Litopenaeus vannamei disease resistance related EST-SSR molecular marker and application thereof

A technology of molecular markers and disease-resistance-related genes, which is applied in the fields of climate change adaptation, DNA/RNA fragments, and microbial detection/inspection, can solve problems that have not been studied or reported, and achieve the effect of broad application prospects

Active Publication Date: 2018-10-02
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no studies or reports on EST-STR markers related to disease resistance of Litopenaeus vannamei

Method used

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  • Litopenaeus vannamei disease resistance related EST-SSR molecular marker and application thereof
  • Litopenaeus vannamei disease resistance related EST-SSR molecular marker and application thereof
  • Litopenaeus vannamei disease resistance related EST-SSR molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Discovery of SSR loci of functional genes related to disease resistance of Litopenaeus vannamei and design of primers for EST-SSR molecular markers

[0051] The inventor used the microsatellite search software SSR Hunter to analyze wxya Gene EST sequence to obtain core repeat sequence (CT) X , x = 10-30, when x = 19, the nucleotide sequence of the LvIRF-SSR is shown in SEQ ID NO.1.

[0052] Use the primer design software PrimerPrimer 5.0 to design primers for the upstream and downstream of the core repeat sequence of the above sequence, and obtain specific amplification containing the SSR site. wxya Specific amplification primers for gene 5'UTR sequence: LvIRF-5'UTR:

[0053] Plus strand 5'-ATCGGGATCCACTCGCAGAT-3' (SEQ ID NO: 2),

[0054] Negative strand 5'-GGCGACCTTAGACCGACGAG-3' (SEQ ID NO: 3); annealing temperature 56°C.

Embodiment 2

[0055] Example 2 Verification of the effect of SSR site polymorphisms on the expression of LvIRF at the cellular level

[0056] 1. Litopenaeus vannamei genomic DNA was extracted according to the instructions of OMEGA Animal Tissue DNA Extraction Kit. Use a Nanovue spectrophotometer to measure the DNA purity and concentration of the extracted DNA. The purity OD260 / 280 is 1.7~1.9, and the concentration is >100 ng / μl. Then it is detected by 1% agarose gel electrophoresis, and the integrity of the DNA fragment is detected by electrophoresis. Qualified DNA samples were stored at -20 °C until use.

[0057] 2. The total RNA of Litopenaeus vannamei was extracted according to the instructions of QIAGEN Animal Tissue RNA Extraction Kit. The purity and concentration of RNA were determined according to the above method, and the purity OD260 / 280 was 1.9-2.0. Qualified RNA samples were synthesized into cDNA using the Takara reverse transcription kit, and the operation was performed accord...

Embodiment 3

[0071] Example 3 Effect of LvIRF SSR site polymorphism on shrimp mortality

[0072] 1. Collect 120 prawns from two different sources from the breeding base, put them in the culture tank for 24 hours, and inject 10 μl of WSSV virus solution after the prawns are in a stable state (the content of WSSV virus is 10 5 copies / μl), the dead shrimps were collected every 4 h after the challenge, the dead shrimp within 96 h after the challenge were marked as group A, and the surviving and stable shrimp after 96 h after the challenge were marked as group B. The genomic DNA of prawns was extracted and the purity and concentration were detected according to the above method.

[0073] 2. Use the primer design software PrimerPrimer 5.0 to design primers for the upstream and downstream of the core repeat sequence of the above sequence, and obtain wxya SSR site primer nucleotide sequence: LvIRF-SSR:

[0074] Plus strand 5'-GATCCACTCGCAGATACAGAT-3' (SEQ ID NO: 8),

[0075] Negative strand...

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Abstract

The invention discloses a litopenaeus vannamei disease resistance related EST-SSR molecular marker and the application thereof. An SSR site is identified in a 5'-UTR area of litopenaeus vannamei disease resistance related genes LvIRF, specific primers are designed according to sequence characteristics of the area, and by virtue of PCR (Polymerase Chain Reaction) amplification and sequence-based typing, a set of analytical method for detecting polymorphism of the site is established. In addition, a number of repeats in a core repeat (CT) sequence in the site is directly associated with diseaseresistance of litopenaeus vannamei, so the site can serve as a rapid, efficient and convenient molecular marker so as to be applied to screening of the litopenaeus vannamei related to disease resistance. Moreover, the molecular marker can be combined with other molecular markers to be used for analyzing a population genetic structure of the litopenaeus vannamei and the like. The litopenaeus vannamei disease resistance related functional gene EST-SSR molecular marker and the application thereof provided by the invention can be directly used for selecting disease-resistant litopenaeus vannamei individuals, can also be used for population genetic structure analysis and molecular mark assisted breeding, and particularly have a wide application prospect in selective breeding of litopenaeus vannamei disease-resistant good varieties.

Description

technical field [0001] The invention belongs to the technical field of molecular biology markers, more specifically, relates to Litopenaeus vannamei ( Litopenaeus vannamei ) EST-SSR molecular markers related to disease resistance and their applications. Background technique [0002] Litopenaeus vannamei ( Litopenaeus vannamei ), commonly known as Penaeus vannamei and white shrimp, belongs to Arthropoda, Crustacea, Decapoda, Penaeidae, and Penaeus in taxonomy. It is native to Central South China A eurythermal and euryalline tropical shrimp of the Americas. The farming areas of Litopenaeus vannamei include the coastal marine aquaculture areas and inland freshwater aquaculture areas of the country. It is the most productive shrimp species in my country. With the expansion of the scale of farming, the outbreak of diseases has become more and more serious. Among them, white spot syndrome virus (WSSV) has seriously hindered the development of the shrimp farming industry beca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156Y02A40/81
Inventor 李朝政何建国尹斌翁少萍
Owner SUN YAT SEN UNIV
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