A USP gene related to the drought tolerance of Pomacea canaliculata, the encoded protein thereof, and applications thereof

Abstract

The present invention discloses a USP gene related to the drought tolerance of Pomacea canaliculata, the encoded protein thereof, and their applications, belonging to the field of biotechnology. The nucleotide sequence of the USP gene is shown as SEQ ID NO:1; this USP gene can be used as a target for pesticide screening and biological control of Pomacea canaliculata. The present invention further provides dsRNA of the USP gene, which can reduce the survival rate of Pomacea canaliculata and the hatching rate of Pomacea canaliculata eggs. The present invention is rapid, effective, and has strong repeatability, and is an important supplement to the USP gene family. The present invention reduces the drought tolerance of Pomacea canaliculata by blocking the expression of the USP gene in Pomacea canaliculata, so as to achieve the purpose of controlling Pomacea canaliculata to overwinter in winter soil and reducing the reproduction and spread of Pomacea canaliculata. The present invention is of great significance to the research and determination of the biology, ecology, and new pesticides of Pomacea canaliculata and the research on the resistance mechanism of Pomacea canaliculata to drought tolerance.

Description

Technical Field

[0001] The present invention belongs to the field of freshwater mollusk biotechnology, and in particular relates to a USP gene related to drought tolerance of golden apple snails, an encoded protein and applications thereof. Background Art

[0002] The golden apple snail (Pomacea), also known as the giant bottle snail and apple snail, is a serious international pest. Native to the Amazon River basin in South America, it was introduced to Asia in the early 1980s. Due to poor management and unpalatable taste, it was abandoned. The snail quickly spread to farmland, causing floods in some areas and severely damaging aquatic crops such as rice and wild rice. The golden apple snail has a strong reproductive capacity and spreads rapidly, becoming a serious agricultural pest in most provinces and regions south of the Yangtze River. Today, the golden apple snail is widely distributed in most provinces and regions south of 30° north latitude in my country, including Zhejiang, Fujian, Guangdong, Hainan, Guangxi, Yunnan, Guizhou, Hunan, Jiangxi, Chongqing, Sichuan, and Anhui provinces. Its population density is extremely high, severely impacting agricultural production.

[0003] A key reason for the widespread distribution of golden apple snails across mid- and low-latitudes across China is their drought tolerance, a key factor in their successful colonization of new habitats. Under drought stress, golden apple snails enter a dormant state to withstand the adverse environment. Once the drought conditions subside, they automatically emerge from dormancy. Currently, there are various methods for controlling golden apple snails. Physical control, such as manual snail removal, egg removal, and co-breeding with ducks and turtles, is not suitable for widespread use due to factors such as stability of results, timing, and cost. Chemical control, using various chemical agents to kill golden apple snails, is currently the primary method for controlling golden apple snails. Chemical control can cause a certain degree of environmental pollution and is highly toxic to other aquatic organisms. Furthermore, in some areas, golden apple snails have developed resistance to chemical pesticides, making control more difficult and costly. Some chemical pesticides can accumulate in the human body through circulation, producing cumulative toxicity and posing a significant threat to human health. Therefore, screening and discovering new targets for controlling golden apple snails and developing new biological pesticides for controlling golden apple snails are of great practical significance for adjusting the current golden apple snail control strategies.

[0004] USP (universal stress protein A-like protein) belongs to an ancient protein family, which is evolutionarily conserved and ancient. It is widely present in bacteria, archaea and eukaryotes. When organisms encounter various external stresses, such as heat shock, oxidative environment, and drought environment, the expression of USP will be stimulated, and then the survival ability of organisms in these environments will be regulated through various molecular mechanisms. There have been many studies on the physiological functions of USP genes in Escherichia coli and Streptomyces, but there is no report on the function of USP genes in Pomacea canaliculata. The USP gene of Pomacea canaliculata obtained by the present invention can lay a foundation for expanding the research on genes and functions related to the physiology of aquatic mollusks, and provide a theoretical basis and practical reference for more effectively controlling the spread and harm of Pomacea canaliculata. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a USP gene related to the drought tolerance of Pomacea canaliculata, the encoded protein and its application. According to the characteristics that the protein encoded by the USP gene is relatively conserved but the nucleic acid sequence has low homology with other organisms, RNA interference is carried out on the target gene, realizing the inhibition of Pomacea canaliculata at the nucleic acid level and significantly reducing the survival rate of Pomacea canaliculata under drought conditions.

[0006] The specific technical solutions are as follows:

[0007] First of all, the present invention provides a USP gene related to the drought tolerance of Pomacea canaliculata, and the nucleotide sequence of the gene is shown in SEQ ID NO:1. This gene plays an important role in maintaining the drought tolerance of Pomacea canaliculata, and the decline of its function will lead to the decrease of the survival rate of Pomacea canaliculata.

