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Real-time quantitative PCR detection primer set and method of differentially expressed acipenser dabryanus gonad gene

A differentially expressed gene, real-time quantitative technology, applied in the field of molecular biology, can solve the problem of not analyzing the differential gene expression of sturgeon gonads

Active Publication Date: 2018-04-03
FISHERIES INST SICHUAN ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the analysis of differential gene expression in the gonads of Acipenser dabryi by quantitative PCR in the prior art

Method used

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  • Real-time quantitative PCR detection primer set and method of differentially expressed acipenser dabryanus gonad gene
  • Real-time quantitative PCR detection primer set and method of differentially expressed acipenser dabryanus gonad gene
  • Real-time quantitative PCR detection primer set and method of differentially expressed acipenser dabryanus gonad gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The selection of embodiment 1 object detection gene and internal reference gene:

[0028] 1. Dabry's sturgeon sample collection and RNA extraction:

[0029] Select healthy, gonad-differentiated Acipenser dabryi from the Sichuan Fisheries Research Institute, dissect their gonads to identify their sex, and extract the remaining samples for total RNA. In a pre-cooled mortar, use liquid to quickly grind into powder, put it into a 1.5mL centrifuge tube, and add 1mL TRIzol; (2) Add 200μL chloroform to the centrifuge tube, shake vigorously for 15s, and stand at room temperature for 3min; (3) 4°C , centrifuge at 12,000r / min for 10min, absorb the supernatant and put it into a new centrifuge tube; (4) add isopropanol equal to the volume of the supernatant, shake gently to mix, and place at room temperature for 5-10min; (5) 4°C, Centrifuge at 12,000r / min for 10min, discard the supernatant, and carefully absorb the remaining isopropanol; (6) Add 1mL of 75% ethanol to wash the prec...

Embodiment 2

[0038] Example 2 Establishment of real-time quantitative PCR detection method for differentially expressed genes in gonads of Acipenser dabfieldii:

[0039] 1. Primer design:

[0040]According to the sequences of SOX9, FST and DF in the transcriptome unigene gene, Primei5.0 software was used to design specific primers suitable for fluorescent quantitative PCR detection. The internal reference gene β-actin also designed primers according to the transcriptome unigene results. The primers are shown in Table 1. Shown:

[0041] Table 1 Specific primers suitable for fluorescent quantitative PCR detection

[0042]

[0043] The result of agarose gel electrophoresis shows that the amplified length is consistent with the expected product length, and the sequence is determined to be the detected target gene (shown in SEQ ID NO.1-4), as shown in Figure 1-4 shown, from figure 1 It can be seen that the melting curve of the SOX9 gene is a single peak, indicating that the primer has go...

Embodiment 3

[0055] Embodiment 3 quantitative PCR result statistics:

[0056] Table 2 below compares the expression levels of the three genes SOX9, DF and FST in quantitative PCR and transcriptome sequencing.

[0057] Calculated as follows:

[0058] ΔCt=Ct(target gene)-Ct(β-actin gene)

[0059] ΔΔCt = ΔCt (male sample) - ΔCt (female sample)

[0060] to 2 -ΔΔCt Values ​​represent relative expression levels of target genes.

[0061] Table 2 Comparison of transcriptome sequencing and quantitative PCR results

[0062]

[0063] FPKM is the expression level of transcriptome sequencing, qPCR is the expression level of quantitative PCR; T: testis; O: ovary.

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Abstract

The invention discloses a real-time quantitative PCR detection primer set and a real-time quantitative PCR detection method of a differentially expressed acipenser dabryanus gonad gene. The real-timequantitative PCR detection primer set comprises an SOX9 primer pair, an FST primer pair, a DF primer pair or a beta-actin primer pair. The real-time quantitative PCR detection method comprises the following steps: extracting RNA from an acipenser dabryanus sample to be detected, using the total RNA as a template, and synthesizing cDNA of the acipenser dabryanus sample to be detected; using the cDNA of the acipenser dabryanus sample to be detected as a template, adding the real-time quantitative PCR detection primer set of the differentially expressed acipenser dabryanus gonad gene, performingreal-time fluorescent quantitative PCR detection, and detecting an amplification product according to an agarose gel electrophoresis result and an amplification product dissolution curve. The primersand the quantitative PCR detection methods of a real-time quantitative PCR target gene and an internal reference gene are suitable for acipenser dabryanus as well as other sturgeons with a similar kinship, and a basis is provided for identifying genders of the sturgeons and researching a gender determining gene and a gender determining mechanism in the future.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a real-time quantitative PCR detection primer set and method for differentially expressed genes in gonads of Acipenser dabryi. Background technique [0002] Dabry's sturgeon (Acipenser dabryanus), commonly known as the Yangtze River sturgeon, belongs to the order Acipenseridae, Acipenseridae, and the genus Acipenser. It is a unique freshwater resident species in the Yangtze River Basin of my country. In recent years, due to elements such as overfishing, habitat destruction, and environmental pollution, the wild population of Dabry's sturgeon has declined sharply. Since 1982, only about 150 Dabry's sturgeons have been captured in the Yangtze River Basin, and since 1995, no wild Dabry's sturgeon has been reported in the lower reaches of Gezhou Dam in the Yangtze River Basin. Therefore, Dabry's sturgeon was listed as a first-class national key protected animal in 1988, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686
CPCC12Q1/686C12Q1/6888C12Q2600/158C12Q2561/113C12Q2545/114
Inventor 陈叶雨刘亚龙治海林珏龚全赖见生杜军宋明江
Owner FISHERIES INST SICHUAN ACADEMY OF AGRI SCI
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