Vannamei alkaline phosphatase gene and its detection method and application

Abstract

The alkaline phosphatase gene of Penaeus vannamei and its detection method and application relate to the field of molecular biology of aquatic animals. The cloning, sequence analysis and expression level detection method of the alkaline phosphatase (ALP) gene of Penaeus vannamei are disclosed. The alkaline phosphatase gene PvALP was successfully cloned by extracting the total RNA of Penaeus vannamei shrimp fry, reverse transcribing it into cDNA, and using specific primers for PCR amplification. qRT-PCR primers were designed based on the gene sequence to detect the transcription level of the alkaline phosphatase gene. The study found that the expression level of the alkaline phosphatase gene is closely related to the ability of Penaeus vannamei to resist the highly lethal vibrio disease HLVD, and the transcription level of this gene in HLVD-resistant shrimp fry is significantly lower than that in ordinary shrimp fry. It provides new technical means and methods for the prevention and control of HLVD in Penaeus vannamei, the selection and breeding of excellent seedlings, and the evaluation of germplasm resources.

Description

Technical Field

[0001] The invention relates to the field of aquatic animal molecular biology, and in particular to a method and application of cloning, sequence analysis and expression level detection of an alkaline phosphatase gene of Penaeus vannamei. Background Art

[0002] Alkaline Phosphatase (ALP) is a zinc metalloenzyme whose catalytic mechanism involves the hydrolysis of phosphate monoesters by forming phosphorylated serine intermediates under alkaline conditions (high pH environment), thus playing a key role in the dephosphorylation process. ALP is widely present in a variety of organisms such as mammals, birds, fish, and crustaceans, and its physiological functions are closely related to the development of bones and liver. Therefore, the activity detection of ALP has become a typical serum biochemical index for the diagnosis of liver diseases and can be used as a biological marker for osteopenia in premature infants. In addition, ALP activity is related to immune response and is regarded as an important immune parameter, playing a key role in the immune defense of crustaceans. In crustaceans, ALP is involved in the digestion and detoxification process and is an important physiological indicator in toxicity research.

[0003] With the large-scale development of shrimp farming, the industry is facing challenges such as environmental deterioration and limitations in farming technology, which have led to frequent shrimp diseases. In particular, the outbreak of Highly lethal Vibrio disease (HLVD) poses a serious threat to young shrimp seedlings. The mortality rate of 6-12-day-old shrimp seedlings after infection can be as high as 90% or more, manifested as decreased vitality, anorexia, empty intestines and lighter color of the hepatopancreas, commonly known as "glass seedlings". The rapid spread, high mortality rate and strong infectivity of HLVD pose a huge threat to the shrimp farming industry. In order to promote the sustainable development of shrimp farming, it is urgent to develop effective prevention and control strategies and technologies to reduce the occurrence and spread of HLVD, which is of great significance to the shrimp farming industry. In this context, the present invention proposes a method for evaluating the ability of shrimp seedlings to resist HLVD by detecting the transcription level of alkaline phosphatase, aiming to provide new technical means for the prevention and control of HLVD, the breeding of shrimp seedlings and the evaluation of germplasm resources. Summary of the invention

[0004] The first object of the present invention is to provide an alkaline phosphatase gene of Penaeus vannamei.

[0005] The second object of the present invention is to provide Penaeus vannamei alkaline phosphatase protein.

[0006] The third object of the present invention is to provide a method for detecting the transcription level of alkaline phosphatase gene of Penaeus vannamei, which can accurately evaluate the expression of alkaline phosphatase gene in shrimp by real-time fluorescence quantitative PCR (qRT-PCR) technology.

[0007] The fourth object of the present invention is to provide an alkaline phosphatase gene of Penaeus vannamei for use in evaluating the ability of shrimp fry to resist highly lethal Vibrio disease (HLVD). This method provides a new tool and strategy for disease resistance assessment, breeding of improved varieties and evaluation of germplasm resources of shrimp fry.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0009] The present invention provides a Penaeus vannamei alkaline phosphatase gene, a nucleotide sequence encoding the Penaeus vannamei alkaline phosphatase, denoted as PvALP, a molecular type of which is cDNA, and sequence characteristics: a length of 1611 bp, a type of nucleic acid, a double-stranded chain, and a linear topological structure. The nucleotide sequence of the Penaeus vannamei alkaline phosphatase gene PvALP is shown in the sequence table SEQ ID No. 1, and is specifically as follows:

[0010] 1 ATGAGACTTC AACTCGCCTT TGTCCTCGCT GTGGCGGCGG TGGCAGCCTC GGCCCGCGTT

[0011] 61 CCACCGCCAA GAGGACCTCA ACGGGAGCCA CTTATCAAGA AGACATATAA AGACCAAGAT

[0012] 121 CACCTGGATG TCATGGGACA CATGCGGGCG AGCAAGAGCG CCAAGAACCG CAATGCTGTA

[0013] 181 GAAGGACTGG GCTACTGGAA TCAGCAAATG CAGGCCGAGC TCCAGGACCA GCTAGCGAAG

[0014] 241 TCCCCCATTG TCAAGCAAGC CAAGAACATC ATCTTCTTCC TCGGAGACGG CACCTCGATC

[0015] 301 TCCACTCTGA CCGCCGCCCG CCTCCTCAAG GGTCATTTGA CAGGCAATGA CGAGCACGAG

[0016] 361 GTCATGGCGT ATGAGAAGTT CCCATATTCG TCACTCATCA AGACCTACAG CGCAGACAAG

[0017] 421 ATCGTCACCG ACTCCGCCGC CAGCGCCACC GCCTACCTGA CCGGCGCCAA GGGCAACCAG

[0018] 481 GCGACCATCG GTGTGGACGC CAACGTGCTG CTCTCAGACT GCGATGCCAT GAACAACCCG

[0019] 541 AGCTACCACA CGCCCTCTGT GCTGAAGAAC TTCCAGGATG CTGGGAAATC CACAGGAATC

[0020] 601 GTGACCGTAA CCCGGGTCAC CCACGCTTCT CCCGCAGGAA ACTATGCCCA CACTGCTGAA

[0021] 661 AGGCACTGGG AAAATGACGA CGATATCAAC GACGTCGAAG GCGACCCCGA GAAGTGTGAC

[0022] 721 GACATCGCCG AACAGCTGGT CTACGGGGCT ACGGGATCCC AGATTAAGGT CATCCTTGGT

[0023] 781 GGAGGACGCA AGAAGTTCAC ACCGAAGGGC GTGGACGATC CCGAGGGAGG CGACGGAGGT

[0024] 841 AAGCGTGACG ACGGCAAGAA CCTCATTCAA ACGTGGATCG ACCAAAAGCA GGTTCTTGGC

[0025] 901 AATGCCTCGT ACGTTTGGCA CCGCGAGGAC CTTCTGGCTT TGGACACCGC AAACACCGAT

[0026] 961 TACCTCATGG GCCTCTTCGA CTGGAGCCAC ATGTCGTTCG CCATTGACGA GGACACGAGT

[0027] 1021 AATCCTTCCC TTGAAGAGAT GACTCGCGCT GCCATCGAGG TCCTGCAGCG AGACGACAAC

[0028] 1081 GGGTTCTTCC TTTTCGTCGA AGGAGGAAAC ATCGATAAGG CTCATCACTT GAACGAGCAC

[0029] 1141 CGTTCTGCTC TGCAAGAAGC ACTGGAATTT GAGAAGGCCA TTGCTTTGGC TGACTCGATG

[0030] 1201 ACTGACCCCG AAGAAACTCT TATCATCGTC ACCGCTGACC ATTCGCAACC CCTCGTCATC

[0031] 1261 AACGGCTATC CTGAGAGGGG GACAGACGTG CGCGATCTCG GAGACTTCTC AGACGTGGAC

[0032] 1321 GGCCTCCCTT TCACGACTCT GATGTACACC AACGGACCAG GGTACAGAGG GGAACCACAC

[0033] 1381 GGCGAGCGAC CAGACCCTTC CGCTGAAAAC TATTACGACC CCCACTACAT GGGCGCCGCC

[0034] 1441 ACCGTGCCCA TGATCGAGTC CCACCACGCC GGCGAGGAGG TGATCCTGTA CGCCCGCGGC

[0035] 1501 CCTCACGCCC ACCTCTTCAC AGGGATCCAC GAGAACGCCT ACATTCCCCA CGCCCTCAGA

[0036] 1561 TACGCTGCCT GTGTCGGCGA CGGACTCCAC TTCTGCCCAT CGGAAGCCTA G

[0037] The nucleotide sequence of the Penaeus vannamei alkaline phosphatase gene of the present invention is obtained by the following method: extracting total RNA from Penaeus vannamei shrimp fry, and reversely transcribing it into first-chain cDNA, using the obtained cDNA as a template, and obtaining the SEQ ID No.1 sequence through PCR amplification.