[0008] Furthermore, the present invention also provides the protein encoded by the above USP gene, which has the amino acid sequence shown in SEQ ID NO:2.

[0009] The present invention also provides the application of dsRNA of a USP gene related to the drought tolerance of Pomacea canaliculata in reducing the survival rate of Pomacea canaliculata. The present invention also provides the application of dsRNA of a USP gene related to the drought tolerance of Pomacea canaliculata in reducing the hatching rate of Pomacea canaliculata eggs. The nucleotide sequence of the above USP gene related to the drought tolerance of Pomacea canaliculata is shown in SEQ ID NO:1.

[0010] Furthermore, according to the preferred embodiment of the present invention, the dsRNA includes the nucleotide sequence shown in SEQ ID NO:3 and the nucleotide sequence shown in SEQ ID NO:4.

[0011] The present invention also provides an application of the USP gene related to the drought tolerance of Pomacea canaliculata, specifically, the application of the USP gene as a target for pesticide screening and biological control of Pomacea canaliculata.

[0012] The beneficial effects of the present invention are as follows: The present invention is rapid, effective, and has strong repeatability, which is an important supplement to the USP gene family. By interfering with the expression of the USP gene of the present invention and inhibiting the function of the USP gene, the drought tolerance of Pomacea canaliculata can be significantly reduced, its survival rate can be decreased, and the control efficiency can be improved, thereby achieving the purpose of controlling the spread of Pomacea canaliculata. The most important point is that by interfering with the expression of the USP gene of the present invention, the hatching rate of Pomacea canaliculata eggs can be significantly reduced, and the hatching duration of the eggs can be prolonged. The present invention is of great significance for the research and determination of the biology, ecology, and new pesticides of Pomacea canaliculata and the research on the resistance mechanism of Pomacea canaliculata to drought tolerance. Description of the Drawings

[0013] Figure 1 USP expression in different tissues under different water contents.

[0014] Figure 2 Effect of RNA interference on the expression level of the USP gene.

[0015] Figure 3 Effect of RNA interference of the USP gene on the survival rate of Pomacea canaliculata. Detailed Embodiments

[0016] The following further elaborates on the present invention in conjunction with specific embodiments. In the following embodiments, materials, methods, or other technical features not mentioned are the same as those described in the invention content.

[0017] Example 1.

[0018] 1 Materials and Methods

[0019] 1.1 Test Pomacea canaliculata

[0020] The selected Pomacea canaliculata are juvenile snails with a shell height of 5.0 - 10.5 mm. Before the experiment, all Pomacea canaliculata individuals were cultured in tanks (40 cm × 25 cm × 28) in a constant-temperature laboratory. The temperature in the constant-temperature room was 26 ± 1°C, the light:dark ratio was 16 h:8 h, and the feed was fresh Chinese cabbage leaves.

[0021] 1.2 Main Reagents

[0022] The akara MiniBest Universal RNA Extraction Kit (TakaRa), TakaRa MiniBEST Agarose Gel DNA Extraction Kit, Taq enzyme, SYBR Premix Ex Taq, and RACE Kit User Manual were purchased from Clontech, USA. Sequencing and primer synthesis were completed by Shanghai Sunny Biotechnology Co., Ltd.

[0023] Cloning of the USP Gene from Pomacea canaliculata

[0024] 1) Take 10 mg - 50 mg of the mantle tissue of Pomacea canaliculata, rinse it 3 - 5 times with DEPC water, and store it in liquid nitrogen.

[0025] 2) Total RNA extraction: Grind the mantle tissue of Pomacea canaliculata into powder under liquid nitrogen conditions, and then extract the total RNA of the mantle tissue using the Takara MiniBest Universal RNA Extraction Kit. The specific steps are carried out according to its instruction manual. Use agarose gel electrophoresis and Nanodrop 2000 (Thermo) to detect the integrity and purity of the RNA.

[0026] 3) Using 1 μg of total RNA as a template, reverse transcribe to synthesize the cDNA of the mantle tissue of Pomacea canaliculata using the PrimeScript RT reagent; store it at -20 °C for later use.

[0027] 4) Design degenerate primers and other primers

[0028] Search for the amino acid sequence of the USP gene in the NCBI database, select the USP amino acid sequences of different species, and then design degenerate primers using the CODEHOP program. The main steps for designing primers using CODEHOP are as follows: Save the amino acid sequences of different species queried above in FASTA format and submit the results to Blockmaker (http: / / blocks.fhcrc.org / blocks / make_blocks.html) for searching for conserved regions. Then submit the obtained conserved regions to the server for primer design. The main parameters for design are: Degeneracy 128; Annealing temperature (Temperature) 60 °C; Genetic code: standard. Try to select primers with a small degeneracy, appropriate Tm value, and suitable target fragment length and send them to the company for synthesis. The designed primer sequences are as follows:

[0029] Upstream primer UP1: 5'-TATTAGgcyatyttyyt-3' (SEQ ID NO: 5),

[0030] Downstream primer UP2: 5'-AGGAGCTATTCAAGCttactnttrtc-3' (SEQ ID NO: 6).

