CN114574597B - SNP marker on chitin binding protein gene related to vibrio parahaemolyticus infection resistance of litopenaeus vannamei and application thereof - Google Patents
SNP marker on chitin binding protein gene related to vibrio parahaemolyticus infection resistance of litopenaeus vannamei and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP marker on chitin binding protein genes related to anti-vibrio parahaemolyticus infection of litopenaeus vannamei and application thereof. The SNP marker is positioned at a 501bp locus of a litopenaeus vannamei chitin binding protein gene sequence shown as SEQ ID NO.1, mutation types are T homozygote, T/A heterozygote and A homozygote, and the dominant genotype of the marker for resisting vibrio parahaemolyticus infection is T homozygote. The invention also discloses an application method of the marker for carrying out auxiliary breeding of the anti-vibrio parahaemolyticus infection character marker by utilizing the marker. The invention provides a method for effectively developing the genetic selection and breeding of the molecular marker-assisted vibrio parahaemolyticus infection resistance character of the litopenaeus vannamei, which has the advantages of high efficiency, simple operation, stable genotype of the selected individuals, no genetic differentiation, capability of remarkably accelerating the progress of disease resistance breeding and great guiding significance for improving the disease resistance breeding of the litopenaeus vannamei.
Description
Technical Field
The invention belongs to the technical field of aquaculture biology, and particularly relates to detection of Single Nucleotide Polymorphism (SNP), in particular to an SNP molecular marker which is generated by causing amino acid sequence change on a litopenaeus vannamei chitin binding protein gene, and application of the SNP molecular marker in anti-vibrio parahaemolyticus infection parent and shrimp larvae identification and disease resistance breeding.
Background
Litopenaeus vannamei (Litopenaeus vannamei, commonly known as Penaeus vannamei Boone) is a main species of first-cultured shrimps in the world and in China, and has great effect on meeting the requirements of people on high-quality protein foods. According to the year's own statistics of 2021, the annual output of Litopenaeus vannamei in China in 2020 is 186 ten thousand tons, which accounts for 86% of the annual output of the national prawns, the output of the young prawns is 15000 hundred million, and the market demand for improved variety is huge. However, in recent years, bacterial diseases frequently outbreak cause great economic losses to the production of the culture; on the other hand, the popularity of bacterial diseases and the shortage of effective prevention and control techniques also cause the misuse and abuse of antibiotics, and generate a plurality of negative effects such as drug residues, environmental pollution, drug-resistant strain spreading and the like. Therefore, starting from the germplasm level, the antibacterial germplasm of the litopenaeus vannamei is deeply excavated and utilized, and the selection of a new excellent variety with strong antibacterial capability and the selection of seedlings with strong antibacterial capability for cultivation production become important points and hot points for prevention and control research and development of bacterial diseases.
Bacterial disease is one of the most serious diseases, while vibriosis is the most common and most harmful bacterial disease in the shrimp culture process. Vibrio parahaemolyticus (Vibrio parahaemolyticus) is the earliest vibrio pathogen found by people and is the main conditional pathogenic bacteria of litopenaeus vannamei. One of the most serious diseases in prawn culture in recent years, "acute hepatopancreatic necrosis syndrome (AHPND)" is caused by Vibrio parahaemolyticus carrying virulence genes Pir A and Pir B. In 2009, the first outbreak of litopenaeus vannamei AHPND in china was reported, followed by successive outbreaks in mexico, ma Laxi subunit, thailand, philippines, vietnam, bangladesh and the united states. The specific symptoms are that the hepatopancreas is pale, the liver is atrophic, the liver is fallen off, the jejunum is empty, the death rate reaches 100% within 1-3 days, and great loss is caused for the breeding production.
Due to the limitation of hard epidermis, crustaceans must undergo molting to develop and grow. The Litopenaeus vannamei is subjected to 35-37 ecdysis, and the immunity decline after each ecdysis is the period that the vibrio is most easy to invade. Chitin as a component of epidermis exists in a combined form with protein, namely chitin binding protein (Chitin binding protein, CBP), which is an important structural protein in the epidermis of crustaceans, and the protein generally contains a characteristic structural domain, and a compound formed by the chitin not only provides an organic scaffold for calcification, but also participates in controlling the degree of calcification, and has an important effect on the vibrio infection resistance of post-ecdysis litopenaeus vannamei.
