participate in the regulation of Dsx . After knocking out Tra-2 , male and female insects die during the embryonic period. Conducting functional research on the sex determination gene Tra-2 of the tomato leafminer helps to deeply understand its role in regulating the growth and development of the tomato leafminer, with a view to providing potential target genes for RNAi control of the tomato leafminer. Summary of the Invention

[0006] The object of the present invention is to provide a sex determination gene Tra-2 of the tomato leafminer.

[0007] Another object of the present invention is to provide the application of the above gene in regulating the development of tomato leafminer larvae.

[0008] According to the specific embodiments of the present invention, the sex determination gene of Tuta absoluta was cloned for the first time Tra-2 , and the encoded amino acid sequence is shown in SEQ ID NO: 1: MSDRERSRSRTRNGSRDPPPKQAVMSRGHSRSRSRTPPPPKSSSRKYRSPSPVVARSPGGRSRSRSASLRRAAAYRASRYSRSRSRSYSPRGSYRRSHSHSPMSSRKRHMGDRKAGHVPENPTPSRCLGVFGLSLYTTEQQINHIFSKYGPVEKTQVVIDAKTGRSRGFCFVYFENQEDAKVAKNECTGMEIDGRRIRVDFSITQRAHTPTPGIYMGKPTSRDNGYDRRGGGGGGRDRDDYYYRGGGGGYRDRERDYYHRSYRHRTPSPHYRRSRRYERERSYSPRRY.

[0009] The present invention provides the application of the above Tuta absoluta Tra-2 . Using CRISPR / Cas9 to induce mutations in the Tuta absoluta Tra-2 gene, the results showed that the pupation time was significantly prolonged, and the pupation rate and adult emergence rate were significantly reduced, indicating that knocking out the Tra-2 gene plays a key role in the growth and development of Tuta absoluta. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Showing the expression analysis of Tuta absoluta Tra-2 at different developmental stages; Figure 2 Showing the effect of the Tuta absoluta Tra-2 gene on the larval phenotype; Figure 3 Showing the effect of the Tuta absoluta Tra-2 gene on the pupation time; Figure 4 Showing the effect of the Tuta absoluta Tra-2 gene on the larval pupation rate Figure 5 Showing the effect of the Tuta absoluta Tra-2 gene on the adult emergence rate. SPECIFIC EMBODIMENTS Example 1: Cloning of the full-length cDNA sequence of the Tra-2 gene of Tuta absoluta

[0011] Put the adult samples of Tuta absoluta into a 1.5 mL centrifuge tube, freeze them in liquid nitrogen for 5 min, then grind and crush them using a grinding rod, extract RNA, and store it at -80 °C for later use. Reverse transcribe the extracted RNA into cDNA using a reverse transcription kit. Design primers and perform PCR amplification with the cDNA as the template. The primers used are as follows: Forward primer: 5' GTGTTCGAAGTGCGCTTGTG 3' (SEQ ID NO:2); Reverse primer: 5' GAGTCAAAGGGGTGTGGTGT 3' (SEQ ID NO:3).

[0012] Perform PCR amplification to obtain Tra-2 the full-length cDNA sequence of the gene, and this gene encodes 288 amino acid sequences as shown in SEQ ID No:2. Example 2: Expression analysis of the Tra-2 gene of Tuta absoluta at different developmental stages

[0013] Collect eggs, larvae from the 1st to 4th instars (L1 to L4), female pupae FP, male pupae MP, female adults F, and male adults M. The extraction of RNA and the synthesis of cDNA are the same as in Example 1.

[0014] Quantitative real-time PCR: Use an ABI 7500 fluorescence quantitative PCR instrument. The relative expression levels of the genes are calculated using the 2 -△△Ct method; perform a significance analysis of the expression levels using SPSS software, and represent significant differences using the letter marking method ( P <0.05).

