CN117568490A - Programme cell death protein molecular marker of penaeus vannamei boone and application thereof - Google Patents
Programme cell death protein molecular marker of penaeus vannamei boone and application thereof Download PDFInfo
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- 239000003147 molecular marker Substances 0.000 title claims abstract description 71
- 241000238553 Litopenaeus vannamei Species 0.000 title claims abstract description 58
- 108090000623 proteins and genes Proteins 0.000 title abstract description 13
- 102000004169 proteins and genes Human genes 0.000 title description 6
- 230000030833 cell death Effects 0.000 title description 2
- 238000012408 PCR amplification Methods 0.000 claims abstract description 17
- 238000009395 breeding Methods 0.000 claims abstract description 16
- 230000001488 breeding effect Effects 0.000 claims abstract description 16
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 claims abstract description 15
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 claims abstract description 15
- 238000000746 purification Methods 0.000 claims abstract description 5
- 210000003205 muscle Anatomy 0.000 claims abstract description 4
- 239000002773 nucleotide Substances 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 8
- 108010006785 Taq Polymerase Proteins 0.000 claims description 3
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- 238000009400 out breeding Methods 0.000 claims description 2
- 238000009394 selective breeding Methods 0.000 claims description 2
- 241000607272 Vibrio parahaemolyticus Species 0.000 abstract description 12
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 238000003782 apoptosis assay Methods 0.000 abstract description 3
- 230000005522 programmed cell death Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 239000003550 marker Substances 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 7
- 230000034994 death Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 238000012216 screening Methods 0.000 description 3
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 2
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- 238000009826 distribution Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
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- 238000012163 sequencing technique Methods 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
- 101710113864 Heat shock protein 90 Proteins 0.000 description 1
- 241000530454 Litopenaeus schmitti Species 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 101710089369 Programmed cell death protein 6 Proteins 0.000 description 1
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Abstract
The invention discloses a molecular marker of a programmed cell death protein of penaeus vannamei boone and application thereof, wherein the molecular marker comprises a molecular marker A, a molecular marker B, a molecular marker C and a molecular marker D which are positioned on a sequence of a Programmed cell death protein-like gene of penaeus vannamei boone; the molecular marker is used as a functional marker of the vibrio parahaemolyticus resistance of the penaeus vannamei, disease-resistant breeding of the penaeus vannamei is improved, specifically, genomic DNA of the penaeus vannamei muscle tissue to be detected is extracted, then the genomic DNA is used as template DNA for PCR amplification and PCR amplification product purification, and then the obtained products are sequenced, so that genotypes of the molecular marker A, the molecular marker B, the molecular marker C and the molecular marker D are determined, and therefore, dominant genotype individuals are selected for breeding the penaeus vannamei, and research and application of disease-resistant breeding of the penaeus vannamei are promoted.
Description
Technical Field
The invention belongs to the technical field of penaeus vannamei boone breeding, and in particular relates to a penaeus vannamei boone programmed cell death protein molecular marker and application thereof.
Background
Penaeus vannamei BooneLitopenaeus Vannamei) The white-skin shrimp, the white-shrimp and the litopenaeus vannamei have higher economic value and nutritive value, are warm in nature and sweet in taste, have the effects of tonifying kidney and strengthening yang, are deeply favored by the masses, and are important cultured shrimps in China at present. In recent years, bacterial diseases are frequently exploded in the cultivation of the penaeus vannamei boone, so that serious economic loss is caused for cultivation production, and the defect of disease prevention and control technology also causes various negative effects such as disordered use and abuse of antibiotics, spreading of drug-resistant strains and the like. Among them, vibriosis caused by vibrio parahaemolyticus (Vibrio parahaemolyticus) is the most common and most harmful bacterial disease in the cultivation process of penaeus vannamei boone.
Programmed death protein as one kind of pro-apoptosis factor and can inhibit protein translation, and is used in immunizing Penaeus vannamei Boone, chitinase, heat shock protein 90, etc. and O 2 The protein plays an important role in transportation, energy metabolism, molting, stress reaction and the like, so that programmed death proteins and the like are very likely to become biomarkers for researching the capability of the penaeus vannamei boone for resisting vibrio parahaemolyticus.