[0038] The present invention provides Penaeus vannamei alkaline phosphatase protein;

[0039] The molecular type of Penaeus vannamei alkaline phosphatase PvALP is protein, and the sequence characteristics are: length of 535 aa, type of amino acid, the protein sequence of Penaeus vannamei alkaline phosphatase PvALP is shown in sequence SEQ ID No.2, specifically as follows:

[0040] 1 MRLQLAFVLA VAAVAASARV PPPRGPQREP LIKKTYKDQD HLDVMGHMRA SKSAKNRNAV

[0041] 61 EGLGYWNQQM QAELQDQLAK SPIVKQAKNI IFFLGDGTSI STLTAARLLK GHLTGNDEHE

[0042] 121 VMAYEKFPYS SLIKTYSADK IVTDSAASAT AYLTGAKGNQ ATIGVDANVL LSDCDAMNNP

[0043] 181 SYHTPSVLKN FQDAGKSTGI VTVTRVTHAS PAGNYAHTAE RHWENDDDIN DVEGDPEKCD

[0044] 241 DIAEQLVYGA TGSQIKVILG GGRKKFTPKG VDDPEGGDGG KRDDGKNLIQ TWIDQKQVLG

[0045] 301 NASYVWHRED LLALDTANTD YLMGLFDWSH MSFAIDEDTS NPSLEEMTRA AIEVLQRDDN

[0046] 361 GFFLFVEGGN IDKAHHLNEH RSALQEALEF EKAIALADSM TDPEETLIIV TADHSQPLVI

[0047] 421 NGYPERGTDV RDLGDFSDVD GLPFTTLMYT NGPGYRGEPH GERPDPSAEN YYDPHYMGAA

[0048] 481 TVPMIESHHA GEEVILYARG PHAHLFTGIH ENAYIPHALR YAACVGDGLH FCPSEA

[0049] According to the nucleotide sequence, suitable primers SEQ ID No. 3 and SEQ ID No. 4 were designed and used for qRT-PCR method to detect the transcription expression level of the gene.

[0050] The primer sequence SEQ ID No.3: ACCTTCTGGCTTTGGA

[0051] The primer sequence SEQ ID No.4: GAACCCGTTGTCGTCT

[0052] The invention relates to a method for detecting the alkaline phosphatase gene (PvALP) of Penaeus vannamei, and the specific steps are as follows:

[0053] 1) Extract total RNA from Penaeus vannamei postlarvae;

[0054] 2) Reverse transcription of the extracted total RNA into complementary DNA (cDNA);

[0055] 3) cDNA was amplified by real-time fluorescence quantitative PCR (qRT-PCR) using specific primers (SEQ ID No.3 and SEQ ID No.4) to detect the transcription level of the alkaline phosphatase gene (PvALP) of Penaeus vannamei.

[0056] The present invention provides an application of an alkaline phosphatase gene of Penaeus vannamei in evaluating the ability of shrimp fry to resist highly lethal Vibrio disease (HLVD). The method provides a new tool and strategy for disease resistance evaluation, improved variety breeding and germplasm resource evaluation of shrimp fry.

[0057] The above detection method is applied to evaluate the resistance of shrimp fry to highly lethal Vibrio disease (HLVD), providing new tools and strategies for disease resistance assessment, breeding of improved varieties and evaluation of germplasm resources of shrimp fry.

[0058] The present invention provides a method for detecting the alkaline phosphatase gene of Penaeus vannamei. The method comprises the following steps: first, extracting total RNA from Penaeus vannamei shrimp fry, and then performing reverse transcription to obtain a cDNA template. Next, using specific primers to perform PCR amplification on the cDNA, thereby identifying the alkaline phosphatase gene. Based on the sequence information of the gene, primers suitable for qRT-PCR are designed and synthesized, providing a basis for detecting the transcriptional expression level of the gene in the shrimp.

