TPR1 GENE RELATED TO LOW-TEMPERATURE TOLERANCE OF POMACEA, CODING PROTEIN AND APPLICATION OF SAME

Abstract

A TPR1 gene related to a low-temperature tolerance of Pomacea, coding protein and application of the gene is disclosed. The disclosure belongs to the field of biotechnology. The present disclosure is rapid, effective and reproducible, and is an important complement to the TPR1 gene family. Pomacea is an important invasive organism, the disclosure is helpful for developing novel biological pesticide, and the low-temperature tolerance of the organism is reduced by blocking the expression of TPR1 gene in Pomacea, so as to control the further northward invasion of Pomacea. Meanwhile, by interfering with the expression of TPR1 gene, the hatching rate of Pomacea eggs can be significantly reduced, and the hatching period of eggs can be prolonged, thereby reducing the harm of Pomacea invasion.

Claims

1. A TPR1 gene related to a low-temperature tolerance of Pomacea, coding protein and application of the gene, wherein the gene has a nucleotide sequence shown in SEQ ID NO:1 in a sequence table, the gene plays an important role in maintaining the normal low-temperature tolerance of Pomacea, and an inhibition of a function of the gene will result in a decrease of a survival rate of Pomacea.

2. A TPR1 gene related to a low-temperature tolerance of Pomacea, coding protein and application of the gene, wherein the protein has an amino acid sequence shown in SEQ ID NO:2 in a sequence table, and an inhibition of a function of the gene will result in a decrease of a survival rate of Pomacea.

3. The application of claim 1, wherein the application is used for pesticide development and biological control of Pomacea.

4. The application of a RNA interference technology for the TPR1 gene of Pomacea in the control of Pomacea of claim 1, wherein the RNA interference technology results in a decrease in the survival rate of Pomacea.

5. The application of claim 2, wherein the application is used for pesticide development and biological control of Pomacea.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is an expression of TPR1 in different tissues under different temperature stress;

[0017] FIG. 2 Effects of RNA interference on TPR1 gene expression;

[0018] FIG. 3 Effects of RNA interference of TPR1 gene on survival rate of Pomacea.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] Hereinafter, that present disclosure will be further described in detail with reference to specific embodiments. In the following embodiments, the materials, methods or other technical features that are not mentioned are consistent with those recorded in the disclosure.

Embodiment 1

[0020] 1 Materials and Methods

[0021] 1.1 Test Pomacea

[0022] Before the experiment, all individuals of Pomacea were cultured in a constant temperature laboratory (40 cm×25 cm×28) at a temperature of 26±1° C. and a light of 16 h: 8 h, and the feed is fresh cabbage leaf.

[0023] 1.2 Main Reagents

[0024] Akara MiniBest Universal RNA Extraction Kit TakaRa, TakaRa MiniBEST Agarosc Gel DNA Extraction Kit, Taq enzyme, SYBR Prefix Ex Taq, RACE Kit User Manual were purchased from Clontech Company. USA, and sequencing and primer synthesis were performed by Shanghai Suni Bio-Express Co Ltd.

[0025] Cloning of TPR1 Gene from Pomacea

[0026] 1) taking 10 mg-50 mg of mantle tissue of Pomacea, washing with DEPC water for 3-5 times, and storing in liquid nitrogen;

[0027] 2) Total RNA extraction: under the condition of liquid nitrogen, grounding the mantle tissue of Pomacea into powder, and then extracting the total RNA of mantle tissue by Takara Minibest Universal RNA Extraction Kit, with specific steps referring to its instructions. Detecting the integrity and purity of RNA by agarose gel electrophoresis and Nanodrop 2000(Thermo).

[0028] 3) Using 1 μg of total RNA as template, synthesizing the cDNA of Pomacea mantle tissue by using PrimeScript RT reagent reverse transcription and storing at −20° C. for future use.

[0029] 4) Design merger primers and other primers

[0030] Searching the amino acid sequence of TPR1 gene in NCBI database, selecting the amino acid sequence of TPR1 of different species, and then using CODEHOP program to design merger primers.

[0031] The main steps of using CODEHOP to design primers are as follows: Saving the amino acid sequences of different species queried above in FASTA format, and submiting the results to Blockmaker (http://blocks.fhcrc.org/blocks/make_blocks.html), searching the conserved region, and then submitting the conserved region to the server for primer design, and the main parameter of the design is Degeneracy 128; the annealing temperature 60° C., the genetic code is standard, and the primer with less degeneracy and suitable Tm value and target fragment length is selected to be sent to the company for synthesis. The designed primer sequences are as follows:

TABLE-US-00001 Upstream primer UP1: 5′-TCTCATgaytayctyyt-3′. Downstream primer UP2: 5′-AGGTCGATACGAGGaacatnaartc-3′.

[0032] Note: n is A, T, C or G: Y is C or T; R is A or G.

