CN108610406B - Oyster cell apoptosis gene caspase3 gene and application thereof in preparation of pathological detection diagnostic reagent - Google Patents
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Abstract
The invention discloses an oyster cell apoptosis gene caspase3 gene and application thereof in preparation of a pathological detection diagnostic reagent. The inventor finds that a marker gene caspase3 gene of crassostrea hongkongensis apoptosis has a nucleotide sequence shown in SEQ ID No.1, is obviously up-regulated after Vibrio alginolyticus and Staphylococcus are infected, can be used as a standard for detecting the apoptosis condition of the crassostrea honggensis, provides a positive and accurate warning signal for preventing crassostrea honggensis diseases, and prevents the expansion of disasters. 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
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 a caspase3 gene screened from a Crassostrea hongkongensis (Crassostreae hongkongensis) blood cell differential hybridization library after vibrio stimulation, a preparation method thereof and application of the gene.
Background art:
the annual yield of oysters in China accounts for more than 80% of the total world yield, and is the largest oyster breeding country in the world. The hong Kong oysters are widely distributed in coastal waters from Guangxi to Fujian and the like, and are the most important oyster cultivation varieties in the coastal areas of south China. Because the cultivation mode is mainly based on traditional cultivation, the method has the limitations of wide cultivation area, high energy consumption and the like, and the yield fluctuates due to various condition changes. With the increasing pollution of marine environment, the risk of oyster cultivation is also increased. The existing oyster disease detection technology in China falls behind and basically belongs to a blank state, so that the oyster health monitoring technology is improved, the disease propagation is prevented in time, and the oyster disease detection method is an effective means for developing and improving the oyster cultivation industry.
Apoptosis is a basic mechanism for cell deletion in the normal development and differentiation process of multicellular organisms, and is closely related to tissue homeostasis, aging and cell damage. Once the mechanism is stressed by diseases and environment, the apoptosis pathway is started, a series of proteases are activated, and the cells are orderly degraded. In this process, a class of enzymes plays a critical role, and their structural features are similar to those of cysteine proteases, and they specifically cleave peptide bonds after Asp, so they are named caspases. Caspase3 is a key execution enzyme of cell apoptosis, is usually a marker gene for whether cell apoptosis is carried out or not, and has important application value in biological pathology detection.
The invention content is as follows:
the first purpose of the invention is to provide a caspase3 gene which is a gene sequence related to the apoptosis of the hong Kong oyster cells.
The present invention relates to a hong Kong oyster cell apoptosis related gene sequence-caspase 3 gene, its nucleotide sequence is shown in SEQ ID NO.1, its coded 296 amino acids, and its amino acid sequence is shown in SEQ ID NO. 2.
The invention discovers a marker gene caspase3 gene of the species apoptosis on the basis of hong Kong giant oyster transcriptome sequencing. By RACE technology, we obtain the full length of the gene, the nucleotide sequence of which is shown as SEQ ID NO.1, and the amino acid sequence of the gene code is shown as SEQ ID NO. 2. Quantitative PCR experiments show that caspase3 gene is obviously up-regulated after Vibrio alginolyticus and Staphylococcus is infected, 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 level of caspase3 gene in the preparation of an oyster disease preventive alert agent.
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 caspase3 gene;
for caspase3 gene:
Chcaspase3-F:5'-GGAGCGGTTTCAGGACTTGG-3'
Chcaspase3-R:5'-AGCATTCCGTCGGTAGCGTA-3'。
the fourth purpose of the invention is to provide the application of the detection primer of the caspase3 gene in the preparation of a reagent for detecting the expression level of the caspase3 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 purpose of the invention is to provide a quantitative detection method of caspase3 gene, which is characterized in that reverse transcription product of total oyster RNA is used as a template, detection primer of the caspase3 gene is used as primer, and quantitative PCR is used for detecting the expression quantity of the caspase3 gene.
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 ℃ for 10s, 58.5 ℃ for 10s, 72 ℃ for 20s, 45 cycles.
The sixth purpose of the invention is to provide the application of the caspase3 gene as an oyster apoptosis marker gene in the preparation of a reagent for detecting the oyster apoptosis.