[0059] Through qRT-PCR technology, the present invention further studies the transcriptional expression differences of alkaline phosphatase gene in common shrimp seedlings and shrimp seedlings resistant to highly lethal vibrio disease (HLVD). The study found that the transcription level of this gene in HLVD-resistant shrimp seedlings was significantly lower than that in common shrimp seedlings, with an average decrease of more than 3 times. This finding shows that the expression level of alkaline phosphatase in Penaeus vannamei is closely related to the ability of shrimp to resist HLVD. Therefore, the present invention provides a new method for evaluating the ability of shrimp seedlings to resist HLVD, and provides important information and methods for the selection and breeding of excellent seedlings and the evaluation of germplasm resources.

[0060] The present invention proposes for the first time the complete gene sequence (Seq ID No. 1) and protein sequence (Seq ID No. 2) of alkaline phosphatase (PvALP) of Penaeus vannamei. It provides a basis for subsequent gene function research, genetic breeding and disease prevention and control. Through real-time fluorescence quantitative PCR (qRT-PCR) technology, the present invention can accurately and quickly detect the transcription level of PvALP gene in Penaeus vannamei. The method is not only highly sensitive, but also highly specific, and can effectively distinguish the expression difference of PvALP gene in different shrimp fry. The present invention finds that the expression level of PvALP gene is closely related to the resistance of Penaeus vannamei to highly lethal vibriosis (HLVD). By detecting the transcription level of PvALP gene, it can be used as an effective method to evaluate the ability of shrimp fry to resist HLVD, providing new tools and strategies for disease resistance evaluation, breeding of improved varieties and evaluation of germplasm resources of shrimp fry. The PvALP gene sequence and the detection method thereof of the present invention are not only applicable to Penaeus vannamei, but can also provide a reference for disease prevention and control and genetic breeding research of other crustaceans. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is the agarose electrophoresis pattern of the qRT-PCR amplification product of the alkaline phosphatase gene of Penaeus vannamei (1: 100 bp DNA Marker; 2: PCR product using shrimp fry cDNA as template).

[0062] Figure 2 This is the melting curve of the qRT-PCR amplification product of the alkaline phosphatase gene of Penaeus vannamei.

[0063] Figure 3 The figure shows the gene transcription level of alkaline phosphatase gene of Penaeus vannamei in normal shrimp fry and HLVD-resistant shrimp fry (A, B, C, XY are normal shrimp fry, 7B-1, anti-B12h, B20 are HLVD-resistant shrimp fry). DETAILED DESCRIPTION

[0064] The present invention will be described in detail below in conjunction with specific implementation cases. It should be noted that these implementation cases are intended to demonstrate the application of the present invention rather than to limit its scope of application. In the absence of special descriptions of experimental conditions, the experimental methods used generally follow the following standards: conventional experimental conditions as described in the Molecular Cloning Laboratory Manual (Sambrook, Russell, translated by Huang Peitang, "Molecular Cloning Laboratory Guide", Science Press, the third edition in 2002), or with reference to the recommendations of the reagent or instrument manufacturer. To achieve the above purpose, the present invention adopts the following technical measures, the specific steps of which are:

[0065] 1. Extraction of Total RNA from Penaeus vannamei Postlarvae

[0066] First, take healthy Penaeus vannamei shrimp fry, freeze them in liquid nitrogen, and quickly grind them into powder. Liquid nitrogen is used to keep the temperature low during the grinding process to prevent RNA degradation; then, add 1 mL of TRIzol reagent to the ground shrimp fry powder, mix well, transfer to a 1.5 mL centrifuge tube, and let it stand at room temperature for 5 minutes to lyse the cells. Next, place the centrifuge tube in a 4 ℃ centrifuge and centrifuge at 12000 g for 15 minutes to precipitate cell debris and protein to the bottom of the centrifuge tube, while RNA is retained in the supernatant. After centrifugation, take the supernatant. Then, add 0.2 mL of chloroform to the supernatant, shake vigorously for 30 seconds, mix the chloroform and supernatant thoroughly, let it stand at room temperature for 5 minutes, and centrifuge it again at 12000 g for 15 minutes. At this point, the solution will be divided into three layers: the upper layer is the aqueous phase (containing RNA), the middle layer is the protein layer, and the lower layer is the organic phase; transfer the upper aqueous phase to another 1.5 mL centrifuge tube without RNase contamination, add 0.5 mL of isopropanol, mix thoroughly and let stand at room temperature for 8 minutes to allow the RNA to fully precipitate. After that, centrifuge at 12000 g for 10 minutes at 4 ° C. Remove the supernatant and add 1 mL of 75% ethanol (prepared with DEPC-treated water) to wash the RNA precipitate to remove residual salts and other impurities while maintaining the integrity of the RNA. Centrifuge again at 7500 g for 5 minutes at 4 ° C. Discard the ethanol, let the centrifuge tube stand to allow the RNA precipitate to dry completely, and then add an appropriate amount of DEPC-treated sterile water to dissolve the RNA precipitate. Finally, use a micro-ultraviolet spectrophotometer to measure the total RNA at OD 230 OD 260 and OD 280 The absorbance at OD was used to assess the concentration and purity of RNA. 260 / OD 280 The OD ratio is used to assess the purity of the RNA, and the ideal ratio is between 1.8 and 2.0. A ratio below 1.8 may indicate protein or phenol contamination; a ratio above 2.0 may indicate that the RNA has been degraded. 230 The absorbance is used to detect whether there is polysaccharide or salt contamination in the solution. In addition, the integrity of RNA is also tested by agarose gel electrophoresis. Intact RNA should show clear bands on the electrophoresis graph, while degraded RNA shows fuzzy or broken bands.

[0067] 2. Reverse transcription of Penaeus vannamei cDNA

[0068] In the reverse transcription system, 2 μg of total RNA was first added as a template, followed by 1 μL of Oligo(dT) 18 Random primers are synthetic oligonucleotides that can bind to the 3' end of the RNA template and provide a starting point for reverse transcriptase. 18 Contains 18 thymine (T) residues, which can complement the 3' polyadenylic acid (polyA) tail of mRNA. Then, the reaction system was fixed to 13.5 μL with sterile water treated with DEPC (diethylpyrocarbonate). Next, the mixture was heated at 70 °C for 10 minutes to denature the RNA template; then the mixture was quickly transferred to an ice bath for cooling for 1 minute and centrifuged for several seconds. Subsequently, the following reagents were added to complete the establishment of the reverse transcription system: 4 μL of 5×M-MLV buffer, 0.5 μL of RRI RNase Inhibitor (40 U / μL), 1 μL of dNTPs (10 mM), and 1 μL of M-MLV reverse transcriptase (200 U / μL). After the reaction system is fully mixed, it is incubated at 42°C for 1 hour. Under the guidance of the primer, the M-MLV reverse transcriptase synthesizes cDNA based on the RNA template. After the reaction is completed, the reaction is terminated by heating at 70°C for 15 minutes.

[0069] 3. PCR amplification of the alkaline phosphatase gene PvALP of Penaeus vannamei

[0070] Vannamei shrimp ( Penaeus vannamei) was used as a template for PCR amplification. PCR amplification was performed using specific primers to successfully obtain the open reading frame (ORF) sequence of the alkaline phosphatase gene. The total system of the PCR reaction was 50 μL, including 1 μL of cDNA template (500 ng), 1 μL of forward and reverse primers (concentration of 20 μM), 4 μL of dNTP mixture, 10 μL of 5× PrimeSTAR Buffer, 0.5 μL of PrimerSTAR HS DNA Polymerase, and the total volume was fixed to 50 μL using ddH2O. The PCR reaction conditions included pre-denaturation at 98 ℃ for 30 s, followed by 30 cycles, each cycle including denaturation at 98 ℃ for 10 s, annealing at 56 ℃ for 15 s, and extension at 72 ℃ for 2 min, and finally a total extension at 72 ℃ for 10 min to ensure that all unfinished chains could be fully extended. After the PCR amplification was completed, the product was purified to remove unbound primers, dNTPs and other possible contaminants. The purified PCR product was connected to the T vector (pMD19-T) to form a recombinant DNA molecule; the recombinant DNA molecule was transformed into the competent E. coli cell Top10. The recombinant DNA molecule was amplified and preserved by the growth and reproduction of bacteria. The transformed E. coli cells were spread on a culture medium containing antibiotics to form a single colony; through colony PCR screening, positive clones containing the target DNA fragment (i.e., the ORF sequence of the PvALP gene) were selected for sequencing to obtain the precise nucleotide sequence of the PvALP gene. Based on the nucleotide sequence SEQ ID No.1 obtained by sequencing, the corresponding amino acid sequence was deduced. The polypeptide consists of 535 amino acids. The specific amino acid sequence is shown in the sequence table SEQ ID No.2. Figure 1 Penaeus vannamei Penaeus vannamei Agarose electrophoresis of qRT-PCR amplification products of alkaline phosphatase gene. 1: 100 bp DNA Marker; 2: PCR product with shrimp fry cDNA as template.

