Macrobrachium rosenbergii anti-bacterial infection immune related gene ATG5 and application thereof

Abstract

The invention discloses a macrobrachium rosenbergii anti-bacterial infection immune related gene ATG5 and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO.1. The invention further discloses a preparation method of the gene ATG5. The invention relates to a macrobrachium rosenbergii anti-bacterial infection immune related gene ATG5 which can respond to aeromonas veronii and has high expression characteristic in the immunoreaction of macrobrachium rosenbergii infected with aeromonas veronii, and the gene plays an important role in the immune defense process of macrobrachium rosenbergii anti-bacterial infection. After healthy macrobrachium rosenbergii is infected with aeromonas veronii, the expression level of the macrobrachium rosenbergii anti-bacterial infection immunity related gene ATG5 is remarkably up-regulated, so that the macrobrachium rosenbergii anti-bacterial infection immunity related gene ATG5 can be used for detecting whether the macrobrachium rosenbergii is infected with aeromonas veronii or not; and the method can be used for identifying the anti-aeromonas veronii level of macrobrachium rosenbergii individuals, so that the screening and cultivation of the macrobrachium rosenbergii anti-aeromonas veronii variety are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic animal molecular biology, and specifically relates to a gene ATG5 related to immunity against bacterial infection of Macrobrachium rosenbergii and an application thereof. Background Art

[0002] Aeromonas veronii belongs to the genus Aeromonas of the family Aeromonadaceae. It is widely distributed in nature and has two biotypes: mild type (A.veronii biovar sobria) and Verona type (A.veronii biovar veronii). It is a Gram-negative pathogen that can cause common diseases in humans, animals and aquatic organisms. So far, Aeromonas veronii has spread to many countries and regions around the world. Because it carries multiple virulence factors and its drug resistance is gradually increasing, it has become an increasing threat to public health in recent years and has also caused great harm to the healthy development of the aquaculture industry. It has been confirmed that Aeromonas vermiformis can infect dozens of marine and freshwater fish, shrimps, crabs, and amphibians, including California bass, grass carp, Chinese sturgeon, Siberian sturgeon, loach, spotted sand loach, yellow catfish, Chinese mitten crab, and vannamei shrimp, often causing symptoms such as bleeding, ascites, and ulcers, resulting in huge economic losses. After people eat aquatic products contaminated by Aeromonas vermiformis, they may suffer from gastroenteritis and sepsis.

[0003] The autophagic protein ATG5 exists in most eukaryotic organisms and is relatively conservative. It plays an important role in the formation of autophagic vacuole (AV). Taking yeast as a model, in the early stage of autophagic vacuole formation (Preautophagosome), the complex formed by ATG12-ATG5-ATG16L binds to its outer membrane. On the one hand, this binding promotes the extension and expansion of the autophagic vacuole, which gradually develops from the initial small vesicle-like and cup-like structure to a semi-ring-like and ring-like structure; on the other hand, the binding of the ATG5 complex to the autophagic vacuole membrane also promotes the recruitment of LC3 (Atg8) to the autophagic vacuole. The localization of the ATG5 complex on the membrane determines the bending direction of the membrane, and the membrane extends in the direction opposite to the ATG5 complex. When the double-layered autophagic vacuole is about to form a ring-shaped closed structure or has just closed, the ATG5 complex detaches from the membrane, leaving only the membrane-bound LC3-II (ATG8-PE) located on the autophagic vacuole membrane. Cellular autophagy has been found in eukaryotic organisms such as plants, yeast, worms, mice and humans. It is closely related to a variety of physiological and pathological processes and has become one of the hot topics in the field of life sciences. However, given that there are no reports on the innate immune response of Macrobrachium rosenbergii's anti-bacterial infection immune-related gene ATG5 against Aeromonas veroni, it is necessary to characterize and study the related effects of Macrobrachium rosenbergii's anti-bacterial infection immune-related gene ATG5 in order to further study its involvement in the innate immune system of Macrobrachium rosenbergii and its related immune signaling pathways and molecular mechanisms. Summary of the invention