[0015] The primers for real-time fluorescence quantitative PCR are shown in Table 2. The results show that Tra-2 the gene mRNA is expressed in each period, and is highly expressed in the egg stage ( Figure 1 ). The primers for real-time quantitative PCR Tra-2 are as follows: Gene Tra-2: q Tra-2c-F: 5'GGTGTCTAGGCGTGTTTGGA3′ (SEQ ID NO:4); q Tra-2-R: 5'GACCTGTGTCTTCTCGACGG3′ (SEQ ID NO:5); Gene Rpl5: qRpl5-F: 5'CAGTCGTCGAGCCAGCAACA3′ (SEQ ID NO:6); qRpl5-R: 5'TCCCGCATTGAAGGAGACCA3′ (SEQ ID NO:7). Example 3: Identification of the function of the Tra-2 gene in Tuta absoluta

[0016] sgRNAs were designed, and the editing efficiency and potential off-target effects were evaluated simultaneously. Finally, 2 sgRNAs were selected, and the sgRNA sequences are shown below: Tra-2 sgRNA1: 5'CCCCGTGGCTCCTACCGCCGCTC3′ (SEQ ID NO:8); Tra-2 sgRNA2: 5'TCCCGCTCGTACTCGCCCCGTGG3′ (SEQ ID NO:9).

[0017] Total RNA extraction and cDNA synthesis were the same as in Example 1. The sgRNAs were synthesized and purified using a kit, and the operations were carried out according to the kit instructions.

[0018] The Cas9 protein was purchased to prepare the injection solution. The total volume of the system was 10 μL, including 0.75 μL of the synthesized Cas9 protein at 4 mg / mL (final concentration 300 ng / μL) and Pamasc the sgRNAs (total final concentration 300 ng / μl, 150 ng / μL for each sgRNA), 1.5 μL each of sgRNA-Pamasc-1 and sgRNA-Pamasc-2, and the rest was made up with ddH2O.

[0019] The eggs within two hours were collected from tomato leaves, sequentially adhered to the cover glass with thin double-sided tape, and longitudinally arranged in a line. The cover glass with the adhered eggs was placed on a glass slide, and the injection solution was injected into the egg embryos using a microinjection operation platform (Eppendorf, Germany). After injection, marks were made on the cover glass where the eggs were located, and it was transferred to a 150 mm disposable culture dish, a water cotton ball for moisturizing was placed in it, and it was cultured in an incubator.

[0020] After injecting the target gene, compared with the control, the larvae had retarded development, smaller body size ( Figure 2 ), and a significantly prolonged pupation time ( Figure 3 ), as well as a significantly reduced pupation rate ( Figure 4 ) and adult emergence rate ( Figure 5 ).

[0021] The above examples are only used to understand the technical solutions of this application and do not limit the protection scope of this application.

Claims

1. Sex-determining gene of tomato leafminer Tra-2 , characterized in that, Tomato leafminer sex determination gene Tra-2 The encoded amino acid sequence is as shown in SEQ ID NO:

1.

2. A tomato leafminer sex determination gene comprising the tomato leafminer sex determination gene according to claim 1. Tra-2 recombinant expression vector.

3. The sex determining gene of tomato leafminer according to claim 1 Tra- 2. Application for prevention and control of tomato leafminer.

4. The use according to claim 3, characterized in that: By silencing the tomato leafminer sex determination gene Tra- 2 to control tomato leafminer.

5. The use according to claim 4, characterized in that: By silencing the sex-determining gene of the tomato leafminer Tra- 2 Used to control the following characteristics of tomato leafminer: It slows down the development of larvae, makes the insect body smaller, prolongs the pupation time, reduces the pupation rate or reduces the adult emergence rate.

6. A method for preventing and controlling tomato leafminer, characterized in that: The method comprises silencing the tomato leafminer sex determination gene in tomato leafminer Tra- 2. The step of expressing, wherein the tomato leafminer sex determination gene Tra-2 The encoded amino acid sequence is as shown in SEQ ID NO:

1.

7. The method for controlling tomato leafminer according to claim 6, characterized in that: Silencing of the sex-determining gene of the tomato leafminer by CRISPR-Cas9 Tra- 2Expression.

8. The method for controlling tomato leafminer according to claim 7, characterized in that: The following sgRNAs were used in the CRISPR-Cas9 method: Tra-2 sgRNA1:5'CCCCGTGGCTCCTACCGCCGCTC3′; Tra-2 sgRNA2:5'TCCCGCTCGTACTCGCCCCGTGG3′。

Images