Soybean reference gene as well as detection primer and application thereof

A technology for internal reference genes and detection primers, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of lack of screening of internal reference genes and lack of systematic research on internal reference genes

Active Publication Date: 2021-03-02
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In conclusion, there is a lack of systematic research on internal reference genes currently used for the analysis of soybean circadian clock-regulated gene expressio...

Method used

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  • Soybean reference gene as well as detection primer and application thereof
  • Soybean reference gene as well as detection primer and application thereof
  • Soybean reference gene as well as detection primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Screening of Internal Reference Genes for Detecting Soybean Endogenous Rhythmic Expression Genes

[0042] In order to screen the most suitable internal references for analyzing the rhythmic expression of soybean circadian clock-regulated genes, the Genevestigator (www.genevestigator.com) database was used for preliminary screening of soybean internal reference genes. In the absence of target genes, soybean RNA-seq and microarray databases were used to select the top 20 recommended internal reference genes at different expression levels, combined with RNA-seq data obtained from soybean single leaf time course under constant conditions, and in RNA-seq data of different tissues and organs and developmental stages, screening candidates with less expression differences in soybean roots, stems, cotyledons, single leaves and compound leaves, and more stable expression in soybean seedlings, vegetative growth and reproductive growth and other developmental stages Refer...

Embodiment 2

[0050] Example 2 Gm GAPDHa is suitable for analysis of gene expression related to circadian rhythm and drought stress

[0051] The internal reference gene in Example 1 was analyzed for its rhythmic expression in soybean single leaves and compound leaves grown under short-day conditions and long-term drought conditions. Soybean WS82 material was cultivated under 12 hours of light / 12 hours of darkness at 25°C. After the seeds germinated for 5 days, the control group maintained sufficient water, and the experimental group was subjected to drought stress by stopping watering. After 10 days of treatment, ZT1 began to collect samples every 3 hours. The expression of candidate internal reference genes in soybean single leaf and compound leaf were detected by qRT-PCR.

[0052] The experimental results showed that the newly screened internal reference genes GmGAPDHa, GmRAB5C and GmPIP7a were relatively stable in short-day conditions with sufficient water and drought stress conditions. ...

Embodiment 3

[0057] Example 3 Detection of Drought Stress Response Gene GmDREB5 Expression

[0058] GmGAPDHa, GmRAB5C, GmGAPDHb, GmH3.3 and GmPIP7a were actually used for qRT-PCR to detect the expression of the drought stress response gene GmDREB5. It is known that drought stress induces the expression of GmDREB5, Figure 5To use GmGAPDHa, GmRAB5C, GmGAPDHb, GmH3.3, GmPIP7a or GmUBQ as internal reference genes, qRT-PCR was used to detect the rhythmic expression of soybean GmDREB5 under drought and non-drought conditions (GmDREB5 gene ID: Glyma12g33020; forward primer: 5' -TCTGCATGGTTCAATGCTATTCCT-3'; reverse primer: 5'-AGGAATGTGTGATTGGCAAAGAAG-3').

[0059] The experimental results showed that under non-drought stress conditions, using GmGAPDHa, GmRAB5C, GmGAPDHb, GmH3.3 or GmPIP7a as internal reference genes, the results of analyzing the relative expression of GmDREB5 were consistent: the expression level of GmDREB5 was very low under non-drought conditions; Under these conditions, the e...

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Abstract

The invention discloses a soybean reference gene as well as a detection primer and application thereof. The soybean reference gene is a group of reference genes used for analyzing gene recent rhythm expression, circadian rhythm expression and drought stress response in a single leaf and a compound leaf of soybeans, and comprises GmGAPDHa and GmRAB5C suitable for analyzing the three conditions in the single leaf and the compound leaf, and GmGAPDHb, GmH3.3 and GmPIP7a suitable for analyzing gene recent rhythm expression in the single leaf and the compound leaf. The reference gene provides favorable support for analyzing the stability and reliability of related research results of gene recent rhythm expression, circadian rhythm expression and drought stress response in soybean single leaf andcompound leaf, and particularly fills the blank of reference genes in soybean recent rhythm research.

Description

technical field [0001] The present invention relates to the technical field of plant genetic engineering, in particular, relates to soybean internal reference genes and their detection primers and applications, in particular to internal reference genes suitable for analyzing circadian rhythm expression, circadian rhythm expression and drought stress response in single leaves and compound leaves Genes GmGAPDHa and GmRAB5C; GmGAPDHb, GmH3.3 and GmPIP7a for analysis of circadian expression of genes in simple and compound leaves. Background technique [0002] There are currently many methods available for detecting gene expression, among which Northern Blot is not conducive to accurate quantification and high-throughput detection of gene expression; DNA chips can achieve high-throughput detection of gene expression, but the chip is not sensitive enough to weak gene expression differences, and Specificity is flawed. Real-time fluorescent quantitative PCR (qRT-PCR) is currently a...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/11C12Q1/6851C12Q1/6895
CPCC07K14/415C12Q1/6851C12Q1/6895C12Q2600/166C12Q2600/158C12Q2531/113C12Q2545/101C12Q2563/107C12Q2561/113
Inventor 徐小冬袁力谢芷欣张思远
Owner HENAN UNIVERSITY
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