[0004] Technical problem to be solved: In response to the above technical problems, the present invention provides a gene ATG5 related to anti-bacterial infection immunity of Macrobrachium rosenbergii and its application. By analyzing its immune function against Aeromonas veronii, it provides a reference for exploring the function of ATG5 in the anti-bacterial infection immunity of Macrobrachium rosenbergii, and enriches the basic theoretical research on ATG5 function and antibacterial immunity.

[0005] Technical solution: In the first aspect, the present invention provides the anti-bacterial infection immunity-related gene ATG5 of Macrobrachium rosenbergii, the nucleotide sequence of which is shown in SEQ ID NO.1:

[0006] SEQ ID NO.1:

[0007] ATGGCAGAAGACAGGGAAATTTTACGGGAAATATGGGATGGTCGTGTCCCAGTATGTGTCCAGCTTGCAAGTGAAGATTGCAACACTCTTAGTGCTCCTGATCCATATTACTTGATGGTTCCAAGGCTGTCGTATTTCCCATTAGTTATGGATAAGGTGAGGAAACATTTGCTACGGTTTATTCCTCCTGATCTTCAGGAAGCAGAAATGTGGTTTGAATGTGATGGATATCCTTTAAAATGGCATTATCCAGTAGGAGTATTATTTGATCTTCATTGTGGTGGAACAGCTTTGCCATGGACTATTACTGCACATTTTTCTCATTTTCCTGAAAAAGAATTAATAAGATGTCCTTCAAGGGAAGTAGTAGAGTCACAGTTTATGTCATCCTTGAAAGAAGCCGATGGATTGAAGCACCGTGGACAAGTCATAACGAACATGCAAAGTAAAGATCATAAACAGTTGTGGATGGGTCTTTGCAATGATAAATTTGACCAATTTTGGGCTATTAATAGAAAACTTATGGAATGTTGTTCTGATGAGGGATTTAAACACATTCCATTCCGTCTTCACACACTTTACCGACCACCCATACAAAAACTTATAAAACCCCTCACTGATGATGGGAGGAAAGTCACCTTGGCAGATCTCCTTCATGATGCCTTACCAGATACCATAGGGGAAGGTGACCGAATCGTGATCCAAGGTATTGAAACTCCCCTAGATACACCAGTACAATGGCTCAGTGAACATCTCAGCCATCCTGACAACTTTCTTCATATAAGTTATATTCCTGCTCAAGCCTGA;

[0008] The amino acid sequence deduced from the nucleotide sequence is shown in SEQ ID NO.2:

[0009] SEQ ID NO.2:

[0010] MAEDREILREIWDGRVPVCVQLASEDCNTLSAPDPYYLMVPRLSYFPLVMDKVRKHLLRFIPPDLQEAEMWFECDGYPLKWHYPVGVLFDLHCGGTALPWTITAHFSHFPEKELIRCPSREVVESQFMSSLKEA DGLKHRGQVITNMQSKDHKQLWMGLCNDKFDQFWAINRKLMECCSDEGFKHIPFRLHTLYRPPIQKLIKPLTDDGRKVTLADLLHDALPDTIGEGDRIVIQGIETPLDTPVQWLSEHLSHHPDNFLHISYIPAQA.

[0011] In a second aspect, the present invention provides an expression vector comprising the Macrobrachium rosenbergii anti-bacterial infection immunity-related gene ATG5 described in claim 1.

[0012] In a third aspect, the present invention provides a recombinant bacterium comprising the expression vector described in the second aspect.

[0013] In a fourth aspect, the present invention provides a recombinant Escherichia coli comprising the expression vector described in the second aspect.

