CN108586596B - Oyster apoptosis gene SMAC gene and application thereof in preparation of pathological detection diagnostic reagent - Google Patents
Oyster apoptosis gene SMAC gene and application thereof in preparation of pathological detection diagnostic reagent Download PDFInfo
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Abstract
The invention discloses an oyster apoptosis gene SMAC gene and application thereof in preparation of a pathological detection diagnostic reagent. The invention discovers a marker gene SMAC gene of the apoptosis of the species, and the nucleotide sequence of the marker gene SMAC gene is shown as 50 bp to 1006bp of SEQ ID NO. 1. The amino acid sequence (the amino acid sequence is shown as SEQ ID NO. 2) coded by the gene has high species specificity, the similarity of the amino acid sequence in the oyster is 60-88%, the evolutionary relationship is relatively close, and the similarity of the amino acid sequence and other species homologous proteins is below 30%, so the evolutionary relationship is relatively far. Quantitative PCR experiments show that the SMAC gene is obviously up-regulated after Vibrio alginolyticus and Staphylococcus (Staphylococcus haemolyticus) infection, can be used as a standard for detecting oyster cell apoptosis, provides a positive and accurate warning signal for oyster disease prevention, and prevents disaster expansion.
Description
The technical field is as follows:
the invention relates to a molecular mechanism of shellfish immunity and apoptosis, in particular to a nucleotide sequence and an amino acid sequence of an SMAC (second dinitrogendria-derived activator of caspases) gene screened from a Vibrio-stimulated Crassostrea hongkongensis (Crassostingkongensis) blood cell differential hybridization library, a preparation method thereof and application of the gene.
Background art:
china is the biggest oyster cultivation country in the world, and according to statistics, the annual output of China accounts for more than 80% of the total world output. According to the statistics of the annual yearbook of fishery statistics in 2015 China, the breeding area of oysters in the year reaches 14.15 ten thousand hectares, and the yield is about 457.34 ten thousand tons, so that the oyster breeding occupies a great position in the development of coastal economic society in China, and is an important prop industry; the crassostrea hongkongensis is mainly distributed in the coastal estuary and bay areas from the west to the Fujian in China, and is the most important oyster cultivation variety on the coast of south China. The development of marine shellfish in China is mainly based on traditional culture, the culture area is wide, the energy consumption is high, the yield fluctuates due to various condition changes, and the risk of oyster culture is increased along with the expansion of culture scale due to the lack of effective disease detection means. In oyster cultivation, large-scale death events frequently occur, the death rate is up to 70% -80%, and almost all death of some farms occurs. The oyster death is caused by a plurality of reasons, wherein the oyster death is aggravated by pathogenic bacteria and parasite invasion, and severe environmental stress is also an important factor for inducing oyster death. Therefore, establishing a real-time oyster cultivation disease prevention and monitoring mechanism is one of the problems to be solved urgently.
Apoptosis is an important mark for detecting the survival state of an individual, and any reaction stressed in a body can activate a physiological mechanism of apoptosis, so that the selection of apoptosis marker genes has important significance for establishing a mechanism prevention mechanism. SMAC (second mitochondria-derived activator of Caspases) is a marker gene for apoptosis and refers to a second protein found in mitochondria that regulates apoptosis by activating the cysteine protease Caspases, in addition to cytochrome C. The cells contain a large amount of Caspases, and the active sites of the Caspases contain cysteine residues, so that peptide bonds on aspartic acid residues of target proteins can be specifically cut, and apoptosis can be induced. Under normal conditions, caspase protein in cytoplasm is combined with apoptosis inhibitor protein IAPs (inhibitor apoptosis proteins), so that stable structure of cells is maintained, and apoptosis of cells is not activated. In some cases, upon receipt of an apoptotic signal, SMAC is released from the mitochondria to the cytoplasm, binds to IAPs, deprives caspase inhibitory activity, activates caspase proteins, and thereby initiates apoptosis of the cell, and thus detection of intracellular SMAC is the gold standard for determining apoptosis.
The invention content is as follows:
the invention provides a shellfish cell apoptosis related gene sequence-SMAC gene.
The nucleotide sequence of the shellfish cell apoptosis related gene sequence SMAC gene is shown as 50 bp to 1006bp of SEQ ID NO.1, 318 amino acids are coded by the gene sequence, and the amino acid sequence is shown as SEQ ID NO. 2.