[0071] 4. Detection of the transcription level of alkaline phosphatase gene PvALP in Penaeus vannamei

[0072] The detection of the transcription level of the alkaline phosphatase gene PvALP in Penaeus vannamei was achieved by real-time fluorescence quantitative PCR (qRT-PCR) experiment, in which specific primers were used to amplify the specific region of the PvALP gene.

[0073] Forward primer sequence: ACCTTCTGGCTTTGGA (SEQ ID No. 3)

[0074] Reverse primer sequence: GAACCCGTTGTCGTCT (SEQ ID No. 4)

[0075] The primers are designed based on the known sequence of the PvALP gene, and can specifically bind to the target DNA sequence and guide the DNA polymerase to extend the chain during the PCR process.

[0076] In the qRT-PCR experiment, the total reaction volume was 20 μL, including 2 μL of cDNA template, 6 μL of nuclease-free water, 10 μL of 2× TransStart ® Top Green qPCR superMix, as well as 1 μL of forward primer (concentration of 20 μM) and 1 μL of reverse primer (concentration of 20 μM). The PCR reaction program included: 94 ℃ pre-denaturation for 30 seconds, followed by 30 cycles, each cycle including 94 ℃ denaturation for 5 seconds, 56 ℃ annealing for 15 seconds, and 72 ℃ extension for 10 seconds. During the annealing and extension steps of each cycle, the fluorescent dye binds to the double-stranded DNA and emits light, and this light signal is captured and recorded by the real-time fluorescence quantitative PCR instrument. By monitoring the changes in the intensity of the fluorescent signal, the generation of PCR products can be evaluated in real time. In this experiment, the transcription level of the housekeeping gene 40S rRNA of Penaeus vannamei was used as an internal reference control.

[0077] In order to accurately evaluate the transcription level of the PvALP gene, the housekeeping gene 40S rRNA of Penaeus vannamei was used as an internal reference in the experiment. Housekeeping genes are a type of gene that is universally expressed in all cells, and their transcription levels are usually not affected by experimental conditions. By comparing the fluorescence signal intensity of the target gene (PvALP) and the internal reference gene (40S rRNA), the relative transcription level of the PvALP gene can be calculated.

[0078] After the experiment, the software provided by the real-time fluorescence quantitative PCR instrument was used to process and analyze the collected fluorescence signal data. By calculating the cycle threshold (Ct value) of the target gene and the internal reference gene, the relative transcription level of the target gene can be evaluated. The Ct value refers to the number of cycles required for the fluorescence signal intensity to reach a predetermined threshold, which is inversely proportional to the initial template concentration of the target gene. Therefore, by comparing the Ct value difference between the target gene and the internal reference gene, the relative expression of the PvALP gene can be calculated. Figure 2 This is the melting curve of the qRT-PCR amplification product of the alkaline phosphatase gene of Penaeus vannamei.

[0079] 5. Correlation between the alkaline phosphatase gene of Penaeus vannamei and its resistance to HLVD

