Invasive pest tomato leaf miner trehalase Tre gene and effect thereof in high temperature tolerance and invasion improvement

Abstract

The invention relates to the technical field of agricultural biology, in particular to a trehalase Tre gene of an invasive pest tomato leaf miner and an effect of the trehalase Tre gene in high temperature resistance and invasion improvement. The tomato leaf miner trehalase Tre gene encodes a protein with an amino acid sequence as shown in SEQ ID NO: 2. By feeding the target gene dsRNA, the survival rate of the second-instar larvae of the tomato leaf miner under high temperature stress is obviously reduced and is only 32.92%.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and specifically to the trehalase Tre gene of the invasive pest Tuta absoluta and its role in tolerating high temperature and enhancing invasiveness. Background Art

[0002] Tuta absoluta belongs to Gelechiidae of Lepidoptera and is one of the most destructive quarantine pests in the world, seriously harming various solanaceous crops.

[0003] In the face of external high-temperature stress environment, insects will respond by a series of physiological and metabolic regulations, such as increasing the evaporation rate of body water to lower the body surface temperature, changing the cell structure and the spatial conformation and synthesis rate and content of macromolecular substances. At the same time, a series of gene stress regulations will occur in the insect body to cope with external adversity stress. Trehalase (Tre) is a key enzyme in the trehalose metabolism in insects by specifically decomposing trehalose into glucose, and plays an important role in the energy metabolism and growth and development of insects.

[0004] RNA interference (RNAi) widely exists in the biological world and is a commonly used tool for studying gene functions. In insects, the target dsRNA mainly enters the body through feeding, soaking or microinjection. Then, the double-stranded RNA (dsRNA) with a homologous complementary sequence to the transcription product mRNA of the target gene can specifically degrade the mRNA after being introduced into the cell, thereby resulting in the deletion of the corresponding functional phenotype. Among them, the method of feeding dsRNA is simple and easy to operate, which promotes its wide application in the research of tiny insects. Summary of the Invention

[0005] The purpose of the present invention is to provide the trehalase Tre gene of Tuta absoluta.

[0006] Another purpose of the present invention is to provide the role of the above gene in the high-temperature tolerance of Tuta absoluta.

[0007] According to the specific embodiments of the present invention, the trehalase Tre gene of Tuta absoluta is cloned for the first time, and the encoded amino acid sequence is shown in SEQ ID NO: 1:

[0008] MYYIWGFVFVMAVVGADRSSLPPACDSLIYCHGPLLDTVQMASLYEDSKTFVDMKLKDPANITLTRFQEMMNRTGHKPTKADVLEFVNRNFDPAGSEFEEWRPDDWKENPAFLRRIKDPLLHQWASDLNQLWLELGRKMKEEVRNHQELYSIIYVDNPVIVPGGRFREFYYWDSYWIIKGLLLSEMHHTARGMVSNFLDIVERIGFIPNGGRIYYAMRSQPPLLIPMVQLIMDDSYDEAFLRQHIHTLDKEYDYWMTNHTLEIEHNGKRYKLARYSDMSQGPRPESYKEDIDCARHFDSNDKKEELYAELKAAAESGWDFSTRWFILNGTNKGNLTNLKTRSIIPVDLNAIMCGNAELMAQFHQKLGNYEKVQYYKDLHAKFMIAIDEVLWHEDVGVWLDYSLESGRRRDYFYPSNIAPLWTNAYDKARKDYYVNRVINYLDKVKVDIFEGGIPTTYEHSGEQWDYPNAWPPLQHIVVSGLSQTGVPEAERLASELATKWVRSNFAVWKKKTAMLEKYDATIFGGVGGGGEYVVQTGFGWTNGVIMALLDHYGDTISVADTFGSGTAESAAIVGAQVGASGVVTSLLVVFASLAAGTLGLMVYRKRQGYAPLAGYEDVRLLGRKAYTELKSLNGASQSAATRLR。

[0009] The present invention also provides a recombinant expression vector containing the above-mentioned tomato leafminer trehalase Tre gene.

[0010] The present invention also provides the use of the above-mentioned tomato leafminer trehalase Tre gene for controlling tomato leafminers.

[0011] According to the application of the present invention, wherein the tomato leafminer is controlled by inhibiting the expression level of the tomato leafminer trehalase Tre gene.

[0012] According to the method for controlling tomato leafminers of the present invention, the method includes the step of inhibiting the expression level of the tomato leafminer trehalase Tre gene, wherein the tomato leafminer trehalase Tre gene encodes a protein with an amino acid sequence as shown in SEQ ID NO:2.

[0013] According to the method for controlling Tuta absoluta of the present invention, the nucleotide sequence of the trehalase Tre gene of Tuta absoluta is as shown in SEQ ID NO: 1.

[0014] According to the method for controlling Tuta absoluta of the present invention, prepare the dsRNA of the trehalase Tre gene of Tuta absoluta, and feed the dsRNA to Tuta absoluta.

[0015] According to the method for controlling Tuta absoluta of the present invention, amplify the trehalase Tre gene of Tuta absoluta by the following primer pair to obtain dsRNA.

[0016] T7+dsEcR-F: 5’taatacgactcactatagggAGGAGCAGTTTACCACC3’;

[0017] T7+dsEcR-R: 5’taatacgactcactatagggAGCAGCAGACCCTTGA3’.