The invention discovers a marker gene SMAC gene of the species apoptosis on the basis of the hong Kong giant oyster transcriptome sequencing. By RACE technology, we obtained 1153bp cDNA total length (shown as SEQ ID NO.1 specifically), 49bp 5 'non-coding region length, 147bp 3' non-coding region length and containing poly A tailing signal (ATTAAA) and poly A tail, and the nucleotide sequence of open reading frame is shown as 50-1006 bp of SEQ ID NO.1 and named as SMAC gene. The protein sequence (the amino acid sequence is shown as SEQ ID NO. 2) containing 318 amino acids coded by the gene is named as SMAC protein, has high species specificity, has the similarity of the amino acid sequence in oyster of 60-88 percent and is relatively close in evolutionary relation, and has the similarity of less than 30 percent with other species homologous proteins, so the evolutionary relation is relatively far. Quantitative PCR experiments show that the SMAC gene is obviously up-regulated after Vibrio alginolyticus and staphylococcus (staphylococcus), can be used as a standard for detecting oyster cell apoptosis, provides a positive and accurate warning signal for oyster disease prevention, and prevents disaster expansion.
Therefore, a second object of the present invention is to provide use of a detection reagent for detecting an expression amount of SMAC gene in preparing an oyster disease-preventive and surveillance reagent.
The oyster disease prevention and warning reagent is a reagent for detecting the apoptosis condition of oyster cells.
The oyster is Crassostrea hongkongensis.
The reagent for detecting the oyster cell apoptosis condition is preferably a detection reagent for detecting the oyster cell apoptosis condition after the oyster is infected by vibrio alginolyticus and/or staphylococcus.
The third purpose of the invention is to provide a detection primer of the SMAC gene;
the detection primer of the SMAC gene is as follows:
ChSMAC-F:5'-AAAACTAGCTCTGAGGCTGATAATA-3'
ChSMAC-R:5'-ATAACGAATACAAGAACAGGCATAA-3'。
the fourth purpose of the invention is to provide the application of the detection primer of the SMAC gene in the preparation of a reagent for detecting the expression level of the SMAC gene.
Preferably, the kit further comprises a detection primer of a reference gene GAPDH, and the detection primer specifically comprises:
ChGAPDH-F:5'-GGATTGGCGTGGTGGTAGAG-3'
ChGAPDH-R:5'-GTATGATGCCCCTTTGTTGAGTC-3'。
the fifth object of the present invention is to provide a method for quantitatively detecting an SMAC gene, which is characterized in that a reverse transcription product of total RNA of oyster is used as a template, a detection primer of the SMAC gene is used as a primer, and the expression level of the SMAC gene is detected by quantitative PCR.
Preferably, the gene GAPDH is also used as an internal control.
Preferably, the reaction system of the quantitative PCR is as follows:
the reaction procedure is as follows: 5min at 95 ℃ for 1 cycle; 95 ℃ 10s, 56.5 ℃ 10s, 72 ℃ 20s, 45 cycles.
The sixth purpose of the invention is to provide the application of the SMAC gene as an oyster apoptosis marker gene in the preparation of a reagent for detecting oyster apoptosis.
The inventor finds that a marker gene-SMAC gene of crassostrea hongkongensis apoptosis is obviously up-regulated after Vibrio alginolyticus and Staphylococcus (Staphylococcus aureus) infection, can be used as a standard for detecting oyster apoptosis, provides a positive and accurate warning signal for oyster disease prevention, and prevents disaster expansion. The application of the technology can further improve the germplasm innovation capability of shellfish culture and support the high-efficiency sustainable development of the industry.
Description of the drawings:
figure 1 is a structural analysis of SMAC protein;
figure 2 is the tissue distribution of SMAC protein. The real-time quantitative PCR experiment result shows that the SMAC protein is expressed in each tissue, and the expression level is the highest in blood cells. Different lower case letters indicate significant differences between samples.
FIG. 3 shows that Vibrio alginolyticus stimulates the induction of SMAC gene expression. The expression of the SMAC gene in blood cells was significantly upregulated following pathogenic stimulation. Asterisks indicate that the differences from the control group were extremely significant.