With the development of molecular biology and genotyping technologies, a molecular marker technology based on a character-related functional gene is one of key technologies for aquatic genetic breeding. Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP), i.e.DNA sequence polymorphism caused by variation of a single nucleotide at the genomic level. SNP has the advantages of large quantity, wide distribution, strong representativeness, good genetic stability, convenient high-throughput and high-automation detection analysis, etc. In the breeding of penaeus vannamei boone, SNP markers have been applied, molecular markers related to ammonia nitrogen resistance, nitrate resistance, growth characteristics and the like have been reported, but SNP markers related to vibrio parahaemolyticus resistance have been reported rarely.
Disclosure of Invention
Aiming at the defects, the invention discloses a molecular marker of programmed cell death protein of penaeus vannamei and application thereof, wherein the molecular marker is obtained by screening a related SNP (single nucleotide polymorphism) molecular marker based on programmed death protein gene (Programmed cell death protein-like) of penaeus vannamei, and the molecular marker is used as a functional marker of the capability of resisting vibrio parahaemolyticus of penaeus vannamei and is used for disease-resistant breeding of penaeus vannamei.
The invention is realized by adopting the following technical scheme:
the molecular marker of the programmed cell death protein of the penaeus vannamei comprises a molecular marker A, a molecular marker B, a molecular marker C and a molecular marker D;
the molecular marker A is positioned at a 4689 bp locus of a nucleotide sequence shown in a sequence 1 in a sequence table, and is marked as D.4689G > A, the base of the locus is G or A, and the mutation types are G/G homozygosity, A/G heterozygosity and A/A homozygosity;
the molecular marker B is positioned at a 4698 bp locus of a nucleotide sequence shown in a sequence 1 in a sequence table, and is marked as D.4698T > A, the base of the locus is T or A, and the mutation types are T/T homozygote, T/A heterozygote and A/A homozygote;
the molecular marker C is positioned at a 4742 bp locus of a nucleotide sequence shown in a sequence 1 in a sequence table, and is marked as D.4742T > G, the base of the locus is T or G, and the mutation type is T/T homozygosity and T/G heterozygosity;
the molecular marker D is positioned at a 4756 bp site of a nucleotide sequence shown in a sequence 1 in a sequence table, and is marked as D.4756G > T, the base of the site is G or T, and the mutation type is G/G homozygosity, G/T heterozygosity and T/T homozygosity.
The nucleotide sequence described in the sequence table 1 is the nucleotide sequence of the gene Programmed cell death protein-like.
The application of the molecular marker of the programmed cell death protein of the penaeus vannamei, wherein the molecular marker A, the molecular marker B, the molecular marker C and the molecular marker D are used for selective breeding of the penaeus vannamei, specifically, genomic DNA of the penaeus vannamei muscle tissue to be detected is extracted, then the genomic DNA is used as template DNA for PCR amplification and PCR amplification product purification, and then the obtained products are sequenced to determine genotypes of the molecular marker A, the molecular marker B, the molecular marker C and the molecular marker D;
when the genotype of the molecular marker A is AA genotype, selecting the individual as a backup parent to carry out the breeding of the penaeus vannamei boone variety; when the genotype of the molecular marker B is AA genotype, selecting the individual as a backup parent to carry out the breeding of the penaeus vannamei boone variety; when the genotype of the molecular marker C is TT genotype, selecting the individual as a backup parent to carry out breeding of the penaeus vannamei boone variety; when the genotype of the molecular marker D is GG genotype, selecting the individual as a backup parent to carry out the breeding of the penaeus vannamei boone variety.
In the PCR amplification and PCR amplification product purification process, a primer group for detecting the molecular marker of the programmed cell death protein of the penaeus vannamei boone comprises a primer F and a primer R;
the sequence of the primer F is as follows: GAATGTTTGACAAAAAAGGCTCCG (sequence 2 in the sequence Listing);
the primer R has a sequence of ATGGAGGACAATACAGCACTGAA (sequence 3 in the sequence table).
The PCR amplification system consists of the following components: 2.0. Mu.L of 10 XTaq buffer, 0.4. Mu.L of dNTP (10 mmol/L each), 0.2. Mu.L of Taq DNA polymerase (5U/. Mu.L), 0.5. Mu.L of primer F, 0.5. Mu.L of primer R, 14.4. Mu.L of ddH 2 O, 2.0. Mu.L of template DNA.
The reaction procedure for PCR amplification comprises the following steps:
s1, pre-denaturation at 95 ℃ for 5min;
s2, carrying out denaturation at 95 ℃ for 30S, annealing at 60 ℃ for 30S and extension at 72 ℃ for 30S, and carrying out 34 cycles;
s3, extending for 5min at 72 ℃.
