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A method for establishing technical indicators of coral free and endoparasitic symbiodinium abundance and coral bleaching warning coefficient h

A technical indicator and technology of symbiotic algae, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as interference, lack of representativeness, and lack of classification characteristics of symbiotic algae

Active Publication Date: 2019-10-29
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditionally, microscopic counting is the main method for observing and counting the number of Symbiodinium cells, but because Symbiodinium lacks obvious classification characteristics in morphology and is easily disturbed by other algae
In addition, microscopy techniques can only count 1ul seawater samples, which is statistically unrepresentative

Method used

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  • A method for establishing technical indicators of coral free and endoparasitic symbiodinium abundance and coral bleaching warning coefficient h
  • A method for establishing technical indicators of coral free and endoparasitic symbiodinium abundance and coral bleaching warning coefficient h
  • A method for establishing technical indicators of coral free and endoparasitic symbiodinium abundance and coral bleaching warning coefficient h

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1: the collection of coral sample and the preparation culture test of symbiotic algae

[0070] Collection of coral samples in the wild: Collect healthy helmet coral samples in the east and west waters of Hainan Island (for the GPS location of the samples, see figure 1 ), some samples were crushed or cut into small pieces, and stored in liquid nitrogen.

[0071] Collection of aquarium samples: Helmet coral samples that have been cultivated in the aquarium environment for about half a year, collected healthy and bleached coral samples, cut into small pieces, and stored in liquid nitrogen at low temperature.

[0072] In addition, 100ml of seawater samples from the aquarium were collected, and 6 repetitions were set up as the test material for detecting free symbiotic algae.

[0073] Pure culture of Symbiodinium: Coral Symbiodinium strains ITO 10 (Clade A), WZD 17 (Clade C), SGA1 (Clade D) and Symka (Clade F) were cultured under light in f / 2 medium, 27°C, photop...

Embodiment 2

[0074] The standard extraction of embodiment 2.DNA sample

[0075] figure 2 Diagram of standard DNA extraction methods for corals, seawater samples and symbiotic algae cultures. according to figure 2 The standard extraction of DNA samples can be carried out according to the steps shown.

[0076] The marine biological DNA extraction kit (Beijing Tiangen Biotechnology Co., Ltd.) was used as the implementation of the DNA extraction method. The extraction steps are as follows: take the coral sample collected in Example 1, cut into ≈5mm 2 , 0.1-0.2g of coral tissue, take several pieces and put them into 2.0ml EP tubes, then add 500μl of lysis solution GA to obtain coral samples.

[0077] The algal cells collected by centrifugation were transferred into a 2.0ml EP tube, and 500 μl of lysate GA was added to obtain a Symbiodinium sample.

[0078] Take 500ml of seawater sample and filter it with a nylon membrane with a diameter of 0.45μm. After filtering, remove the filter membr...

Embodiment 3

[0080] Embodiment 3: the standard curve of quantitative PCR

[0081] Table 1. The combination of primers and probes for coral hosts and symbiotic algae involved in the experiment

[0082]

[0083]

[0084] Note: where Y and W are bases and combined bases, Y stands for C or T, W stands for A or T.

[0085] The 5' and 3' ends of the Taqman probes were labeled with a FAM reporter fluorescent dye and a TAMRA quencher, respectively. The total volume of the amplification reaction is 20 μL, including upstream and downstream primers (Forward, Reverse) 200 nM, probe (probe) 100 nM, 10 μl qPCR Probe Master Mix (Vazyme, Nanjing), template 1 μl DNA of ITO 10 (Clade A), WZD 17 (Clade C), SGA1 (Clade D) and Symka (Clade F) serially diluted by 10 times, (the above algae strains are all preserved In the China Marine Microbial Culture Collection Management Center (MCCC), MCCC is a national public welfare microbial resource sharing platform), and the rest was filled with double distille...

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Abstract

The invention discloses a method for establishing technical indicators of abundance of free and parasitic symbiotic algae of a coral and a coral bleaching warning coefficient H. The method comprises the following steps: establishing an abundance indicator of free-state symbiotic algae in a coral growth sea area ocean environment, establishing an abundance indicator of parasitic-state symbiotic algae in the body of the coral, and establishing the coral bleaching warning coefficient H. According to the method disclosed by the invention, the sea water which is collected from various types of field seawater samples and experimental samples of aquariums can be determined accurately; time and space dynamics can be monitored for a long time and a short time; therefore, the method has a broad application prospect.

Description

technical field [0001] The invention relates to the evaluation technology and application field of marine coral ecological environment, and relates to a method for establishing the technical index H of coral free and endoparasitic symbiotic algae abundance and coral bleaching warning coefficient H. Background technique [0002] Symbiodinium can parasitize in corals, because it can perform photosynthesis, synthesize essential amino acids and provide energy support and necessary nutrients for coral calcification, as the primary producer, it is indispensable in coral growth and coral reef building a missing role. Due to the pollution of the marine environment, extreme climate changes, the frequent occurrence of El Niño and the destruction of human activities, the degradation of coral reefs and coral "bleaching" are caused. Because the symbiotic algae are sensitive to the marine environment, the occurrence of coral "bleaching" triggers the occurrence of symbiotic algae. The "es...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851
CPCC12Q1/6851C12Q2563/107C12Q2545/114
Inventor 林镇跃陈建明陈明谅
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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