FIG. 4 is a graph showing that staphylococcal stimulation induced SMAC gene expression. The expression of SMAC in blood cells was significantly upregulated following pathogenic stimulation. Asterisks indicate that the differences from the control group were extremely significant.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
RNA extraction and reverse transcription
a) After adding Trizol to crassostrea hongkongensis cells or tissues, grinding the cells or tissues by using a tissue grinder, and standing the ground cells or tissues at room temperature for 5min to fully crack the cells or tissues.
b) Centrifuge at 12,000rpm for 5min and discard the pellet.
c) Chloroform was added to 200ul of chloroform/ml of Trizol, followed by shaking and mixing, and then allowed to stand at room temperature for 15 min.
d) Centrifugation at 12,000g for 15min at 4 ℃.
e) The upper aqueous phase was aspirated into another centrifuge tube.
f) Adding 0.5ml of isopropanol/ml of Trizol into the isopropanol, mixing uniformly, and standing at room temperature for 5-10 min.
g) Centrifugation at 12,000g for 10min at 4 ℃ was carried out, the supernatant was discarded, and RNA was precipitated at the bottom of the tube.
h) Add 75% ethanol to 1ml of 75% ethanol/ml Trizol, gently shake the centrifuge tube, and suspend the precipitate.
i) Centrifuge at 8,000g for 5min at 4 ℃ and discard the supernatant as much as possible.
j) Drying at room temperature or vacuum drying for 5-10 min.
k) RNA samples can be lysed with 50ul H2O, TE buffer or 0.5% SDS at 55-60 ℃ for 5-10 min.
l) after 15min incubation at room temperature, 250 μ LDNase Stop Solution (DSA) was added and centrifuged at 13,000 × g for 1 min.
m) repeating steps a-j, adding 100. mu.L of nuclease-free water, then centrifuging at 13,000 Xg for 1min, discarding the spin column, measuring the concentration of RNA in the collected solution using a spectrophotometer, dispensing, and storing at-80 ℃.
n) Synthesis of first Strand cDNA
1) DNase I digestion
Adding the RNA of each group into a PCR centrifuge tube according to the following components
| RNA | 1μg |
| 10XDNaseIbuffer | 1.2μL |
| DNaseI | 1μL |
| SterileH2O | Upto12μL |
2) Lightly blowing and beating the mixture by using a gun head, and slightly centrifuging the mixture; digesting DNA in RNA at 37 ℃ for 15min
3) Adding 1 μ L EDTA, mixing, inactivating DNase I at 65 deg.C for 10min
4) The following components were added to the centrifuge tube in sequence:
lightly blowing and uniformly mixing by using a gun head, slightly centrifuging, and placing at 37 ℃ for 30 min;
5) reverse transcriptase was inactivated at 85 ℃ for 5s and then stored at 4 ℃ for a short period of time and at-20 ℃ for a long period of time.
2. Subtractive hybrid library construction
The Subtraction hybridization library was constructed using the PCR-Select cDNA transcription Kit (Clontech, USA) Kit. After 8 hours of Vibrio hongkongensis stimulation, blood cells were collected, total RNA from blood cells was extracted according to the above method, and total RNA from control group (not treated with Vibrio) blood cells was extracted using the same method. Subtractive hybridization was carried out using crassostrea hongkongensis as the tester and the control as the driver. The differentially hybridised cDNAs were subcloned into pGEM-Teasy vector (Promega, USA) and transformed into Escherichia coli JM109(Promega, USA) competent cells. 2000 clones were randomly picked and sequenced.
Cloning of the full Length of the SMAC Gene
According to the clone sequencing obtained in step 2, the sequence of SMAC is analyzed and determined by biological software such as BLASTEN, and then 5'/3' RACE of the gene is carried out by GeneRacer TM RACE Ready cDNA kit (Invitrogen, USA) by taking the sequence as a template, and the specific operation is referred to the instruction. After obtaining RACE sequence, the full-length cDNA of the gene (the nucleotide sequence is shown as SEQ ID NO. 1) is obtained through splicing, the coding region is analyzed, the nucleotide sequence of the coding region is shown as 50 bp to 1006bp of SEQ ID NO.1 and is named as SMAC gene, the coded amino acid sequence is shown as SEQ ID NO.2 and is named as SMAC protein, and the coded amino acid sequence is analyzed through SMART online software (http:// smart.embl-heidelberg. de /), and the analysis shows that the protein coded by the gene has a characteristic structural domain of the SMAC protein family (figure 1).