[0018] In the present invention, the cDNA of the trehalase (Tre) Tre gene was cloned from Tuta absoluta, and by feeding the dsRNA of the target gene, the survival rate of the 2nd instar larvae of Tuta absoluta under high temperature stress was significantly reduced, only 32.92%. The results of the present invention lay a foundation for clarifying the role of the Tre gene in the temperature tolerance of Tuta absoluta, and are expected to provide a basis for further studying the temperature adaptation mechanism of Tuta absoluta and the method for controlling the damage of Tuta absoluta through temperature adaptation in the future. Brief Description of the Drawings

[0019] Figure 1 Showing the changes in the expression profile of the trehalase Tre gene under different temperature stresses;

[0020] Figure 2 Showing the changes in the expression level of Tre under the conditions of feeding dsRNA of the Tre gene and feeding dsEGFP;

[0021] Figure 3 Showing the effect of dsRNA treatment of the Tre gene on the mortality of Tuta absoluta larvae under high temperature stress. Detailed Description of the Invention

[0022] Example 1: Cloning of the full-length cDNA sequence of the Tre gene of Tuta absoluta

[0023] Four Tuta absoluta larvae were placed into a 1.5 mL centrifuge tube, frozen in liquid nitrogen, ground into powder using a grinding rod, and then RNA was extracted and stored at -80 °C for later use. According to the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit, the extracted RNA was reverse transcribed to synthesize cDNA. Using the cDNA as a template, primers were designed for PCR amplification. The designed primers are shown in Table 1:

[0024] Table 1 Primer sequences for cloning the full-length cDNA of the Tre gene

[0025]

[0026] Using the sequences in Table 1, through PCR amplification, the full-length cDNA sequence of the Dopa gene was obtained, which is 1935 bp, and this gene encodes 644 amino acid sequences as shown in SEQ ID No: 1.

[0027] Detection of the expression changes of the trehalase Tre gene under different temperature stresses, the results are as Figure 1 shown.

[0028] Example 2: Analysis of the effect of the Tre gene on the high-temperature tolerance of Tuta absoluta larvae

[0029] 2.1 Synthesis of dsRNA

[0030] Primer sequences with the T7 promoter (the underlined sequences) were designed and synthesized:

[0031] T7+dsEcR-F: taatacgactcactatagggAGGAGCAGTTTACCACC;

[0032] T7+dsEcR-R: taatacgactcactatagggAGCAGCAGACCCTTGA.

[0033] Total RNA extraction and cDNA synthesis: The same as in Example 1. T7 primer PCR amplification and product purification were performed, and the purified PCR product was the template for synthesizing dsRNA. The dsRNA was synthesized and purified using a kit, and the operations were carried out according to the kit instructions.

[0034] 2.2 dsRNA feeding

[0035] Fresh tomato leaves were picked and dried for 1 hour. Subsequently, the petioles were soaked in a solution containing dsTre at a concentration of 25 μg / mL. After the leaves had fully absorbed the solution containing dsTre for about 4 hours, 25 second-instar larvae that had been starved for 3 hours were gently transferred back to the soaked leaves for them to feed for 48 hours. The leafminers that had completed feeding were divided into two groups. One group was frozen with liquid nitrogen and stored to detect the silencing efficiency of the target gene, and the other group was used for observing the high-temperature tolerance phenotype. For the specific observation of the high-temperature tolerance phenotype, the larvae after 48 hours of feeding were placed in 1.5 mL centrifuge tubes and exposed to a high temperature of 40 °C for 1 hour. After the temperature stress, they were placed at room temperature of 26 °C for 1 hour, and then the number of surviving larvae was recorded and the mortality rate was calculated. Feeding dsEGFP was used as a control. There were five biological replicates for each experiment.

[0036] The relative expression levels of the genes were calculated by the 2-ΔΔCT method, and the results were as Figure 2 shown. Feeding dsTre could significantly knockdown the expression of the Tre gene. The mortality rates of the tomato leafminer larvae under high-temperature stress after feeding different solutions were analyzed using SPSS statistical software, and the results were as Figure 3 shown. The mortality rate of the tomato leafminer fed with dsRNA of the Tre gene was significantly higher than that of the group fed with dsEGFP (P < 0.05). Therefore, the present invention demonstrates that the Tre gene plays a key role in the high-temperature tolerance of tomato leafminer larvae.

[0037] 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 Trehalase gene from the tomato leafminer, characterized in that: The tomato leafminer trehalase Tre gene encodes a protein whose amino acid sequence is shown in SEQ ID NO:

1.

2. A recombinant expression vector comprising the Trehalase Tre gene of the tomato leafminer according to claim 1.

3. Use of the trehalase Tre gene of tomato leafminer according to claim 1 for controlling tomato leafminer.

4. The use according to claim 3, characterized in that: The tomato leafminer is controlled by inhibiting the expression of the tomato leafminer trehalase Tre gene of the tomato leafminer.

5. A method for controlling tomato leafminer, characterized in that: The method comprises the step of inhibiting the expression level of a tomato leafminer trehalase Tre gene of a tomato leafminer, wherein the tomato leafminer trehalase Tre gene encodes a protein whose amino acid sequence is shown in SEQ ID NO:

1.

6. The method for controlling tomato leafminer according to claim 5, characterized in that: The dsRNA of the trehalase Tre gene of the tomato leafminer is prepared, and the tomato leafminer is fed with the dsRNA.

7. The method for controlling tomato leafminer according to claim 6, characterized in that: The dsRNA was obtained by amplifying the Trehalase gene of the tomato leafminer using the following primer pair: T7+dsEcR-F:5'taatacgactcactataggggAGGAGCAGTTTACCACC3'; T7+dsEcR-R:5'taatacgactcactataggggAGCAGCCAGACCCTTGA3'.

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