[0080] The experiment selected multiple Vannamei shrimp strains from shrimp hatcheries, including unselected common shrimp A, B, C, XY, and selected HLVD-resistant shrimp 7B-1, anti-B12h, B20, etc. In order to ensure the stability of the experiment, all shrimp fry used in the experiment were pre-cultured under the same culture conditions, which were cultured in circulating water at 21 ° C and a salinity of about 11% for 1 to 2 days to reduce experimental errors caused by environmental factors. After the shrimp fully adapted to the new environment, 3 shrimp fry were randomly selected from each strain as experimental samples, which were quickly frozen in liquid nitrogen to maintain their biological activity, and total RNA was extracted. Subsequently, RNA was converted into cDNA by reverse transcription, and the transcriptional expression level of alkaline phosphatase gene in common shrimp and disease-resistant shrimp was detected by qRT-PCR technology. The results showed that the expression level of alkaline phosphatase gene in disease-resistant shrimp was generally lower than that in common shrimp (such as Figure 3 ), which indicates that this gene may be related to the resistance of shrimp fry to HLVD. Therefore, given the low expression level of alkaline phosphatase gene in disease-resistant shrimp, this gene and its detection method are expected to become a new indicator for evaluating the ability of shrimp fry to resist HLVD. By monitoring the expression level of this gene, the disease resistance of shrimp fry can be preliminarily judged, providing strong support for the breeding of disease-resistant varieties.

[0081] The present invention comprises extracting total RNA from shrimp fry, synthesizing cDNA as a PCR template by reverse transcription, using specific primers for PCR amplification to clone the alkaline phosphatase gene, and designing primers for real-time fluorescence quantitative PCR (qRT-PCR) based on the gene sequence. The transcription level of the alkaline phosphatase gene is detected by qRT-PCR technology, aiming to evaluate the resistance of shrimp fry to highly lethal Vibrio disease (HLVD), and provide technical means and methods for the selection and breeding of excellent seedlings, evaluation of germplasm resources and other research. The ability of shrimp fry to resist HLVD can be more effectively evaluated, thereby breeding excellent seedlings with strong disease resistance, reducing the disease risk in the breeding process, improving breeding benefits, and promoting the sustainable development of the shrimp breeding industry.

[0082] The above embodiments are only preferred embodiments of the present invention and cannot be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. Penaeus vannamei alkaline phosphatase gene, characterized in that The nucleotide sequence encoding the alkaline phosphatase of Penaeus vannamei is recorded as PvALP; the molecular type of the alkaline phosphatase gene PvALP of Penaeus vannamei is cDNA, and the sequence characteristics are: length is 1611 bp, type is nucleic acid, chain is double-stranded, and topological structure is linear. The sequence of the alkaline phosphatase gene PvALP of Penaeus vannamei is shown in the sequence table SEQ ID No.

1.

2. The Penaeus vannamei alkaline phosphatase gene according to claim 1, characterized in that The nucleotide sequence is obtained by the following method: extracting total RNA from Penaeus vannamei shrimp fry, synthesizing the first-chain cDNA by reverse transcription of the obtained RNA, using the cDNA as a template for PCR amplification to obtain the SEQ ID No.1 sequence; and obtaining the polypeptide SEQ ID No.2 encoded by the nucleotide sequence by prediction.

3. Penaeus vannamei alkaline phosphatase protein, characterized in that The molecular type is protein, the sequence length is 535 aa, and the type is amino acid. The protein sequence of Penaeus vannamei alkaline phosphatase PvALP is shown in the sequence table SEQ ID No.

2.

4. A method for detecting the alkaline phosphatase gene PvALP of Penaeus vannamei, characterized in that The following steps are involved: A procedure for extracting total RNA from Penaeus vannamei shrimp larvae; A procedure for reverse transcription of Penaeus vannamei cDNA; A procedure for detecting the transcription level of alkaline phosphatase gene in Penaeus vannamei.

5. The method for detecting the alkaline phosphatase gene PvALP of Penaeus vannamei as claimed in claim 4, characterized in that Real-time fluorescence quantitative PCR amplification of cDNA was performed using specific primers to detect the transcription level of the alkaline phosphatase gene. The sequences of the specific primers are shown in Seq ID No.3 and Seq ID No.

4.

6. The method for detecting the alkaline phosphatase gene PvALP of Penaeus vannamei according to claim 4, characterized in that The method is used to evaluate the ability of shrimp fry to resist the highly lethal vibriosis HLVD, and is specifically evaluated by comparing the difference in transcription levels of alkaline phosphatase genes between HLVD-resistant shrimp fry and ordinary shrimp fry.

7. A tool for evaluating the ability of shrimp seedlings to resist highly lethal vibriosis HLVD, characterized in that: The tool comprises the nucleotide sequence of the Penaeus vannamei alkaline phosphatase gene of claim 1 or the Penaeus vannamei alkaline phosphatase protein sequence of claim 3, and a reagent or device for detecting the expression level of the gene or protein.

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