4. Real-time quantitative PCR
The kit used for real-time quantitative PCR is LightCycler 480SYBR Green I (Roch), the used instrument is LightCycler 480 System (Roche), the used template is a reverse transcription product of total RNA, the used internal reference GAPDH, and the used primers are as follows:
for the SMAC gene: ChSMAC-F5'-AAAACTAGCTCTGAGGCTGATAATA-3'
ChSMAC-R:5'-ATAACGAATACAAGAACAGGCATAA-3'
For the internal control GAPDH gene: ChGAPDH-F:5'-GGATTGGCGTGGTGGTAGAG-3'
ChGAPDH-R:5'-GTATGATGCCCCTTTGTTGAGTC-3'
qRT-PCR experiments the reaction System was carried out using the Light-Cycler 480II System (Roche) as follows:
the thermal cycling conditions were as follows: 5min at 95 ℃ for 1 cycle; 95 ℃ 10s, 56.5 ℃ 10s, 72 ℃ 20s, 45 cycles. Use 2－ΔΔCTThe method performs relative quantification of transcripts. The experimental and control groups were set up for 3 parallel reactions.
5. Tissue distribution and sample processing
Respectively extracting total RNA (shown in step 1) of each tissue of 5 crassostrea hongkongensis and carrying out reverse transcription to obtain cDNA (complementary deoxyribonucleic acid), and detecting the distribution condition of the SMAC gene in lymphocytes, gills, touch lips, gonads, digestive glands, mantle, pericardial cavity and adductor muscles of the crassostrea hongkongensis by adopting a Real-time PCR (polymerase chain reaction) technology. The results showed that it was widely expressed in each tissue, with the lowest expression in the mantle and the highest expression in the haemolymph (FIG. 2).
Injecting normal Crassostrea hongkongensis into 1.0 × 109Live pathogens were sampled in groups of 5 at different time points, using PBS as a reference, and total RNA from blood cells was extracted and reverse transcribed into cDNA, see step 1. And performing Real-time PCR detection on the sample by taking the template as a template, and finding that infection of vibrio alginolyticus (figure 3) and staphylococcus (figure 4) causes remarkable up-regulation expression of the SMAC gene.
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
<120> oyster apoptosis gene SMAC gene and application thereof in preparation of pathological detection diagnostic reagent
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<213> Crassostera hongkongensis (Crassostra hongkongensis)
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ggcgtctttt tctggtggtc gtaccttcct caagatcttt acagggtcgt caatgacagt 180
gtgtataatg ccggtggtct gtgcaatgga ggttcccaat gttgatgact atgctatgtc 240
caagctcctc caaaactcaa gtgtggcaag tgctgaggca gcgtcagcat ttctgtcata 300
tgtcaccatg gctctatata acacagagaa ggaatacaca aaggaggttt acaccctgat 360
aaagctgata gagatgagat tggagaatcc cggccatccg gatcgggaca agatggagga 420
gcttattgta gagaccagaa tccaggtcaa gtccctgaag aagaagagac aggacttgat 480
gatgctgcta tctgtatgtc agagacttgt gaatgacgcc gcagaggcag cctacatggc 540
aggagcagag tttgctggct atacaactaa caacaaattg acaagctccg agagttttct 600
gtcgccatgt gaaatcgagc gtgtgattgc agagaggcat ttagcagagc ttgaagctcg 660
tgtcattgag gtggaatcca aacgtgccaa agaagaggag gaaagagaaa caaaacagga 720
agaagtgaag gatgatggga attctaggga ggggaatgaa ggtgtgaatt ctaacagcaa 780
tactagagac aagaaagaag atgtgaaatc tgatggaagt acagcagagg cgaagggaga 840
taagtcagtc gatgaagaga cagaggaaac tgacattgcc cagatagatg caagtacttc 900
aacaaaaact agctctgagg ctgataataa ggaattcata cccagaatgg actatgtaga 960
aacattcagt ataggttcca aggatgaatc attagaggac gattgatttg tcaacaatta 1020
ggacctttaa tatggttgtg aatgaattta tgcctgttct tgtattcgtt ataattgtta 1080
tgatagcgtc tttgtaaatg ataaatttta tgtatattta atataaaata ttaaaaaatg 1140
caaaaaaaaa aaa 1153
<210>2
<211>318
<212>PRT
<213> Crassostera hongkongensis (Crassostra hongkongensis)
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Met Ile Arg Asn Thr Trp Val Arg Ile His Ala Leu Arg Ser Cys Val
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Val Val Cys Ala Met Glu Val Pro Asn Val Asp Asp Tyr Ala Met Ser
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Lys Leu Leu Gln Asn Ser Ser Val Ala Ser Ala Glu Ala Ala Ser Ala
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Phe Leu Ser Tyr Val Thr Met Ala Leu Tyr Asn Thr Glu Lys Glu Tyr