[0031] Note: n is A, T, C or G; y is C or T; r is A or G;

[0032] Meanwhile, 3'-RACE and 5'-RACE primers were designed, and the sequences are as follows:

[0033] USP-3F: 5'-TACCTTATAAATGTCACAAAAGGAATG-3' (SEQ ID NO: 7); USP-3R: 5'-ATGGAATATTACAGTGTTTRCCTTAC-3' (SEQ ID NO: 8);

[0034] USP-5F: 5'-TCATCATGAACAGCAGAAGAAGGAT-3' (SEQ ID NO: 9);

[0035] USP-5R: 5'-CTAGTAGTACCAGTAGTACTTGTCG-3' (SEQ ID NO: 10).

[0036] 5) Cloning of the USP gene of Pomacea canaliculata

[0037] The degenerate primers UP1 and UP2 were used for PCR amplification to obtain a 357-bp USP intermediate fragment. The PCR reaction system was 25 μL: 10x rTaq buffer 2.5 μL, dNTPs (10 nmol / L each) 0.5 μL, MgCl2 (25 mM) 1.5 μL, cDNA 1 μL, Taq enzyme 0.5 μL, and supplemented with ddH2O to 25 μL. The PCR reaction program was: 95°C for 30 s, 56°C for 45 s, 72°C for 1 min, 35 cycles; 72°C for 10 min.

[0038] The 3'-terminal fragment of 1200 bp was amplified using the USP-3F and USP-3R primer pair.

[0039] The 5'-terminal fragment of 545 bp was amplified using the USP-5F and USP-5R primer pair.

[0040] The fragments obtained by the above amplification were spliced using DNAman software to obtain the full-length sequence of the USP gene of Pomacea canaliculata, which is 516 bp in length, as shown in SEQ ID NO: 1. The amino acid sequence encoded by this sequence is shown in SEQ ID NO: 2.

[0041] Example 2. Analysis of USP expression in different tissues under different drought stresses

[0042] Total RNA was extracted from different tissues, and the tissue expression of the USP gene under different temperature stresses was detected by qPCR. The reaction system was 25 μL. The reaction program was: pre-denaturation at 95 °C for 1 min; then 95 °C for 30 s, 58 °C for 45 s, 72 °C for 1 min, for 35 cycles; all data were statistically analyzed using Excel 2010. According to the 2 -ΔΔCt method, the relative expression level of the USP gene was analyzed. All data were marked in the form of mean ± SE, and the results are shown in Figure 1 .

[0043] From Figure 1 it can be seen that when the soil water content is greater than 50%, the expression of USP in each tissue does not change much. When the soil water content ≤ 5%, the expression of USP in all detected tissues increases significantly (P < 0.05), and the highest expression is in the gill tissue. This indicates that under drought stress conditions, the USP gene is an anti-drought metabolite of Pomacea canaliculata and plays an important role in improving the drought tolerance of Pomacea canaliculata.

[0044] Example 3: Synthesis of Pomacea canaliculata USP-dsRNA

[0045] Based on the USP gene finally obtained in Example 1, the present invention designed its dsRNA, which includes the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4. In the example, this dsRNA was named USP-dsRNA.

[0046] The synthesized dsRNA sequence is as follows:

[0047]

[0048] Note: The underlined sequence is the T7 promoter sequence.

[0049] The synthesis process of USP-dsRNA is as follows:

[0050] The kit MEGAscript RNAi Kit from Thermo scientific company was used for the synthesis and purification of dsRNA. The specific operation steps are as follows:

[0051] (1) Preparation of washing solution: Add 12 mL of absolute ethanol to 2X Wash Solution, mix well, and place at room temperature.

[0052] (2) Transcription reaction: Take out T7 EnzymeMix and ATP, CTP, GTP, UTP solution in the kit from the refrigerator, dissolve them on ice, and centrifuge briefly.

[0053] Prepare the reaction system according to the components in Table 1 below:

[0054] Table 1 Synthesis system of dsRNA

[0055]

[0056]

[0057] Flick the PCR tube gently with your finger to mix, centrifuge briefly, and place in a 37°C water bath or react in a PCR instrument for more than 4 h.