Compared with the prior art, the technical scheme has the following beneficial effects:
the invention provides a SNP molecular marker closely related to the anti-vibrio parahaemolyticus character of penaeus vannamei, which can be applied to the breeding of new anti-vibrio parahaemolyticus varieties of penaeus vannamei, and is beneficial to promoting the research and application of disease-resistant cultivation of penaeus vannamei. The method for breeding the vibrio parahaemolyticus-resistant penaeus vannamei boone has the advantages that the bred individuals are stable in genotype and do not generate genetic differentiation, the breeding efficiency and accuracy are high, and a good foundation is provided for the breed cultivation and improvement research of the penaeus vannamei boone.
Drawings
FIG. 1 shows the chi-square test results of SNP locus genes and genotype frequencies of the apoptosis proteins of the invention.
Description of the embodiments
The invention is further illustrated by the following examples, which are not intended to be limiting. The specific experimental conditions and methods not specified in the following examples are generally conventional means well known to those skilled in the art.
Examples: the invention discloses screening of a molecular marker of programmed cell death protein of penaeus vannamei boone, which comprises the following specific processes:
(1) 245 penaeus vannamei boone weighing about 20 g are selected for 5 days temporary rearing, and then the penaeus vannamei boone individual injection concentration is 7 multiplied by 10 6 cfu/mL of Vibrio parahaemolyticus 100. Mu.L; in order to eliminate death caused by injection factors, beginning to record dead individuals after 6 hours of toxicity attack, and respectively selecting 60 penaeus vannamei boone which die first and survive after 96 hours as samples of a vibrio parahaemolyticus sensitive group and a vibrio parahaemolyticus tolerant group of the penaeus vannamei boone;
(2) Randomly selecting 5 penaeus vannamei boone from a sensitive group and a tolerant group respectively, extracting muscle tissues, extracting genome DNA by adopting a conventional phenol imitation extraction method, and storing the obtained genome DNA at a temperature of-20 ℃ for later use;
(3) Designing a primer F and a primer R according to a gene sequence (gene accession number: LOC 113805132) of the penaeus vannamei boone Programmed cell death protein, and then amplifying and screening SNP loci positioned in Programmed cell death protein 6 genes;
the sequence of the primer F is as follows: GAATGTTTGACAAAAAAGGCTCCG;
the sequence of the primer R is ATGGAGGACAATACAGCACTGAA;
the PCR amplification system consists of the following components: 2.0. Mu.L of 10 XTaq buffer, 0.4. Mu.L of dNTP (10 mmol/L each), 0.2. Mu.L of Taq DNA polymerase (5U/. Mu.L), 0.5. Mu.L of primer F, 0.5. Mu.L of primer R, 14.4. Mu.L of ddH 2 O, 2.0. Mu.L of template DNA;
the reaction procedure for PCR amplification comprises the following steps:
s1, pre-denaturation at 95 ℃ for 5min;
s2, carrying out denaturation at 95 ℃ for 30S, annealing at 60 ℃ for 30S and extension at 72 ℃ for 30S, and carrying out 34 cycles;
s3, extending for 5min at 72 ℃;
(4) Purifying and sequencing the PCR amplified product after 1% agarose gel electrophoresis detection, and comparing and analyzing the sequencing result by DNAstar software, wherein the comparison of nucleotide sequences and peak diagram analysis are included, and relevant SNPs loci are screened out; the PCR amplification product sequence of one of the samples is shown below, where position 212 is D.4689G > A, position 221 is D.4698T > A, position 265 is D.4742T > G, and position 279 is D.4756G > T:
GAATGTTTGACAAAAAAGGCTCCGGAACAATTAACTTTGATGAATTTGGAGCGGTCTGGAAGTATGTAACAGACTGGCAGCAGTGCTTTAGGTCATTTGATCGAGATGGTTCTGGCAACATTAGTAAAGATGAATTAAAGACTGCTATCACTACATTTGGTTACAGGTGAGTGAAAATGTCTAATCACAAATGCATCTTCATATTTTAGATGTTTGATTGTAGTGTAAAAAGGGAAAATTGGATTAATAGCTATGCATTTTAGTTTGTGACAGTTACAGTTATTTGCAGTTGTATATGTTTAGAGCATTGTACATGATACATTACTTTTAGTTGATACAATTTATTATGTGCGGAAATTATTTTTTTATCATTATTTCATTAACAAAATACATTTAAAGATCCACTGTTTTGTGCTAATGTATTTTTATATTTATTTTTAGGTTTTCAGAGAAGTTCTATGATGTCCTCATGCAACGTTTTGACAGAACTGGAAGAGGTGGAGTTTACTTCGATGACTTCATTCAGTGCTGTATTGTCCTCCAT；
(5) According to the screened SNPs loci, detecting and genotyping the penaeus vannamei boone in the sensitive group and the tolerant group respectively according to the method, counting samples of different SNP loci in the sensitive group and the tolerant group, calculating genotype frequency and allele frequency, and carrying out independent test by chi-square analysis, wherein the specific result is shown in figure 1.
