Tomato leafminer beta-fructofuranosidase gene suc and application thereof

Abstract

The present application relates to the field of agricultural biotechnology, in particular to tomato leaf roller beta-furan fructosidase gene Suc and application thereof. The present application clones tomato leaf roller beta-furan fructosidase gene Suc, and induces mutation of tomato leaf roller Suc gene by using CRISPR / Cas9. The results show that the survival rate of F0 generation adults in the Suc mutant group within 7 days is significantly decreased by 64% compared with the control group, and the eggs produced by the surviving F1 generation cannot be normally hatched.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to the β-fructofuranosidase gene Suc from the tomato leafminer moth and its applications. Background Technology

[0002] The tomato leafminer, *Tuta absoluta* (Meyrick), is native to Peru in South America and is a global quarantine pest.

[0003] Horizontal gene transfer, also known as transverse gene transfer, refers to the transfer of genetic material between distantly related species that overcome reproductive isolation. Horizontal gene transfer is considered a common phenomenon observed in the genomes of bacteria, fungi, plants, and animals. Through the transmission and integration of horizontally transferred genes, beneficial traits can be provided, playing an important role in the evolution of organisms. The horizontally transferred gene β-fructofuranosidase is widely found in Lepidoptera and sporadically in Coleoptera and Hymenoptera species. Mutants lacking this gene have a lower body weight than controls, and their food intake is generally reduced.

[0004] RNAi, or dsRNA-induced gene silencing, is crucial for achieving lethal and effective target genes. CRISPR-Cas9, on the other hand, is a reliable, efficient, and rapid method for gene knockout. Gene editing using CRISPR / Cas can achieve durable and stable results, making it an effective genetic tool for studying gene function.

[0005] Studies have reported that the β-fructofuranosidase gene can promote metabolic homeostasis in the midgut and silk glands of the silkworm, indicating that it enhances the digestion and metabolic adaptation of the silkworm. Mutant individuals lacking this gene have a lower body weight than the control group, and the food intake of the mutants is generally lower than that of the control group.

[0006] Functional studies of the β-fructofuranosidase gene, a horizontal transfer gene of the tomato leafminer, will help to gain a deeper understanding of its regulatory role in the growth and development of the tomato leafminer, provide a research basis and target gene for the control of the tomato leafminer, and are of great significance for the use of RNAi to control the damage caused by the tomato leafminer. Summary of the Invention

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

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

[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 full-length cDNA 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] This invention provides the application of the above-mentioned tomato leafminer Suc.

[0014] This invention cloned the β-fruc-furanosidase gene of the tomato leafminer moth, and induced mutations in the Suc gene of the tomato leafminer moth using CRISPR / Cas9. The results showed that the survival rate of F0 generation adults in the Suc mutant 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. Attached Figure Description

[0015] Figure 1 Expression analysis of Suc, a tomato leafminer, at different developmental stages;

[0016] Figure 2 This study demonstrates the effect of the Suc gene in the tomato leafminer moth on the survival rate of F0 generation adults.

[0017] Figure 3 This study demonstrates the effect of the Suc gene in the tomato leafminer moth on the hatching rate of F1 generation eggs. Detailed Implementation

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

[0019] Tomato leafminer adult samples were placed in 1.5 mL centrifuge tubes, frozen in liquid nitrogen for 5 min, then ground and broken up using a grinder to extract RNA, which was stored at -80℃ for later use. cDNA was synthesized from the extracted RNA using a reverse transcription kit. Primers were designed, and PCR amplification was performed using the cDNA as a template. 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] PCR amplification yielded a full-length cDNA sequence of 1458 bp for the Suc gene. The resulting gene has the nucleotide sequence shown in SEQ ID No:1. This gene encodes 495 amino acid sequences as shown in SEQ ID No:2, contains the Glyco_32 protein domain, and belongs to the glycoside hydrolase family.

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

[0026] Eggs, 1st-4th instar larvae, pupae, female adults, and male adults were collected. RNA extraction and cDNA synthesis were performed as in Example 1.

[0027] The results of quantitative real-time PCR were obtained using The relative expression levels of the gene were calculated using the method described above; SPSS software was used for expression significance analysis, and significant differences were expressed using letter notation (P < 0.05). The primers for real-time quantitative PCR detection are shown in Table 1. The results showed that the Suc gene mRNA was expressed at all stages, with the highest expression in male adults. Figure 1 ).

[0028] Table 1. Primers for real-time quantitative PCR Suc

[0029]

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

[0031] sgRNAs were designed, and their editing efficiency and potential off-target effects were evaluated. Finally, three sgRNAs were selected, and their sequences are shown below:

[0032] Suc-sgRNA1:GTTTGGGGTCCGATGCACTGGGG,

[0033] Suc-sgRNA2:GAGCAAGGATTTAGTCACATGGG,

[0034] Suc-sgRNA3:ATTGCCTACAGCCCTCATCCCGG.

[0035] Synthesize and purify sgRNA using a kit, following the kit instructions. Purchase Cas9 protein and prepare an injection solution of 10 μL, including 0.75 μL of synthesized Cas9 protein (4 mg / mL, final concentration 300 ng / μL) and sgRNA from Suc (final total concentration 300 ng / μL, 150 ng / μL per sgRNA), 1.5 μL each of sgRNA-TaSuc-1, sgRNA-TaSuc-2, and sgRNA-TaSuc-3, with the remainder made up with ddH2O.

[0036] Eggs collected from tomato leaves within two hours were sequentially attached to coverslips with thin strips of double-sided tape, arranged longitudinally in a line. The coverslips with the eggs were placed on a slide, and the injection solution was injected into the embryos. The coverslips containing the injected eggs were marked, and the embryos were transferred to 150mm disposable culture dishes. Water-soaked cotton balls were placed in the dishes for humidification, and the dishes were incubated in an incubator.

[0037] After injection of the target gene, the survival rate of F0 generation adults within 7 days was only 20%, a significant decrease of 64% compared to the control group. 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 solutions of this application and do not limit the scope of protection of this application.

Claims

1. A method of controlling Tuta absoluta, characterized by, The method comprises a step of silencing a beta-fructofuranosidase gene of Tuta absoluta, said beta-fructofuranosidase gene encoding a protein of amino acid sequence as set forth in SEQ ID NO:

2.

2. A method of controlling Tuta absoluta according to claim 1, characterized in that, The method mutates a beta-fructofuranosidase gene of Tuta absoluta by: injecting an sgRNA fragment of the beta-fructofuranosidase gene of Tuta absoluta into eggs of Tuta absoluta.

3. A method of controlling Tuta absoluta according to claim 2, characterized in that, The sgRNA fragment is: Suc - sgRNA1: 5' GTTTGGGGTCCGATGCACTGGGG 3', Suc - sgRNA2: 5' GAGCAAGGATTTAGTCACATGGG 3', and Suc - sgRNA3: 5' ATTGCCTACAGCCCTCATCCCGG 3'.

Images