[0058] (3) Removal of DNA and ssRNA by nuclease

[0059] Prepare according to the system in Table 2 below and operate on ice;

[0060] Table 2 Removal system of DNA and ssRNA

[0061]

[0062] Incubate at 37°C for 1 h

[0063] (4) Purification of dsRNA

[0064] a) Take a 1.5 mL Eppendorf tube and add the relevant reagents according to the components in Table 3 below

[0065] Table 3 Purification system of dsRNA

[0066]

[0067] b) Transfer the above 500 μL of the mixed solution to a Filter Cartridge filter cartridge, place the filter cartridge in the Tube provided in the kit, centrifuge at 12000 r / min for 2 min, discard the waste liquid, and put the filter cartridge back into the Tube.

[0068] c) Add 500 mL of washing solution to the Filter Cartridge filter cartridge in step (b) above, centrifuge at 12000 r / min for 1 min; add 500 mL of washing solution and repeat the washing once, centrifuge at 12000 r / min for 2 min and discard the waste liquid.

[0069] d) Place the Filter Cartridge that has been washed twice into another clean Tube; add 50 - 100 μL of sterilized DEPC ddH2O to the center of the membrane and centrifuge at maximum speed for 2 min;

[0070] e) Take 5 μL for agarose gel electrophoresis detection, and store the remaining samples in a -80 °C refrigerator for later use.

[0071] Example 4: Effect of Pomacea canaliculata USP-dsRNA on the mRNA expression of target genes

[0072] (1) Transfect Pomacea canaliculata with the Pomacea canaliculata USP-dsRNA (0.1 μM) obtained in the present invention by injection method, and place it in soil with a water content of 5% for 5 consecutive days.

[0073] (2) At 0, 6, 12, 24, 72, and 120 hours after transfection, take the gill tissues of Pomacea canaliculata, extract total RNA, and detect the mRNA expression level of the USP gene of Pomacea canaliculata by Real-time quantitative PCR.

[0074] The results are as Figure 2 shown. The expression level of USP in the experimental group decreased significantly 6 hours after transfection, reached the lowest point 24 hours after transfection. As the transfection time increased, the interference effect decreased, and the expression level of USP in the experimental group began to rise, but the expression level of USP in the experimental group was still significantly lower than that in the control group (P < 0.05). This indicates that the small interfering RNA expression vector of Pomacea canaliculata USP constructed in the present invention was successfully constructed. It can be seen from Figure 3 this that as the transfection time prolonged, the expression level of USP gradually decreased, Pomacea canaliculata lost the protection of USP, and the survival rate gradually decreased. As the dsRNA-USP interference failed, the survival rate of Pomacea canaliculata slowly increased again.

[0075] Example 5: Effect of Pomacea canaliculata USP-dsRNA on the hatching rate of Pomacea canaliculata eggs

[0076] The mature female and male Pomacea canaliculata were transfected with the USP-dsRNA (0.1 μM) of Pomacea canaliculata obtained in the present invention by injection method. The female and male snails were placed in an environment of 26 °C and continuously fed for 6 days. After the female snails laid eggs, the egg masses were transferred to an incubator, and the hatching of the egg masses was observed and recorded. The light in the incubator was L:D = 13:11, and the hatching temperature was 26 °C. The hatching of the egg masses in each treatment was observed every day, and the hatching rate and hatching duration were counted. The results showed that the average hatching duration of the egg masses of normally fed Pomacea canaliculata was 18.8 days, and the average hatching rate was 60.5%. While the average hatching duration of the egg masses produced by Pomacea canaliculata transfected by injection method was 26.5 days, and the average hatching rate was 20.3%. It shows that by interfering with the expression of the USP gene of Pomacea canaliculata, the hatching rate of Pomacea canaliculata eggs can be significantly reduced, and the hatching duration of the eggs can be prolonged, which provides a theoretical basis and practical reference for controlling the further invasion of Pomacea canaliculata and developing green and environmentally friendly biogenic pesticides.

[0077] The above embodiments only represent several implementation modes of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. Use of dsRNA of USP gene related to drought tolerance of Pomacea canaliculata in reducing the survival rate of Pomacea canaliculata, characterized in that, The nucleotide sequence of the USP gene related to the drought tolerance of Pomacea canaliculata is shown as SEQ ID NO:

1.

2. Application of dsRNA of USP gene related to drought tolerance of Pomacea canaliculata in reducing hatching rate of Pomacea canaliculata eggs, characterized in that, The nucleotide sequence of the USP gene related to the drought tolerance of Pomacea canaliculata is shown as SEQ ID NO:

1.

3. The application according to claim 1 or 2, characterized in that, The dsRNA includes the nucleotide sequence shown as SEQ ID NO: 3 and the nucleotide sequence shown as SEQ ID NO:

4.

4. Application of a USP gene related to the drought tolerance of Pomacea canaliculata, characterized in that Application of the USP gene as a target for pesticide screening and biological control of Pomacea canaliculata, wherein the nucleotide sequence of the USP gene related to the drought tolerance of Pomacea canaliculata is shown as SEQ ID NO: 1.

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