With the rapid transformation of modern biotechnology and genetic technology in the field of aquatic products, a molecular marker technology based on character-related functional genes becomes one of key technologies for aquatic product genetic breeding. SNP is used as a genetic marker, has the advantages of high occurrence frequency, stable inheritance, easy automatic detection and the like, and has difference in SNP distribution among different species and varieties. In the breeding of aquatic animal seedlings and genetic breeding, SNP markers have been used for many years, and some molecular markers related to growth, propagation, disease resistance and the like are successively screened. The invention aims at establishing a method for molecular marker assisted disease resistance breeding of litopenaeus vannamei by exploring SNP markers related to the anti-vibrio parahaemolyticus infection characters of litopenaeus vannamei and applying the markers.
Disclosure of Invention
The invention aims to provide an SNP marker on a chitin binding protein gene related to the anti-vibrio parahaemolyticus infection character of litopenaeus vannamei and application thereof, thereby realizing molecular marker disease resistance breeding of litopenaeus vannamei.
The first object of the invention is to provide a SNP molecular marker related to the anti-vibrio parahaemolyticus infection character of litopenaeus vannamei.
The invention discovers polymorphism of the locus of the litopenaeus vannamei chitin binding protein gene (the nucleotide sequence of which is shown as SEQ ID NO. 1). The locus is positioned at 501bp of a chitin binding protein gene, the base is T or A, the survival rate of Litopenaeus vannamei homozygous for T (T/T) is obviously higher than that of T/A heterozygosis (T/A) under the infection condition of vibrio parahaemolyticus, and the T/A heterozygosis is obviously higher than that of A homozygosis (A/A). The SNP locus is a Litopenaeus vannamei individual with a T/T genotype, has good capability of resisting vibrio parahaemolyticus infection, and can be stably inherited in offspring; the SNP locus is a Litopenaeus vannamei individual with a T/A genotype, has better anti-vibrio parahaemolyticus infection capacity, but the character can not be inherited stably in offspring; the SNP locus is of an A/A genotype of a litopenaeus vannamei individual, and has poor capability of resisting infection of vibrio parahaemolyticus. Therefore, the dominant genotype of the SNP molecular marker against vibrio parahaemolyticus infection is T homozygosity (T/T).
Therefore, the invention provides SNP molecular markers for distinguishing the infection capability of Litopenaeus vannamei against vibrio parahaemolyticus, which are positioned at 501bp sites of Litopenaeus vannamei chitin binding protein genes (the nucleotide sequences of which are shown as SEQ ID NO. 1), and are homozygous for mutation types T (T/T), T/A heterozygous (T/A) and A homozygous (A/A).
The second object of the invention is to provide a detection primer for distinguishing SNP molecular markers related to the anti-vibrio parahaemolyticus infection character of litopenaeus vannamei, which comprises the following primers:
LvCBP-SNP-F：5'-CCAAGGCAATGAAACCCTCAGACA-3'；
LvCBP-SNP-R：5'-TCATTCATCCATCACCATAAGT-3'。
the detection primer has stable amplification and good repeatability. The nucleotide sequence of the amplified product is shown as SEQ ID NO.2, the SNP locus is located at the 324bp position of the amplified sequence, and is a mutation from T to A, and the marked dominant genotype of resisting the infection of the vibrio parahaemolyticus is T homozygosity (T/T).
The third object of the invention is to provide the application of the SNP molecular labeled product in the preparation of the reagent for distinguishing the anti-vibrio parahaemolyticus infection capability of the litopenaeus vannamei.
The fourth object of the invention is to provide the application of the SNP molecular marker or the detection primer in preparing a detection kit for distinguishing the anti-vibrio parahaemolyticus infection capability of litopenaeus vannamei.
The fifth object of the invention is to provide a detection kit for distinguishing the infection capacity of Litopenaeus vannamei against Vibrio parahaemolyticus, which comprises the detection primer.
The sixth object of the invention is to provide the application of the SNP molecular marker, the detection primer or the detection kit in distinguishing the anti-vibrio parahaemolyticus infection capability of the litopenaeus vannamei.
The anti-vibrio parahaemolyticus infection capability of the litopenaeus vannamei with the SNP molecular marker being of a T homozygous genotype is obviously higher than that of the litopenaeus vannamei with the SNP molecular marker being of a T/A heterozygous genotype, and the anti-vibrio parahaemolyticus infection capability of the litopenaeus vannamei with the SNP molecular marker being of a T/A heterozygous genotype is also obviously higher than that of the litopenaeus vannamei with the SNP molecular marker being of an A homozygous genotype.
The seventh object of the invention is to provide the application of the SNP molecular marker, the detection primer or the detection kit in the identification of the parent shrimp of the litopenaeus vannamei with the anti-vibrio parahaemolyticus infection character, the group breeding or the maintenance of the high-resistance strain of the litopenaeus vannamei with the anti-vibrio parahaemolyticus infection character.