[0014] In a fifth aspect, the present invention provides a primer set for detecting bacterial infection of Macrobrachium rosenbergii, wherein the primer set is used to detect the expression level of the anti-bacterial infection immunity-related gene ATG5 of Macrobrachium rosenbergii described in the first aspect; the bacterial infection includes infection with Aeromonas veronii, and the nucleotide sequence of the primer set is shown in SEQ ID NO.3-SEQ ID NO.4,

[0015] SEQ ID NO.3: catatcgaaggtagg catatg ATGGCAGAAGACAGGGAAATTTT,

[0016] SEQ ID NO.4: cttgaattcggatcc ctcgag TCAGGCTTGAGCAGGAATATAACTT.

[0017] In a sixth aspect, the present invention provides a kit for detecting bacterial infection of Macrobrachium rosenbergii, comprising the primer set described in the fifth aspect.

[0018] In a seventh aspect, the present invention provides a kit for identifying the ability of Macrobrachium rosenbergii to resist infection by Aeromonas veseri, comprising the primer set described in the fifth aspect.

[0019] In an eighth aspect, the present invention provides the use of the Macrobrachium rosenbergii anti-bacterial infection immunity-related gene ATG5 described in the first aspect, or the expression vector described in the second aspect, or the recombinant bacteria described in the third aspect, or the recombinant Escherichia coli described in the fourth aspect in the preparation of an agent for controlling Aeromonas veronii of Macrobrachium rosenbergii.

[0020] Beneficial effects: The present invention provides a Macrobrachium rosenbergii anti-bacterial infection immune-related gene ATG5 that can respond to Aeromonas verni and has a high expression characteristic in the immune response of Macrobrachium rosenbergii infected with Aeromonas verni, and the gene plays an important role in the immune defense process of Macrobrachium rosenbergii against Aeromonas verni. When healthy Macrobrachium rosenbergii is infected with Aeromonas verni, the expression level of Macrobrachium rosenbergii anti-bacterial infection immune-related gene ATG5 is significantly upregulated, so Macrobrachium rosenbergii anti-bacterial infection immune-related gene ATG5 can be used to detect whether Macrobrachium rosenbergii is infected with Aeromonas verni, and can be used to identify the level of anti-Aeromonas verni of Macrobrachium rosenbergii individuals, thereby realizing the screening and cultivation of Macrobrachium rosenbergii resistant to Aeromonas verni. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the identification results of the recombinant cloned plasmid pCold-SUMO-ATG5; wherein, M1 represents DNA Marker2000Plus, and lanes 1 and 2 represent the amplified products obtained by PCR using the plasmid as a template;

[0022] Figure 2 The nucleotide sequence and deduced amino acid sequence analysis results of the anti-bacterial immunity-related gene ATG5 of Macrobrachium rosenbergii, where the three domains of the ATG5 protein are drawn with black, red and yellow horizontal lines respectively;

[0023] Figure 3 The amino acid sequence analysis results of the anti-bacterial infection immune-related gene ATG5 of Macrobrachium rosenbergii. Among them, A is the hydrophilicity analysis; B is the signal peptide prediction; C is the transmembrane domain prediction; D is the ATG5 protein secondary structure prediction, H represents α-helix, e represents β-fold, t represents β-turn, and c represents random coil; E is the ATG5 protein tertiary structure prediction, α-helix is ​​marked in blue, β-fold is marked in green, and random coil is marked in gray;

[0024] Figure 4 The results of multiple sequence comparison analysis of the amino acid sequence of ATG5, a gene related to immunity against bacterial infection in Macrobrachium rosenbergii;

[0025] Figure 5 The phylogenetic tree analysis results of ATG5, a gene related to immunity against bacterial infection in Macrobrachium rosenbergii;

[0026] Figure 6The distribution of ATG5, a gene related to immunity against bacterial infection, in different tissues of healthy Macrobrachium rosenbergii;

