USP gene related to drought tolerance of pomacea canaliculata, encoded protein and application of USP gene

Abstract

The invention discloses a USP gene related to the drought tolerance of pomacea canaliculata, an encoded protein and application of the USP gene, and belongs to the technical field of biology. The nucleotide sequence of the USP gene is as shown in SEQ ID NO: 1; the USP gene can be used as a target for pesticide screening and biological control of ampullaria gigas. The invention further provides dsRNA of the USP gene, and the dsRNA can reduce the survival rate of ampullaria gigas and the hatching rate of ampullaria gigas eggs. The method is rapid, effective and high in repeatability, and is an important supplement to the USP gene family. The drought tolerance of the ampullaria gigas is reduced by blocking the expression of the USP gene in the ampullaria gigas, so that the purposes of controlling overwintering of the ampullaria gigas in soil in winter and reducing propagation and diffusion of the ampullaria gigas are achieved. The application disclosed by the invention has important significance on research and determination of biology, ecology and novel insecticides of the ampullaria gigas and research on a resistance mechanism of the ampullaria gigas on drought tolerance.

Description

Technical Field

[0001] The invention belongs to the field of freshwater mollusk biotechnology, and in particular relates to a USP gene related to drought tolerance of Pomacea canaliculata, a coded protein and an application thereof. Background Art

[0002] The golden apple snail (Pomacea), also known as the big bottle snail and apple snail, is an internationally vicious pest. The snail is native to the Amazon River Basin in South America and was introduced to Asia in the early 1980s. It was abandoned due to poor management and bad taste. The golden apple snail quickly spread to the fields, causing floods in some areas and causing serious damage to aquatic crops such as rice and wild rice. The golden apple snail has a very strong reproductive capacity and spreads and spreads quickly. It has now become 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, Anhui Province, etc. The population density is very large, causing serious impacts on agricultural production.

[0003] The important reason why golden apple snails are widely distributed in all middle and low latitudes of the country is inseparable from their tolerance to drought, which is also an important reason why golden apple snails can successfully colonize in new habitats. Under drought stress, golden apple snails will enter a dormant state to resist adverse environments. When drought conditions disappear, golden apple snails will automatically wake up from dormancy. At present, there are many ways to control golden apple snails. Physical control, such as manual snail picking, manual egg removal, duck and turtle co-breeding, etc., is not suitable for large-scale use due to factors such as stability of effect, choice of implementation timing and cost. Chemical control uses various chemical agents to kill golden apple snails. At present, the control of golden apple snails is mainly based on chemical pesticides. Chemical control will cause a certain degree of pollution to the environment and is also highly toxic to other aquatic organisms. In addition, golden apple snails in some areas have developed resistance to chemical pesticides, making control more difficult and costly. Some chemical pesticides can accumulate in the human body after circulation, producing cumulative toxicity, which poses great harm to human health. Therefore, screening and discovering new control targets for golden apple snails and developing new biological pesticides for controlling golden apple snails are of great practical significance for adjusting the current control strategies for golden apple snails.

[0004] USP (universal stress protein A-like protein) belongs to an ancient protein family, which is a relatively conservative and ancient protein family in evolution. It is widely present in bacteria, archaea and eukaryotes. When organisms encounter multiple external pressures, 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 multiple molecular mechanisms. There are many studies on the physiological functions of USP genes in Escherichia coli and Streptomyces, but there is no report on the functional study of USP genes in Pomacea canaliculata. The Pomacea canaliculata USP gene obtained by the present invention can lay the foundation for expanding the research on physiological related genes and functions of aquatic molluscs, and provide theoretical basis and practical reference for more effectively controlling the spread 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, encoded protein and application thereof related to drought tolerance of Pomacea canaliculata. Based on the characteristics that the protein encoded by the USP gene is relatively conservative but the nucleic acid sequence has low homology with other organisms, the present invention performs RNA interference on the target gene, achieves inhibition of Pomacea canaliculata at the nucleic acid level, and significantly reduces the survival rate of Pomacea canaliculata under drought conditions.

[0006] The specific technical solutions are as follows:

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

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

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

[0010] Further, according to a preferred embodiment of the present invention, the dsRNA comprises 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 above-mentioned USP gene related to 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: the present invention is fast, effective, and highly repeatable, and 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 USP gene function, the drought tolerance of the golden apple snail can be significantly reduced, the survival rate can be reduced, and the control efficiency can be improved, thereby achieving the purpose of controlling the spread of the golden apple snail. The most important point is that by interfering with the expression of the USP gene of the present invention, the hatching rate of the golden apple snail eggs can be significantly reduced and the hatching period of the eggs can be extended. The present invention is of great significance to the research and determination of the biology, ecology, and new pesticides of the golden apple snail and the research on the resistance mechanism of the golden apple snail to drought tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0014] Figure 2 This is the effect of RNA interference on the expression of USP gene.

[0015] Figure 3 This is the effect of RNA interference of the USP gene on the survival rate of Pomacea canaliculata. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below in conjunction with specific embodiments. In the following embodiments, materials, methods or other technical features not mentioned are consistent with those described in the summary of the invention.

[0017] Example 1.

[0018] 1 Materials and methods

[0019] 1.1 Tested Pomacea canaliculata

[0020] The selected golden apple snails were young snails with a height of 5.0-10.5 mm. Before the experiment, all golden apple snails were cultured in a constant temperature laboratory (40 cm × 25 cm × 28), with a constant temperature of 26 ± 1 ° C, a light: dark ratio of 16h:8h, and fresh cabbage leaves as feed.

[0021] 1.2 Main Reagents

[0022] TakaRa MiniBest Universal RNA Extraction Kit (TakaRa), TakaRa MiniBESTAgarose 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 Biotech Express Co., Ltd.