The inventor finds that a caspase3 gene which is a marker gene of crassostrea hongkongensis apoptosis is obviously up-regulated after Vibrio alginolyticus and Staphylococcus (Staphylococcus aureus) are infected, 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:
FIG. 1 shows the tissue distribution of caspase3 protein, and the results of real-time quantitative PCR experiments show that caspase3 protein is expressed in all tissues, and has the lowest content in heart and the highest expression level in blood cells. Different lower case letters indicate significant differences between samples.
FIG. 2 shows the stimulation of the expression of caspase3 gene by Vibrio alginolyticus. After the stimulation of pathogenic bacteria, the expression of caspase3 gene in blood cells is obviously up-regulated. Asterisks indicate that the differences from the control group were extremely significant.
FIG. 3 is a graph showing the stimulation of caspase3 gene expression by Staphylococcus. After the stimulation of pathogenic bacteria, the expression of caspase3 gene in blood cells is obviously up-regulated. 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 the cells or tissues of crassostrea hongkongensis, 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 |
| 10XDNase I buffer | 1.2μL |
| DNase I | 1μL |
| Sterile H2O | Up to 12μL |
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 caspase3 Gene
Sequencing the clone obtained in step 2, analyzing and determining caspase3 sequence by BLASTEN and other biological software, and performing 5'/3' RACE of the gene by GeneRacer TM RACE Ready cDNA kit (Invitrogen, USA) with the sequence as template, wherein the specific operation is described in the specification. After obtaining RACE sequence, obtaining the full-length cDNA of the gene through splicing, analyzing the coding region, wherein the nucleotide sequence of the coding region is shown as SEQ ID NO.1 and named as caspase3 gene, and the coded amino acid sequence is shown as SEQ ID NO.2 and named as caspase3 protein.
4. Real-time quantitative PCR
The kit used for real-time quantitative PCR is LightCycler 480SYBR Green I (Roch), the used instrument is LightCycler 480System (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 caspase3 gene: chcaspase3-F:5'-GGAGCGGTTTCAGGACTTGG-3'
Chcaspase3-R:5'-AGCATTCCGTCGGTAGCGTA-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 ℃ for 10s, 58.5 ℃ for 10s, 72 ℃ for 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
Total RNA of each tissue of 5 crassostrea hongkongensis is respectively extracted (see step 1) and is reversely transcribed into cDNA, and the distribution condition of caspase3 genes in lymphocytes, gills, touch lips, gonads, digestive glands, mantle, pericardial cavity and adductor muscles of the crassostrea hongkongensis is detected by adopting a Real-time PCR technology. The results showed that it was widely expressed in various tissues, with the lowest expression in the mantle and the highest expression in the haemolymph (FIG. 1).
Injecting normal Crassostrea hongkongensis into 1.0 × 109Live pathogens were randomly sampled at different time points (5 in one group) using the PBS injection group as a reference, total RNA from blood cells was extracted and reverse transcribed into cDNA, see step 1. The expression quantity of caspase3 gene is detected by Real-time PCR of a sample by taking the template as a template, and the result shows that the infection of vibrio alginolyticus (figure 2) and staphylococcus (figure 3) can cause the obvious up-regulation expression of the caspase3 gene in blood cells.