[0027] Figure 7 The expression of anti-bacterial infection immune-related gene ATG5 in the hepatopancreas of Macrobrachium rosenbergii at different time points after infection with Aeromonas veronii;

[0028] Figure 8 The expression of anti-bacterial infection immune-related gene ATG5 in the gills of Macrobrachium rosenbergii at different time points after infection with Aeromonas veroni;

[0029] Fig. 9 The expression of anti-bacterial infection immune-related gene ATG5 in the intestine of Macrobrachium rosenbergii at different time points after Aeromonas veronii infection;

[0030] Fig.10 The expression of ATG5, an immune-related gene against bacterial infection, in the muscle of Macrobrachium rosenbergii at different time points after infection with Aeromonas veroni. DETAILED DESCRIPTION

[0031] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0032] Example 1: Construction of recombinant cloning plasmid of Macrobrachium rosenbergii anti-bacterial infection immunity-related gene ATG5

[0033] The average body weight of healthy Macrobrachium rosenbergii was 1.5-2g, and it was provided by Yangzhou Fuyuan Aquatic Products Co., Ltd. The shrimp used was tested to be free of pathogens. Before the experiment, Macrobrachium rosenbergii was temporarily kept in a 0.5m×1m PP bucket, the water temperature was 22°C, the dissolved oxygen was greater than 5 mg / L, and it was fed once in the morning and evening. Twelve juvenile individuals were randomly selected, and after anesthesia, the hemolymph, gills, liver, muscle, intestine and heart tissues were taken respectively, and they were quickly frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator at -80°C for later use. Total RNA was extracted from hepatopancreatic tissue using Trizol Reagent (NoviZan, 411-02-AA) and according to the manufacturer's instructions. The quality of total RNA was detected by 1% agarose gel electrophoresis, the concentration of total RNA was determined using Nanodrop ND-2000 spectrophotometer, and PrimeScript TM II First-strand cDNA synthesis kit was used to generate the first-strand cDNA, and the cDNA product was stored at -20℃ for future use.

[0034] The Macrobrachium rosenbergii ATG5 gene was retrieved from the transcriptome database uploaded by the laboratory (SRA accession number: PRJNA1164130), and the primers for the ORF sequence of the Macrobrachium rosenbergii ATG5 gene were designed using Primer-BLAST (restriction sites were NdeⅠ (catatg) and XhoⅠ (ctcgag)). The primers were synthesized by Nanjing Qingke Biotechnology Co., Ltd. (as shown in SEQ ID NO.3-SEQ ID NO.4 in Table 1). The full-length sequence of the CDS region of ATG5 was amplified using the above hepatopancreas cDNA as a template. The specific operation steps are as follows: PCR reaction system (50 μL): PrimeSTAR Max Premix (2×) 25 μL, upstream and downstream primers (both at a concentration of 10 μmol) 1 μL each, cDNA 2 μL, enzyme-free water 21 μL. PCR reaction conditions: ① 98℃, 10s; ② 60℃, 15s; ③ 72℃, 2min; a total of 35 cycles. The PCR amplification product was verified by agarose gel electrophoresis, and part of the product was sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. Use the SanPrep column PCR product purification kit to purify and recover the PCR product according to its instructions, use a micro-spectrophotometer to measure the DNA concentration of the recovered product, and store it at -20°C for later use. Follow the instructions of the TAKARA ligation kit to connect the PCR amplification product to the T vector (19-T). The recombinant product was transformed into DH5α competent cells, cultured overnight at 37°C, and the positive clones were picked for colony PCR detection, and then sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0035] Table 1 Experimental related primers

[0036]

[0037] Note: The capital letters in SEQ ID NO.3 and SEQ ID NO.4 represent the full-length amplified sequence of the CDS region of the ATG5 gene, the lowercase letters represent the terminal homologous sequences of the vector (pCold-SUMO) used for homologous recombination, and the underlined lowercase letters represent the restriction enzyme sites of NdeⅠ (catatg) and XhoⅠ (ctcgag).