According to FIG. 1Analysis, D.4689G>A、D.4698 T>A、D.4742 T>G and D.4756G>The distribution of the 4 different genotypes of AA, TT and GG corresponding to the four T sites was significantly correlated with the presence of Vibrio parahaemolyticus resistance (x 2 =6.2206，P=0.0446）、（x 2 =8.0000，P=0.0183）、（x 2 =8.6195，P=0.0033）、（x 2 =6.2471, p= 0.0440), and the 4 genotypes of individuals have better anti-vibrio parahaemolyticus ability compared with other genotypes, and can be preferentially used as parents for breeding of penaeus vannamei vibrio parahaemolyticus resistance.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (5)
1. A molecular marker for programmed cell death protein of penaeus vannamei boone, which is characterized in that: the molecular marker of the programmed cell death protein of the penaeus vannamei comprises a molecular marker A, a molecular marker B, a molecular marker C and a molecular marker D;
the molecular marker A is positioned at a 4689 bp locus of a nucleotide sequence shown in a sequence 1 in a sequence table, and the base of the locus is G or A;
the molecular marker B is positioned at a 4698 bp locus of a nucleotide sequence shown in a sequence 1 in a sequence table, and the base of the locus is T or A;
the molecular marker C is positioned at a 4742 bp locus of a nucleotide sequence shown in a sequence 1 in a sequence table, and the base of the locus is T or G;
the molecular marker D is positioned at the 4756 bp locus of the nucleotide sequence shown in the sequence 1 in the sequence table, and the base of the locus is G or T.
2. The use of the molecular marker of programmed cell death protein of penaeus vannamei boone as claimed in claim 1, wherein: the application is that the molecular marker A, the molecular marker B, the molecular marker C and the molecular marker D are used for selective breeding of the penaeus vannamei, specifically, genomic DNA of the penaeus vannamei muscle tissue to be detected is extracted, then the genomic DNA is used as template DNA for PCR amplification and PCR amplification product purification, and then the obtained products are sequenced to determine genotypes of the molecular marker A, the molecular marker B, the molecular marker C and the molecular marker D;
when the genotype of the molecular marker A is AA genotype, selecting the individual as a backup parent to carry out the breeding of the penaeus vannamei boone variety; when the genotype of the molecular marker B is AA genotype, selecting the individual as a backup parent to carry out the breeding of the penaeus vannamei boone variety; when the genotype of the molecular marker C is TT genotype, selecting the individual as a backup parent to carry out breeding of the penaeus vannamei boone variety; when the genotype of the molecular marker D is GG genotype, selecting the individual as a backup parent to carry out the breeding of the penaeus vannamei boone variety.
3. The use of the molecular marker of programmed cell death protein of penaeus vannamei boone as claimed in claim 2, wherein: in the PCR amplification and PCR amplification product purification processes, the primer group for detecting the molecular marker of the programmed cell death protein of the penaeus vannamei boone comprises a primer F and a primer R;
the sequence of the primer F is as follows: GAATGTTTGACAAAAAAGGCTCCG;
the sequence of the primer R is ATGGAGGACAATACAGCACTGAA.
4. The use of the molecular marker of programmed cell death protein of penaeus vannamei boone as claimed in claim 2, wherein: the PCR amplification system consists of the following components: 2.0. Mu.L of 10 XTaq buffer, 0.4. Mu.L of dNTP, 0.2. Mu.L of Taq DNA polymerase, 0.5. Mu.L of primer F, 0.5. Mu.L of primer R, 14.4. Mu.L of ddH 2 O, 2.0. Mu.L of template DNA.
5. The use of the molecular marker of programmed cell death protein of penaeus vannamei boone as claimed in claim 2, wherein: the reaction procedure for PCR amplification comprises the following steps:
s1, pre-denaturation at 95 ℃ for 5min;
s2, carrying out denaturation at 95 ℃ for 30S, annealing at 60 ℃ for 30S and extension at 72 ℃ for 30S, and carrying out 34 cycles;
s3, extending for 5min at 72 ℃.
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