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
<120> oyster cell apoptosis gene caspase3 gene and application thereof in preparation of pathological detection diagnostic reagent
<160> 2
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<211> 891
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<213> Crassostera hongkongensis (Crassostra hongkongensis)
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aaagccagcg gagaccgcag atcggttcgt ccaggtgccc catcggagcc aaatgagtac 120
aactttaact atcacaaaag aggccttttt atcattatca ataacaagaa tttccatccg 180
tccaccggca aagagtcacg cgaagggact gacgtagacg cggagagact ggaggagcgg 240
tttcaggact tggggttcga tgttcgacgc tacaacgacg tgtcgtcatc caaactattg 300
caacttatga acgaagcctc acagcttgac cattccgatt ccgactgttt cggttgtgcc 360
attctgagtt atggaattga ggggagggtc tacgctaccg acggaatgct gcctttagac 420
gtcctcatcg ttcccttcaa gggagacaag tgcccgatgt tggttggcaa acccaaactt 480
ttttttctac agtcttgtcg ctgtccaaac ttagaacagt ctgttgaaga ctcggtgtcc 540
tataaaagct tcctgtccga ggattccgag cggagtttct ccgggatgag gaggatccca 600
gttgaggctg acttcttgtt cttctattcc acagtacctg gtttctattc atggcggaat 660
caccaggaag gatcctggct tatccaagcc ttgtgtatcg ttttggagaa ctatggttcc 720
aaaatggaac ttctgcacat gctcactcaa gtcaatcgca tggcggcgta tgaattcgag 780
tcgtgttcgg atgaacattt tacggatgag gtcaaacaga tgccttgtat tgtgtccatg 840
cttaccagat atgtgtactt ccggcctaag aaaccggata tccgggacta a 891
<210> 2
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<213> Crassostera hongkongensis (Crassostra hongkongensis)
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Leu Phe Ile Ile Ile Asn Asn Lys Asn Phe His Pro Ser Thr Gly Lys
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Glu Ser Arg Glu Gly Thr Asp Val Asp Ala Glu Arg Leu Glu Glu Arg
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Phe Gln Asp Leu Gly Phe Asp Val Arg Arg Tyr Asn Asp Val Ser Ser
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Ser Lys Leu Leu Gln Leu Met Asn Glu Ala Ser Gln Leu Asp His Ser
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Asp Ser Asp Cys Phe Gly Cys Ala Ile Leu Ser Tyr Gly Ile Glu Gly
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Arg Val Tyr Ala Thr Asp Gly Met Leu Pro Leu Asp Val Leu Ile Val
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Pro Phe Lys Gly Asp Lys Cys Pro Met Leu Val Gly Lys Pro Lys Leu
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Phe Phe Leu Gln Ser Cys Arg Cys Pro Asn Leu Glu Gln Ser Val Glu
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Asp Ser Val Ser Tyr Lys Ser Phe Leu Ser Glu Asp Ser Glu Arg Ser
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Phe Ser Gly Met Arg Arg Ile Pro Val Glu Ala Asp Phe Leu Phe Phe
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Tyr Ser Thr Val Pro Gly Phe Tyr Ser Trp Arg Asn His Gln Glu Gly
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Ser Trp Leu Ile Gln Ala Leu Cys Ile Val Leu Glu Asn Tyr Gly Ser
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Lys Met Glu Leu Leu His Met Leu Thr Gln Val Asn Arg Met Ala Ala
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Tyr Glu Phe Glu Ser Cys Ser Asp Glu His Phe Thr Asp Glu Val Lys
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Gln Met Pro Cys Ile Val Ser Met Leu Thr Arg Tyr Val Tyr Phe Arg
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Pro Lys Lys Pro Asp Ile Arg Asp
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Claims (4)
1. The application of a detection reagent for detecting the expression quantity of a caspase3 gene in preparing a reagent for preventing the oyster from being infected by alert vibrio alginolyticus or staphylococcus is disclosed, wherein the nucleotide sequence of the caspase3 gene is shown as SEQ ID NO.1, and the oyster is crassostrea hongkongensis.
2. The use of claim 1, wherein the agent is an agent for detecting oyster apoptosis.
3. The use of claim 2, 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.
The application of caspase3 gene as oyster apoptosis marker gene in preparing reagent for detecting apoptosis of crassostrea hongkongensis caused by infecting vibrio alginolyticus or staphylococcus, wherein the nucleotide sequence of the caspase3 gene is shown as SEQ ID NO. 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016065048A (en) * | 2014-09-17 | 2016-04-28 | 御木本製薬株式会社 | Caspase-14 expression accelerator |
| CN107540738A (en) * | 2017-04-10 | 2018-01-05 | 清远职业技术学院 | A kind of huge oyster Galectins ChGalectin in Hong Kong and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016065048A (en) * | 2014-09-17 | 2016-04-28 | 御木本製薬株式会社 | Caspase-14 expression accelerator |
| CN107540738A (en) * | 2017-04-10 | 2018-01-05 | 清远职业技术学院 | A kind of huge oyster Galectins ChGalectin in Hong Kong and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| ACCESSION：XP_011445227：PREDICTED: caspase-3 isoform X2 [Crassostrea gigas];Zhang G等;《Genbank》;20170202;Features和Origin * |
| Cloning, characterization and expression analysis of a caspase-8 like gene from the Hong Kong oyster, Crassostrea hongkongensis;Zhiming Xiang等;《Fish & Shellfish Immunology》;20130925;第35卷(第6期);1797-1803 * |
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