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Thr Lys Glu Val Tyr Thr Leu Ile Lys Leu Ile Glu Met Arg Leu Glu
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Asn Pro Gly His Pro Asp Arg Asp Lys Met Glu Glu Leu Ile Val Glu
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Thr Arg Ile Gln Val Lys Ser Leu Lys Lys Lys Arg Gln Asp Leu Met
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Met Leu Leu Ser Val Cys Gln Arg Leu Val Asn Asp Ala Ala Glu Ala
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Ala Tyr Met Ala Gly Ala Glu Phe Ala Gly Tyr Thr Thr Asn Asn Lys
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Leu Thr Ser Ser Glu Ser Phe Leu Ser Pro Cys Glu Ile Glu Arg Val
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Ile Ala Glu Arg His Leu Ala Glu Leu Glu Ala Arg Val Ile Glu Val
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Glu Ser Lys Arg Ala Lys Glu Glu Glu Glu Arg Glu Thr Lys Gln Glu
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Glu Val Lys Asp Asp Gly AsnSer Arg Glu Gly Asn Glu Gly Val Asn
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Ser Asn Ser Asn Thr Arg Asp Lys Lys Glu Asp Val Lys Ser Asp Gly
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Ser Thr Ala Glu Ala Lys Gly Asp Lys Ser Val Asp Glu Glu Thr Glu
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Glu Thr Asp Ile Ala Gln Ile Asp Ala Ser Thr Ser Thr Lys Thr Ser
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Ser Glu Ala Asp Asn Lys Glu Phe Ile Pro Arg Met Asp Tyr Val Glu
290 295 300
Thr Phe Ser Ile Gly Ser Lys Asp Glu Ser Leu Glu Asp Asp
305 310 315
Claims (10)
- The SMAC protein is characterized in that an amino acid sequence is shown as SEQ ID NO. 2.
- 2. A gene encoding the SMAC protein of claim 1.
- 3. The coding gene according to claim 2, characterized in that it is a SMAC gene, the nucleotide sequence is shown in the 50 th to 1006bp of SEQ id No. 1.
- 4. Use of a detection reagent for detecting the expression level of the SMAC gene of claim 3 in the preparation of a preventive or preventive remedy for oyster diseases caused by infection with Vibrio alginolyticus or Staphylococcus.
- 5. The use according to claim 4, wherein the oyster disease prevention and surveillance agent is an agent for detecting oyster apoptosis.
- 6. The use according to claim 4 or 5, wherein the oysters are Crassostrea hongkongensis.
- 7. The use of claim 5, wherein the oyster apoptosis assay reagent is an oyster apoptosis assay reagent for detecting oyster apoptosis after infection of oysters with Vibrio alginolyticus and/or Staphylococcus.
- 8. A detection primer for the SMAC gene of claim 3;the detection primer of the SMAC gene is as follows:ChSMAC-F:5'-AAAACTAGCTCTGAGGCTGATAATA-3'ChSMAC-R:5'-ATAACGAATACAAGAACAGGCATAA-3'。
- 9. use of the detection primer for the SMAC gene according to claim 8 for preparing a reagent for detecting the expression level of the SMAC gene according to claim 3.
- 10. The use of the SMAC gene of claim 3 as a marker gene for oyster apoptosis in the preparation of a reagent for detecting oyster apoptosis caused by vibrio alginolyticus or staphylococcus infection.
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| CN105859862A (en) * | 2016-05-19 | 2016-08-17 | 清远职业技术学院 | Crassostrea hongkongensis antimicrobial peptide sa-lectin as well as recombinant expression method and application thereof |
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| CN105859862A (en) * | 2016-05-19 | 2016-08-17 | 清远职业技术学院 | Crassostrea hongkongensis antimicrobial peptide sa-lectin as well as recombinant expression method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| ChBax/Bak as key regulators of the mitochondrial apoptotic pathway:cloned and characterized in Crassostrea hongkongensis;Zhiming Xiang等;《Fish & Shellfish Immunology》;20141120;第225-232页 * |
| Identification and function of an evolutionarily conserved signaling intermediate in Toll pathways(ECSIT) from Crassostrea hongkongensis;F Qu等;《Developmental & Comparative Immunology》;20151231;第53卷(第1期);第244-252页 * |
| 香港牡蛎(Crassostrea hongkongensis) Commd1基因的分子克隆及其在盐度胁迫下的表达分析;王富轩等;《热带海洋学报》;20170115;第36卷(第1期);第48-55页 * |
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