The eighth object of the invention is to provide a breeding method of a variety of Litopenaeus vannamei for resisting Vibrio parahaemolyticus infection, which comprises the following steps:
a) Extracting genomic DNA of Litopenaeus vannamei to be detected;
b) Performing PCR amplification on genomic DNA of litopenaeus vannamei to be detected by using the detection primer LvCBP-SNP-F/LvCBP-SNP-R as set forth in claim 2;
c) Performing Sanger sequencing on the amplified product by using LvCBP-SNP-F primer, and performing SNP typing on the relevant sites of the anti-vibrio parahaemolyticus infection character according to a sequencing peak diagram;
d) Female shrimps and male shrimps homozygous for T are selected as parents, and mating is performed to generate a next-generation breeding population.
Preferably, the PCR amplification reaction system comprises 25 mu L: does not contain Mg 2+ 10 XPCR buffer 2.5. Mu.L, 25mM MgCl 2 2.0. Mu.L, 10mM dNTP 2.0. Mu.L, high-fidelity PCR enzyme 1U 0.5. Mu.L, 10. Mu.M forward primer 0.5. Mu.L, 10. Mu.M reverse primer 0.5. Mu. L, DNA template 12.5ng 0.5. Mu.L, the remainder being made up to 25. Mu.L by sterile double distilled water.
Preferably, the PCR amplification is performed by the following reaction procedures: pre-denaturation at 95 ℃ for 5 min; denaturation at 95℃for 30 seconds, annealing at 60℃for 30 seconds, extension at 72℃for 30 seconds, 35 cycles total; the extension was carried out at 72℃for a further 6 minutes.
Preferably, the PCR amplification product is subjected to Sanger sequencing using an ABI 3730xl DNA Analyzer sequencer.
The invention provides a method for effectively developing the genetic selection and breeding of the molecular marker-assisted vibrio parahaemolyticus infection resistance character of the litopenaeus vannamei, which has the advantages of high efficiency, simple operation, stable genotype of the selected individuals, no genetic differentiation, capability of remarkably accelerating the progress of disease resistance breeding and great guiding significance for improving the disease resistance breeding of the litopenaeus vannamei.
Drawings
FIG. 1 is a T-homozygote, A-homozygote and T/A heterozygote peak map of the litopenaeus vannamei chitin binding protein genes LvCBP-SNP-F and LvCBP-SNP-R primer pairs at positions 324bp (three sequencing peak maps at positions 324bp are shown as 280, 290 and 300bp, respectively, because the reading of tens of bases before capillary Sanger sequencing is inaccurate).
FIG. 2 is an alignment of the amino acid sequences encoded by two genotypes of the chitin-binding protein gene of Litopenaeus vannamei.
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention will be further described in detail with reference to examples. It should be understood that the embodiments described in this specification are only for explaining the present invention, and are not intended to limit the present invention, and parameters, proportions, etc. of the embodiments may be selected according to the circumstances without materially affecting the results. The examples are, unless otherwise indicated, all the reagents and method steps conventional in the art.
Example 1
In a Zhanjiang certain prawn farm, a plurality of tails of litopenaeus vannamei are collected, the swimming feet are respectively taken to extract genome DNA of each litopenaeus vannamei (the tissue is taken to not influence the survival of the litopenaeus vannamei), and LvCBP-SNP-F and LvCBP-SNP-R primer pairs are utilized to amplify, and the specific method is as follows:
amplification primers:
LvCBP-SNP-F：5'-CCAAGGCAATGAAACCCTCAGACA-3'；
LvCBP-SNP-R：5'-TCATTCATCCATCACCATAAGT-3'。
amplification system:
the PCR amplification reaction procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95℃for 30 seconds, annealing at 60℃for 30 seconds, extension at 72℃for 30 seconds, 35 cycles total; the extension was carried out at 72℃for a further 6 minutes. The PCR amplified products were sequenced using LvCBP-SNP-F primers using the ABI 3730xl DNA Analyzer sequencer for Sanger sequencing.
The result shows that: the litopenaeus vannamei bred in the place contains 3 mutation types at 501bp positions (the positions of the LvCBP-SNP-F and the LvCBP-SNP-R primer pair amplification 324 bp) of the chitin binding protein gene, wherein the mutation types are respectively T homozygosity, A homozygosity and T/A heterozygosity, and the sequencing peak diagram is shown in figure 1. When the site is T, the 153 th amino acid of the coding protein sequence is asparagine (N); when this position is A, the 153 th amino acid of the encoded protein sequence is isoleucine (I). The two amino acid sequences are aligned as shown in FIG. 2.