[0038] The electrophoresis identification results of the obtained recombinant clone plasmid pCold-SUMO-ATG5 are as follows Figure 1 As shown: Among them, M1 represents DNAMarker2000P1us, lanes 1 and 2 represent the amplified products obtained by PCR using the recombinant cloning plasmid pCold-SUMO-ATG5 as a template, and the size of the amplified product is about 807 bp, which indicates that the recombinant plasmid has been successfully constructed.

[0039] Example 2: Functional analysis of the antibacterial immunity-related gene ATG5 of Macrobrachium rosenbergii

[0040] Expasy (http: / / www.au.expasy.org / ) online bioanalysis software was used to translate the sequence of ATG5 gene, predict and analyze the physicochemical properties, including relative molecular weight, theoretical isoelectric point, amino acid composition, protein instability index, fat coefficient and average hydrophilicity coefficient, etc. The secondary structure was predicted using the S0MPA online program (https: / / np sa-prabi.ibcp.fr / cgi-bin / npsaautomat.pl?page=npsa%20sopma.html), the protein structure and function domains were analyzed using the NCBI conserved domain (CDD) database and SMART tools (https: / / smart.embl.de / ), and the three-dimensional structure was analyzed using the SWISS-MODEL website (https: / / swissmodel.expasy.org / ). Hydrophilicity analysis was performed using ProtScale (https: / / web.expasy.org / protscale / ). The signal peptide was predicted using the SignalP 4.1 website (https: services.healthtech.dtu.dk / services / SignalP-4.1 / ). The transmembrane region of ATG5 was predicted using the DeepTMHMM 1.0 program (https: / / services.healthtech.dtu.dk / services / DeepTMHMM-1.0 / ). Amino acid multiple sequence alignment analysis was performed using ClustalW (https: / / www.genome.jp / tools-bin / clustalw) and ESPript 3.0 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi). The evolutionary tree was constructed using the neighbor-joining (NJ) method of MEGA7.0. All ATG5 gene alignment sequence information was obtained from the GenBank database. The experimental results are shown in Figure 2. Figure 2-Figure 6 As shown:

[0041] Figure 2 The results of the analysis of the nucleotide sequence of the antibacterial immunity-related gene ATG5 of Macrobrachium rosenbergii (as shown in SEQ ID NO.1) and its deduced amino acid sequence (as shown in SEQ ID NO.2), wherein the black underline is the ATG5-UbIA domain, the red underline is the ATG5-HBR domain, the yellow underline is the ATG5-UbIB domain, and - indicates a stop codon. Figure 3 These are the results of amino acid sequence analysis of the antibacterial immunity-related gene ATG5 of Macrobrachium rosenbergii, where A is the hydrophilicity analysis; B is the signal peptide prediction; C is the transmembrane domain prediction; D is the secondary structure prediction of the ATG5 protein, where H represents α-helix, e represents β-sheet, t represents β-turn, and c represents random coil; and E is the tertiary structure prediction of the ATG5 protein, where α-helix is ​​marked in blue, β-sheet is marked in green, and random coil is marked in gray. Figure 4 This is the analysis result of multiple sequence alignment of the amino acid sequence of ATG5, a gene related to antibacterial immunity of Macrobrachium rosenbergii. Figure 5 This is the result of phylogenetic tree analysis of ATG5, a gene related to immunity against bacterial infection in Macrobrachium rosenbergii.

