Internal reference gene for melon fruit genet PCR expression analysis, and stability verification method thereof

An internal reference gene and stability technology, applied in the field of plant genetic engineering, can solve the problems of poor stability and poor accuracy of melon gene expression analysis, and achieve the effects of wide application range, wide applicability and high expression stability

Inactive Publication Date: 2017-10-03
武汉市农业科学技术研究院作物科学研究所
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Problems solved by technology

At present, most of the traditional internal reference genes are used in the gene expression analysis of melon. These traditional internal re

Method used

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  • Internal reference gene for melon fruit genet PCR expression analysis, and stability verification method thereof
  • Internal reference gene for melon fruit genet PCR expression analysis, and stability verification method thereof
  • Internal reference gene for melon fruit genet PCR expression analysis, and stability verification method thereof

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[0030] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments:

[0031] The internal reference gene used for PCR expression analysis of muskmelon fruit gene of the present invention is the MELO3C007745T1 gene, and the nucleotide sequence of the internal reference gene is shown in SEQ ID NO: 3.

[0032] The nucleotide sequence of the PCR amplification primer of the MELO3C007745T1 gene is:

[0033] Forward primer sequence: TCTGGAGGAGTAGGTCGGATACC (shown in SEQ ID NO: 1);

[0034] Reverse primer sequence: CGATCAATGACGCAACAAGGCA (shown in SEQ ID NO: 2).

[0035] A method for verifying the stability of the aforementioned internal reference gene, which includes the following steps:

[0036] Step 1: At five sampling time points 15, 20, 25, 30 and 35 days after pollination of Xuelihong melon and flavor 4 melon, three biologically repeated Xuelihong melons and three biological species were selected respectively Learn the repeated...

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Abstract

The present invention discloses an internal reference gene for melon fruit genet PCR expression analysis, and a stability verification method for the internal reference gene. According to the present invention, the expression level and the stability of the gene are compared at the whole genome level by using melon transcriptome data so as to select eight stably-expressing genes, two traditional internal reference genes commonly used in melon are selected as controls, the expression stability of the 10 genes in two melon variety fruits at different development stages are compared, and it is determined that the expression stability of the MELO3C007745T1 gene is high highest through verification with genes such as Cmain2 and CmSPS1m, wherein the MELO3C007745T1 gene is the optimal internal reference gene for the real-time fluorescence quantitative PCR analysis in the melon fruit gene expression.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to an internal reference gene for PCR expression analysis of muskmelon fruit genes and a method for verifying the stability of the internal reference gene. Background technique [0002] Gene expression analysis is an important means to study gene function and transcriptional regulation. Real-time fluorescent quantitative PCR (qRT-PCR) is widely used in the quantitative analysis of gene expression because of its high sensitivity, high specificity, high accuracy and low cost. In qRT-PCR analysis, in order to obtain the real difference in the expression level of the target gene, it is necessary to eliminate the differences in the RNA yield, quality and reverse transcription efficiency of different samples. To eliminate the difference between samples, the internal reference gene is usually used for correction and standardization, that is, by measuring the expression o...

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/158C12Q2600/166
Inventor 汤谧任俭刘晔张娜孙玉宏程维舜曾红霞李煜华杨皓琼
Owner 武汉市农业科学技术研究院作物科学研究所
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