Example 2
500 litopenaeus vannamei boone at 7-8 cm in Zhanjiang prawn farm, extracting genome DNA of each litopenaeus vannamei boone by taking swimming feet, and amplifying and sequencing by using the primer pair described in the invention according to the method of the embodiment 1. Thus, the 202 tail of the T homozygous genotype at 501bp position of the chitin binding protein gene, the 243 tail of the A homozygous genotype and the 55 tail of the T/A heterozygous genotype are obtained through identification.
Vibrio parahaemolyticus is separated from acute hepatopancreatic necrosis syndrome Litopenaeus vannamei hepatopancreas by south China academy of sciences of sea, and sea. The strain is preserved in 25% glycerol at-80 ℃, streaked on LB solid medium for culture, inoculated on LB liquid medium for shake culture at 30 ℃ for 24 hours. The bacterial liquid was centrifuged at 3000g for 10 minutes, and the precipitated bacterial cells were washed 3 times with PBS buffer and suspended in an equal volume. The concentration of the bacterial liquid is measured by a spectrophotometer at 560nm (OD 560), and the concentration of the vibrio parahaemolyticus strain is determined by a plate coating experiment to be: 1.43X 10 per OD560 9 cfu/mL. The 500-tail litopenaeus vannamei with the physical size of 7.42 multiplied by 0.21cm is temporarily cultured for 2 weeks, no disease and death symptoms are found, and the litopenaeus vannamei is fasted for one day before a toxicity attack experiment. Placing Litopenaeus vannamei at 1m 3 In a black large plastic barrel, raising the water with the salinity of 30 per mill and the water temperature of 30 ℃. Adding Vibrio parahaemolyticus into black large plastic barrel containing Litopenaeus vannamei, and soaking to obtain final concentration of 2.86×10 5 cfu/mL, dead individuals immediately remove the water quality for preventing pollution in the process of attacking toxin, and survival rate is calculated after 48 hours of attacking toxin. By the toxicity attack experiment of Vibrio parahaemolyticus, 144 tails of T homozygous genotype survived (survival rate 71.3%), 29 tails of T/A heterozygous genotype survived (survival rate 52.7%), and 80 tails of A homozygous genotype survived (survival rate 32.9%).
The survival rate of the T-homozygous Litopenaeus vannamei is obviously higher than that of T/A heterozygous and the T/A heterozygous is obviously higher than that of A homozygous under the infection condition of vibrio parahaemolyticus, and the marked dominant genotype of resisting the infection of the vibrio parahaemolyticus is T homozygosity.
Example 3
The genome DNA of each litopenaeus vannamei is extracted from the swimming feet of the litopenaeus vannamei parent 25-28cm in the standard of the seed field of the litopenaeus vannamei, and amplified and sequenced by using the primer pair described in the invention according to the method of the embodiment 1. Respectively obtaining a pair of T/T homozygous genotypes and T/A heterozygous genotypes and A/A homozygous genotypes of parent shrimps for breeding. After the first generation of isotactic family juvenile shrimps grow to 3cm, 300 families are taken out for each family to carry out a vibrio parahaemolyticus toxicity attack experiment (same as in example 2). Wherein, the parent T/T homozygous genotype parent shrimp produces a young shrimp survival 143 tail (47.7%); the parent T/A heterozygote genotype parent shrimp produces 75 larvae (25.0%); parent A/A homozygous genotype parent shrimp produced 32 postlarvae (10.7%). Therefore, the SNP locus is a litopenaeus vannamei individual with a T/T genotype, has better anti-vibrio parahaemolyticus infection capacity, and can be stably inherited in offspring; the SNP locus is of a T/A genotype of a litopenaeus vannamei individual, and the infection capability of the offspring against vibrio parahaemolyticus is poor; the SNP locus is the individual of the Litopenaeus vannamei with A/A genotype, and the capability of generating offspring to resist infection of vibrio parahaemolyticus is extremely poor. Therefore, the screening and identification of the Litopenaeus vannamei individual with the SNP locus of T/T genotype has very important application value in the identification of Litopenaeus vannamei parent shrimps with anti-Vibrio parahaemolyticus infection characters, group breeding and maintenance of high-resistance strain of Litopenaeus vannamei with anti-Vibrio parahaemolyticus.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the present invention.