[0033] Designing 3′-RACE and 5′-RACE primers at the same time, and the sequence is as follows:

TABLE-US-00002 TPR1-3F: 5′-AATTCGTACAGGCCAAGGCT-3′ TPR1-3R: 5′-TTATCGCTGTCATCGGCTCC-3′ TPR1-5F: 5′-TGCATGCGACTGACTGAAGA-3′ TPR1-5R 5′-CCCATTCGTAGGAGGGTTCTG-3′

[0034] 5) Cloning of TPR1 Gene from Pomacea

[0035] Using the designed merger primers to amplify UP1 and UP2 by PCR, and the intermediate fragment of TPR1 of 357 bp was obtained.

[0036] The PCR reaction system was 25 μL: 10xrtaq buffer 2.5 μL, dNTPs (10 nmol/L each) 0.5 μL, MgCl.sub.2 (25 mM) 1.5 μL, cDNA 1 μL, Taq enzyme 0.5 μL, supplemented with ddH.sub.2O water to 25 μL. The PCR procedure was 95° C. for 30 s, 56° C. for 45 s, 72° C. for 1 min, 35 cycles, 72° C. extended for 10° C.

[0037] Using TPR1-3F and TPR1-3R primer pairs, the 3′end fragment of 1100 bp was amplified.

[0038] Using TPR1-5F and TPR1-5R primer pairs, the 5′end fragment of 625 bp was amplified.

[0039] In order to obtain the full-length TPR1 gene of Pomacea (873 bp), the amplified fragment was spliced by DNAman software. SEQ ID NO:1 and SEQ ID NO:2 respectively provide the nucleotide sequence and the amino acid sequence of the full-length ORF gene.

Embodiment 2

[0040] Analysis of TPR1 Expression in Different Tissues Under Different Temperature Stress

[0041] Total RNA was extracted from different tissues and the expression of TPR1 gene was detected by qPCR under different temperature stress. The reaction system was 25 μL. The reaction procedure was as follows: 95° C. pre-denatured for 1 min, then 95° C. for 30 s, 58° C. for 45 s, 72° C. for 1 min, 35 cycles; all data were analyzed by Excel 2010. The relative expression level of the TPR1 gene was analyzed according to the Ct method (2-.DELTA.Ct method). All data are labeled as mean ±SE. the results are shown in FIG. 1.

[0042] As can be seen from FIG. 1, under the heat shock conditions (36° C.), there was little change in the individual tissues of TPR1 compared to the control (26° C.). The expression level of TPR1 was significantly increased in all the tested tissues (P<0.05) under the cold shock condition (6° C.), and the expression level was highest in the mantle tissues. Therefore, under the condition of short-term cold stress, TPR1 gene is the cold shock metabolite of Pomacea, which plays an important role in improving the low temperature tolerance of Pomacea.

Embodiment 3: Effect of TPR1-dsRNA from Pomacea on mRNA Expression of Target Gene

[0043] (1) TPR1-dsRNA of Pomacea obtained by the disclosure was transfected into Pomacea by injection method, and was continuously fed at 0° C. for 5 days.

[0044] (2) Total RNA was extracted from the mantle tissues of Pomacea at 0, 6, 12, 24, 72 and 120 hours after transfection, and the expression level of TPR1 gene mRNA was detected by real-time PCR.

[0045] Results as shown in FIG. 2, the expression level of TPR1 in the experimental group decreased significantly 6 hours after transfection, reached the lowest point 24 hours after transfection, and the interference effect decreased with the increase of transfection time, and the expression level of TPR1 in the experimental group began to increase. However, the expression of TPR1 in the experimental group was significantly lower than that in the control group (P<0.05). The construction of the small interfering RNA expression vector of Pomacea TPR1 constructed by the present disclosure is successful. As can be seen from FIG. 3, with the increase of transfection time, the expression level of TPR1 gradually decreased, and the survival rate of Pomacea lost the protection of TPR1. The survival rate of Pomacea increased slowly with the failure of dsRNA-TPR1 interference.

Embodiment 4: Effect of Pomacea TPR1-dsRNA on the Hatchability of Pomacea Eggs

[0046] TPR1-dsRNA of Pomacea obtained by the disclosure was transfected into mature female and male Pomacea snails by injection method, the female and male snails were continuously fed at 26° C. for 6 days, after the female snails lay eggs, the egg pieces were transferred to an incubator, and the hatching of the egg pieces was observed and recorded. The illumination of the incubator is L: D=13:11, and the incubation temperature is 26° C. The hatching rate and incubation period of each egg block were observed every day. The results showed that the average incubation period was 18.8 days and the average hatching rate was 78.3%, while the average incubation period was 23.4 days and the average hatching rate was 25.3%. It shows that interfering with the expression of TPR1 gene of Pomacea can significantly reduce the hatching rate of Pomacea eggs and prolong the hatching period of eggs, which provides theoretical basis and practical reference for controlling the further invasion of Pomacea and developing environmentally friendly bio-source pesticides.