[0023] Cloning of USP Gene from Pomacea canaliculata

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

[0025] 2) Total RNA extraction: The mantle tissue of Pomacea canaliculata was ground into powder under liquid nitrogen conditions, and then the total RNA of the mantle tissue was extracted using the Takara MiniBest Universal RNA Extraction Kit. The specific steps were carried out according to the instructions. The integrity and purity of RNA were tested using agarose gel electrophoresis and Nanodrop 2000 (Thermo).

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

[0027] 4) Design of degenerate primers and other primers

[0028] Search the amino acid sequence of the USP gene in the NCBI database, select the USP amino acid sequence of different species, and then use the CODEHOP program to design primers. The main steps of using CODEHOP to design primers 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) to search for conserved regions, and then submit the obtained conserved regions to the server for primer design. The main design parameters are: Degeneracy (Degeneracy) 128; Annealing temperature (Temperature) is 60℃; Genetic code (Genetic code): standard, try to select primers with small degeneracy, Tm value and target fragment length suitable for the company to synthesize. 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] Simultaneously, 3'-RACE and 5'-RACE primers were designed, and their sequences were 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] UP1 and UP2 were amplified by PCR using the designed degenerate primers to obtain a 357 bp USP middle fragment. The PCR reaction system was 25 μL: 10xrTaq 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 ddH2O water was added to make up to 25 μL. The PCR reaction program was: 95℃30s, 56℃45s, 72℃1min, 35 cycles; 72℃ extension 10℃.

[0038] Using the primer pair USP-3F and USP-3R, a 1200 bp 3' end fragment was obtained.

[0039] Using the primer pair USP-5F and USP-5R, a 545 bp 5' end fragment was amplified.

[0040] The fragments obtained by amplification were spliced ​​using DNAman software to obtain a full-length sequence of the Pomacea canaliculata USP gene of 516 bp, as shown in SEQ ID NO: 1. The amino acid sequence encoded by the 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 qPCR was used to detect the expression of USP gene in different tissues under different temperature stresses. The reaction system was 25 μL. The reaction procedure was: 95℃ pre-denaturation for 1 min; then 95℃ for 30 s, 58℃ for 45 s, 72℃ for 1 min, 35 cycles; all data were statistically analyzed using Excel 2010. According to the Ct method (2 -ΔΔCt The relative expression levels of USP genes were analyzed using the PCR method. All data are presented as mean ± SE. Figure 1 .

[0043] from Figure 1 It can be seen that when the soil moisture content is greater than 50%, the expression of USP in various tissues does not change much. When the soil moisture content is ≤5%, the expression of USP in all tested tissues increases significantly (P<0.05), with the highest expression in gill tissue. This indicates that under drought stress conditions, the USP gene is a drought-resistant 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, the dsRNA is named USP-dsRNA.

[0046] The synthetic 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] Thermo scientific company's kit MEGAscript RNAi Kit was used to synthesize and purify dsRNA. The specific steps are as follows:

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

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

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

[0054] Table 1 dsRNA synthesis system

[0055]

[0056]

[0057] Mix the PCR tube by flicking it with your finger, centrifuge it momentarily, and place it in a 37°C water bath or in a PCR instrument to react for more than 4 hours.

[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 DNA and ssRNA removal system

[0061]

[0062] 37℃ warm bath for 1 hour

[0063] (4) Purification of dsRNA

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

[0065] Table 3 dsRNA purification system

[0066]

[0067] b) Transfer the above 500 μL mixed solution into the Filter Cartridge, place the filter cartridge in the Tube provided by the kit, centrifuge at 12000 rpm 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 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 twice-washed Filter Cartridge in another clean tube; add 50-100 μL of sterilized DEPC ddH2O to the center of the membrane and centrifuge at maximum speed for 2 minutes;

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

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

[0072] (1) The Pomacea canaliculata USP-dsRNA (0.1 μM) obtained by the present invention was transfected into Pomacea canaliculata by injection, and the snails were placed in soil with a water content of 5% for 5 consecutive days.

[0073] (2) Gill tissues of Pomacea canaliculata were collected at 0, 6, 12, 24, 72, and 120 hours after transfection, and total RNA was extracted. Real-time quantitative PCR was used to detect the mRNA expression level of the USP gene in Pomacea canaliculata.

[0074] The results are as follows Figure 2 As shown in the figure, the expression level of USP in the experimental group decreased significantly after 6 hours of transfection and reached the lowest point after 24 hours of transfection. As the transfection time increased, the interference effect decreased and the expression level of USP in the experimental group began to increase, 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 USP small interfering RNA expression vector constructed by the present invention was successfully constructed. Figure 3 It can be seen that as the transfection time increases, the expression of USP gradually decreases, the golden apple snail loses the protection of USP, and the survival rate gradually decreases. As the dsRNA-USP interference fails, the survival rate of the golden apple snail slowly increases.

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

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

[0077] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A USP gene related to drought tolerance of Pomacea canaliculata, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. A protein encoded by the USP gene according to claim 1, characterized in that: The protein has an amino acid sequence as shown in SEQ ID NO:

2.

3. Use of dsRNA of a 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 drought tolerance of Pomacea canaliculata is shown in SEQ ID NO:

1.

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

1.

5. The use according to claim 3 or 4, characterized in that: The dsRNA includes the nucleotide sequence shown in SEQ ID NO:3 and the nucleotide sequence shown in SEQ ID NO:

4.

6. An application of the USP gene related to drought tolerance of Pomacea canaliculata according to claim 1, characterized in that Application of USP gene as a target for pesticide screening and biological control of Pomacea canaliculata.

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