Sequence listing
<110> national academy of sciences of China, south sea and sea research institute
<120> SNP marker on chitin binding protein gene related to anti-Vibrio parahaemolyticus infection of Litopenaeus vannamei and application thereof
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ccgtgcatat cggcttgcac gcggccttgg agggcggagg ggaggagaga ggaaggtgaa 660
ggcggacccg agagaggaag gcggcgaagg gaagcgccgc tgggtcggat ctcttctttc 720
gctcacagtg cttttggtga ggagagagag accgtactgt cacttatggt gatggatgaa 780
tga 783
Claims (5)
1. Application of identifying SNP molecular marker products related to the anti-vibrio parahaemolyticus infection character of litopenaeus vannamei in preparing reagents for distinguishing the anti-vibrio parahaemolyticus infection capability of litopenaeus vannamei; the SNP molecular marker is positioned at a 501bp locus of a litopenaeus vannamei chitin binding protein gene sequence shown as SEQ ID NO.1, and mutation types are T homozygosity, T/A heterozygosity and A homozygosity.
2. The use according to claim 1, wherein the detection primers for the SNP molecular markers are:
LvCBP-SNP-F：5'-CCAAGGCAATGAAACCCTCAGACA-3'；
LvCBP-SNP-R：5'-TCATTCATCCATCACCATAAGT-3'。
3. the use according to claim 1, wherein the anti-vibrio parahaemolyticus infection ability of the litopenaeus vannamei with the SNP molecular marker being the T homozygous genotype is significantly higher than that of the T/A heterozygous genotype, and the anti-vibrio parahaemolyticus infection ability of the litopenaeus vannamei with the SNP molecular marker being the T/A heterozygous genotype is also significantly higher than that of the A homozygous genotype.
4. Application of identifying SNP molecular marker products related to the anti-vibrio parahaemolyticus infection character of the litopenaeus vannamei in group breeding or high-resistance strain maintenance of the anti-vibrio parahaemolyticus infection litopenaeus vannamei; the SNP molecular marker is positioned at a 501bp locus of a litopenaeus vannamei chitin binding protein gene sequence shown as SEQ ID NO.1, and mutation types are T homozygosity, T/A heterozygosity and A homozygosity.
5. A breeding method of a Litopenaeus vannamei anti-vibrio parahaemolyticus infection variety is characterized by comprising the following steps:
a) Extracting genomic DNA of Litopenaeus vannamei to be detected;
b) Using the detection primer LvCBP-SNP-F:5'-CCAAGGCAATGAAACCCTCAGACA-3' and LvCBP-SNP-R:5'-TCATTCATCCATCACCATAAGT-3' PCR amplification is carried out on the genomic DNA of the litopenaeus vannamei to be detected;
c) Carrying out Sanger sequencing on the amplified product by using an LvCBP-SNP-F primer, and typing SNP molecular markers related to the anti-vibrio parahaemolyticus infection character of the litopenaeus vannamei according to a sequencing peak diagram, wherein the SNP molecular markers are positioned at a 501bp site of a chitin binding protein gene sequence of the litopenaeus vannamei as shown in SEQ ID NO.1, and the mutation types are T homozygosity, T/A heterozygous and A homozygosity;
d) Female shrimps and male shrimps homozygous for T are selected as parents, and mating is performed to generate a next-generation breeding population.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103450352A (en) * | 2013-08-30 | 2013-12-18 | 中国农业科学院饲料研究所 | Chitin-binding protein CBP21 and encoding genes and applications thereof |
| CN110791571A (en) * | 2019-11-15 | 2020-02-14 | 中国科学院南海海洋研究所 | SNP marker for distinguishing Vibrio harveyi infection resistance of litopenaeus vannamei, and detection method and application thereof |
| CN112114150A (en) * | 2019-06-19 | 2020-12-22 | 欧蒙医学实验诊断股份公司 | Method and product for diagnosing seafood allergy |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103450352A (en) * | 2013-08-30 | 2013-12-18 | 中国农业科学院饲料研究所 | Chitin-binding protein CBP21 and encoding genes and applications thereof |
| CN112114150A (en) * | 2019-06-19 | 2020-12-22 | 欧蒙医学实验诊断股份公司 | Method and product for diagnosing seafood allergy |
| CN110791571A (en) * | 2019-11-15 | 2020-02-14 | 中国科学院南海海洋研究所 | SNP marker for distinguishing Vibrio harveyi infection resistance of litopenaeus vannamei, and detection method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 凡纳滨对虾不同免疫组织在弧菌和WSSV免疫刺激早期的响应机制;王富轩;中国博士学位论文全文数据库 农业科技辑(第1期);D052-41 * |
| 日本沼虾表皮几丁质结合蛋白基因的克隆以及KK-42对其表达的影响;张宇;王佩;吕艳杰;宁黔冀;;河南师范大学学报(自然科学版)(第05期);全文 * |
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