[0042] Depend on Figure 2 and Figure 3 It can be seen that the nucleotide sequence of the antibacterial immunity-related gene ATG5 of Macrobrachium rosenbergii contains a complete open reading frame (ORF) with a total length of 807bp, encoding 268 amino acid residues. The predicted molecular weight is 25.39kDa, and the theoretical isoelectric point is 6.09. According to SMART and CDD predictions, ATG5 has 1 ATG5-UbIA domain, 1 ATG5-HBR domain, 1 ATG5-UbIB domain and 1 APG5 conserved domain. The hydrophilicity prediction results show that most of the polypeptide chain of the antibacterial immunity-related gene ATG5 of Macrobrachium rosenbergii is a hydrophilic region, indicating that the protein may be a hydrophilic protein. Signal peptide prediction analysis shows that ATG5 does not contain a signal peptide, and it is speculated to be a non-secretory protein. Transmembrane region prediction shows that ATG5 does not have a transmembrane structure. Figure 3 The results of the secondary and tertiary structure prediction of ATG5 protein were obtained by using the SOPMA online tool to predict the secondary structure of ATG5 protein. The results showed that the random coil structure accounted for the highest proportion of 48.51% in the secondary structure of ATG5 protein, which was composed of 130 amino acids; 80 amino acids formed α-helix, accounting for 29.85%; 45 amino acids formed β-fold, accounting for 16.79%; 13 amino acids formed β-turn, accounting for 4.85%. The tertiary structure of ATG5 protein showed that α-helix and random coil had the highest content, which was consistent with the secondary structure prediction results.

[0043] Depend on Figure 4 and Figure 5It can be seen that the amino acid sequence of the anti-bacterial infection-related gene ATG5 of Macrobrachium rosenbergii is 93.9%, 80.3%, 78.9%, 78.5%, 54.1% and 52.7% identical to that of ATG5 of Macrobrachium nipponense, Procambarus clarkii, Penaeus vannamei, Penaeus japonicus, zebrafish and Homo sapiens, respectively. From the phylogenetic tree, it can be seen that the ATG5 of arthropods such as Limulus americanus, Thrips palmi, Asian migratory locust and German cockroach are clustered in one branch, the ATG5 of Penaeus japonicus, Penaeus chinensis, Penaeus vannamei and Penaeus monodon are clustered in one branch, and the ATG5 of Macrobrachium rosenbergii and Macrobrachium nipponense are clustered in one branch.

[0044] The above results show that the antibacterial immunity-related gene ATG5 of Macrobrachium rosenbergii contains a CDS region of 807bp in length, which can encode 268 amino acids, has one APG5 conserved domain, does not contain a signal peptide, does not have a transmembrane structure, and is a hydrophilic protein. The Macrobrachium rosenbergii gene ATG5 is clustered with the Japanese Macrobrachium ATG5 in the phylogenetic tree, and its amino acid sequence has the highest similarity with the Japanese Macrobrachium at 93.9%.

[0045] Example 3: Animal test results of Macrobrachium rosenbergii anti-bacterial infection immune-related gene ATG5

[0046] Infection experiment: 120 healthy Macrobrachium rosenbergii were selected and kept for one week for artificial infection. A control group and an experimental group were set up. Each Macrobrachium rosenbergii was injected intramuscularly with 0.1 mL PBS, and each Macrobrachium rosenbergii was injected with 0.1 mL Aeromonas veroni JDM1-1 suspension (1.8 × 10 6 CFU / mL), 9 Macrobrachium rosenbergii were randomly selected to dissect the hepatopancreas, intestine, gills and muscle tissues 0, 12, 24, 36 and 48 h after injection, and the tissues of every 3 Macrobrachium rosenbergii were mixed and placed in an EP tube, and three biological parallels were set up. Then the tissues were quick-frozen in liquid nitrogen and then stored in a -80℃ refrigerator for later use.

