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Screening method for real-time fluorescent quantitative PCR internal reference gene of Hibiscus hibiscus

A technology of real-time fluorescence quantification and internal reference genes, applied in the fields of genomics, instrumentation, sequence analysis, etc., can solve the problems that there are no related reports on the internal reference genes of Hibiscus hibiscus

Active Publication Date: 2020-09-29
INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant report on the identification of the internal reference genes of Hibiscus seabiscus

Method used

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  • Screening method for real-time fluorescent quantitative PCR internal reference gene of Hibiscus hibiscus
  • Screening method for real-time fluorescent quantitative PCR internal reference gene of Hibiscus hibiscus
  • Screening method for real-time fluorescent quantitative PCR internal reference gene of Hibiscus hibiscus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1. Selection of internal reference genes

[0081] The present invention selects 10 candidate internal reference genes from the existing Hibiscus transcriptome database, and designs real-time fluorescent quantitative PCR specific primers for each internal reference gene.

[0082] Comparison and analysis of candidate internal reference gene sequences: Submit the candidate gene sequences to BLASTX in NCBI for comparison and analysis. The sequence identity of each internal reference gene sequence in Hibiscus hibiscus with homologous genes of related species is more than 85%, and most All above 90%. The sequence identity with the model plant Arabidopsis is more than 83%. The comparison results are shown in Table 1.

[0083] sheet BLASTX comparative analysis of internal reference genes

[0084]

[0085] Design and verification of real-time fluorescent quantitative PCR primers: using the candidate gene sequence as a template, use GenScript’s online primer desig...

Embodiment 2

[0089] Embodiment 2. Real-time fluorescence quantitative PCR experiment

[0090] The experimental materials were divided into two groups. One group was the tender leaves of sea hibiscus, which were treated with 7 kinds of abiotic stresses including drought, high salt, low temperature, high temperature, abscisic acid, salicylic acid and methyl jasmonate 0, 1, 6, 12, 24 and 48 h; the other group is the tissues and organs, which are the young roots, old leaves, young leaves, receptacles, petals, and stamens of 10-year-old sea hibiscus. The collection of plant treatment materials is as follows: one group of materials used seashore hibiscus seedlings as materials, and simulated drought and high-salt stress with 15% polyethylene glycol 6000 and 400 mM sodium chloride respectively; the seashore hibiscus seedlings were placed in 4°C C and temperature 42°C incubator to simulate low temperature and high temperature stress. The conditions of hormone treatment were as follows: spray Hibi...

Embodiment 3

[0094] Example 3. geNorm, NormFinder and BestKeeper software evaluation of internal reference genes under all samples

[0095] geNorm software

[0096] In the present invention, the download address of the geNorm software is https: / / genorm.cmgg.be / , and its steps and standards are as follows:

[0097] In the present invention, geNorm first realized the ranking of the expression stability of 10 internal reference genes by calculating the average expression stability (M), and the results showed that in all samples, the two most stable internal reference genes were actin ( ACT ) and SKI-associated proteins ( SKIP ) ( image 3 ), the most unstable is tubulin ( TUB ), the stability ranking of each internal reference gene is as follows: Actin ( ACT ) and SKI-associated proteins ( SKIP )> small nuclear RNA U6 ( U6 )> ribosomal protein S13 ( S13 ) > ubiquitin-conjugating enzyme E2 ( UBC )>Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH )>18S ribosomal RNA ( 18S ) > ...

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Abstract

The invention belongs to the technical field of plant gene engineering, and discloses a screening method for real-time fluorescent quantitative PCR reference genes of Hibiscus hamabo Sieb.et Zucc. Thescreening method utilizes a transcriptome database of Hibiscus hamabo Sieb.et Zucc, selects 10 candidate reference genes, and takes 10 candidate reference gene sequences as templates to design real-time fluorescent quantitative PCR specific primers, selects different tissues of Hibiscus hamabo Sieb.et Zucc and Hibiscus hamabo Sieb.et Zucc leaves under various abiotic stresses and hormone treatments as experimental materials for performing a fluorescent quantitative PCR experiment, and finally, performs data analysis by applying geNorm, NormFinder and BestKeeper software. The most stable reference genes in the screening method are ACT and SKIP. The screening method for real-time fluorescent quantitative PCR reference genes of Hibiscus hamabo Sieb.et Zucc establishes a screening system forstable reference genes of hibiscus hamabo, and provides stable reference genes for developing gene function research of Hibiscus hamabo Sieb.et Zucc.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and relates to a method for screening internal reference genes of real-time fluorescent quantitative PCR in different tissues of sea hibiscus and under various abiotic stresses and hormone treatments. Background technique [0002] In forest tree genetics and breeding, the exploration of gene expression patterns is common and important. Real-time fluorescent quantitative PCR is the most commonly used nucleic acid quantitative technology in the study of gene expression patterns, which has higher sensitivity, better specificity and wider detection range. There may be some differences in initial sample volume, RNA integrity, cDNA synthesis efficiency, etc., which will affect the accuracy of real-time fluorescent quantitative PCR results. Therefore, screening suitable internal reference genes is an important prerequisite for real-time fluorescent quantitative PCR. [0003] Many stu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G16B30/00G16B20/00
Inventor 顾春笋王芝权倪龙杰刘凉琴
Owner INST OF BOTANY JIANGSU PROVINCE & CHINESE ACADEMY OF SCI
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