[0047] Real-time fluorescence quantitative PCR: The RT-qPCR method was used to analyze the expression pattern of ATG5 in healthy and infected tissues of Macrobrachium rosenbergii. β-actin was used as the internal reference gene (primers are shown in Table 1), and the qRT-PCR reaction was performed in a real-time fluorescence quantitative PCR instrument (Thermofisher QuantStudio Real-Time PCR System). The 20 μL reaction system was as follows: 0.4 μL each of forward and reverse primers (10 M), 10 μL of ChamQ Universal SYBRqPCR Master Mix (Novozyme), 8.2 μL of ddH2O, and 1 μL of cDNA template; the reaction program was: 95°C for 30 s, 95°C for 5 s, 60°C for 34 s, and 40 cycles. The melting curve was used to determine the primer specificity. Three biological replicates were set, and 2 -ΔΔCt The relative expression of the selected genes was calculated by the method. The experimental results were expressed as mean ± standard deviation (x ± SD). SPSS (17.0) analysis software was used to perform one-way analysis of variance on the data. Duncan's method was used for multiple comparisons. P < 0.05 between treatments was considered to be significantly different. Different lowercase letters indicate statistical significance. Figure 6-10 As shown:

[0048] Depend on Figure 6 It can be seen that RT-qPCR was used to analyze the distribution of ATG5 in different tissues of healthy Macrobrachium rosenbergii. The results showed that ATG5 was widely distributed in 6 tissues, among which the expression level in muscle was the highest, almost 35.6 times that of muscle, followed by the expression levels in hepatopancreas, hemolymph, heart, intestine and gills decreasing in turn. Figure 7 to Figure 10 It can be seen that by intraperitoneal injection of Aeromonas veronii into healthy Macrobrachium rosenbergii, the expression level of ATG5 in the hepatopancreas, gills, intestines and muscles after infection was analyzed. The results showed that the expression level of ATG5 was significantly upregulated after bacterial infection. In the gill tissue, the expression of the ATG5 gene was significantly upregulated at 12h and reached a maximum value, with an increase of 1.23 times. In the intestine, ATG5 mRNA was significantly upregulated and reached a maximum value at 12h, an increase of 1.51 times compared to 0h. In addition, Aeromonas veronii significantly downregulated the expression of ATG5 in the hepatopancreas and muscle. Although it increased at subsequent time points, the overall expression level was still lower than that at 0h.

[0049] The above results show that the anti-bacterial immunity-related gene ATG5 of Macrobrachium rosenbergii is expressed most highly in muscle tissue, followed by hepatopancreas, hemolymph, heart, intestine and gill tissues. After Macrobrachium rosenbergii was infected with Aeromonas vermiformis, the ATG5 gene was upregulated in gill and intestinal tissues, both reaching a peak at 12 hours. In hepatopancreas and muscle tissues, the ATG5 gene was downregulated compared with the control group.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. ATG5, a gene related to immunity against bacterial infection of Macrobrachium rosenbergii, is characterized by: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. An expression vector, characterized in that: The method comprises the anti-bacterial infection immunity-related gene ATG5 of Macrobrachium rosenbergii according to claim 1.

3. A recombinant bacterium, characterized in that: Comprising the expression vector according to claim 2.

4. A recombinant Escherichia coli, characterized in that Comprising the expression vector according to claim 2.

5. A primer set for detecting bacterial infection in Macrobrachium rosenbergii, characterized in that: The primer set is used to detect the expression level of the anti-bacterial infection immunity-related gene ATG5 of Macrobrachium rosenbergii according to claim 1; the nucleotide sequence of the primer set is shown in SEQ ID NO.3-SEQ ID NO.

4.

6. A kit for detecting bacterial infection in Macrobrachium rosenbergii, characterized in that: Comprising the primer set of claim 5.

7. A kit for identifying the ability of Macrobrachium rosenbergii to resist infection by Aeromonas veseri, characterized in that: Comprising the primer set of claim 5.

8. Use of the Macrobrachium rosenbergii anti-bacterial infection immunity-related gene ATG5 according to claim 1, or the expression vector according to claim 2, or the recombinant bacteria according to claim 3, or the recombinant Escherichia coli according to claim 4 in the preparation of an agent for controlling Aeromonas veronii of Macrobrachium rosenbergii.

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