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Okra reference gene and application thereof

An internal reference gene and basic technology, applied in the field of molecular biology, to achieve the effect of improving detection efficiency, improving stability and improving reliability

Inactive Publication Date: 2018-09-28
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the cloning of 18S rRNA gene in okra and the research on it as an internal reference gene in okra.

Method used

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  • Okra reference gene and application thereof
  • Okra reference gene and application thereof
  • Okra reference gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Obtaining of internal reference genes

[0024] Step (1), designing a pair of primers according to the 18S rRNA gene of upland cotton (Gossypium raimondii) and hollyhock (Abelmoschusmanihot (L.) Medicus) on GenBank, and using the clustalx software to perform sequence alignment on the designed primer pair, and The pair of primers were synthesized by Platinum Biotechnology (Shanghai) Co., Ltd., specifically, the pair of primers (as shown in SEQ ID NO: 1, 2): forward primer 5'-TTCACCGGACCATTCAATCGGTA -3',

[0025] Reverse primer 5'-TGATCCTTCCGCAGGTTCACCTA-3'.

[0026] Step (2), DNA extraction: Weigh about 0.1 g of okra leaves, add liquid nitrogen to quickly and fully grind to powder (add 0.1 g of PVP), transfer the powder to a 1.5 mL centrifuge tube; add 600 μL to each tube and preheat to 2×CTAB extraction buffer at 65°C, add 7 μL of 2-mercaptoethanol at the same time, mix thoroughly, put in a water bath at 65°C for 30 min, and invert several times; take out the ...

Embodiment 2

[0031] Example 2 Real-time fluorescent quantitative PCR design and routine PCR detection

[0032] Based on the nucleotide sequence of the internal reference gene obtained in Example 1, using Primer Premier5.0 software, and following the principles of real-time fluorescent quantitative PCR primer design, a pair of fluorescent quantitative specific primers were designed, and the amplified fragment was 206 bp. The pair of fluorescent quantitative specific primers are the real-time fluorescent quantitative PCR primers (as shown in SEQ ID NO: 4-5): forward primer 5'- TCCATTGGAGGGCAAGTCT -3', reverse primer 5'- GGCAGTAGGGACGAGCAGA -3'.

[0033] The total RNA of okra was extracted, and the first strand of cDNA was synthesized according to the method of PrimeScriptTM 1st Strand cDNA Synthesis Kit, that is, the RNA was reverse transcribed into cDNA; then the obtained cDNA was used as a template and real-time fluorescent quantitative PCR primers were used for PCR amplification. And the ...

Embodiment 3

[0037] Example 3 Real-time fluorescent quantitative PCR primer verification

[0038] Extract the total RNA of okra, and synthesize the first strand of cDNA according to the method of PrimeScriptTM 1st Strand cDNA Synthesis Kit, that is, reverse transcribe the RNA into cDNA; then use the obtained cDNA as a template, follow the instructions of Power SYBR®Green PCR Master Mix in ABI7500 in real time The PCR reaction was carried out on the quantitative PCR instrument, and the reaction system and reaction procedure of the PCR reaction were as follows:

[0039] The reaction system is: the total volume of the reaction system is 25 μL, 12.5 μL Power SYBR® Green PCR MasterMix, 1 μL template, 0.5 μL of the forward primer of the real-time fluorescence quantitative PCR primer in Example 2 (concentration is 10 μmol / L), implement The reverse primer of the real-time fluorescent quantitative PCR primer in Example 2 was 0.5 μL (concentration: 10 μmol / L), and distilled water was added to make t...

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Abstract

The invention belongs to the technical field of molecular biology and provides an okra reference gene and application of the reference gene. A quantitative specific primer is designed on the basis; and an 18S ribosomal RNA (ribonucleic acid) gene (18S r RNA) can be expressed stably in various growth and development stages and tissues of okra and suitable as a reference gene in an okra gene expression research. The 18S r RNA gene is used as the reference gene for okra gene expression analysis for the first time; the stability, reliability and repeatability of the okra gene expression analysis research are improved; the detection primer has specificity; the detection efficiency is greatly improved; and the reliability of a detection result is improved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to an okra internal reference gene and its application. Specifically, a pair of fluorescent quantitative specific primers are designed based on the nucleotide sequence of the okra internal reference gene. Background technique [0002] Okra (Hibiscus esculentus L.), belonging to the genus Okra, is native to Northeast Africa and is widely cultivated in the world. In recent years, the domestic introduction and promotion has been rapid, and the cultivated area has increased year by year. Okra mainly eats tender fruits and is rich in protein, vitamins, mineral salts, sugar polymers and other nutrients. It is a healthy vegetable with high nutritional value and health care functions. With the continuous deepening and development of its molecular biology research, gene expression analysis is gradually being applied to reveal the gene regulation mechanism of okra. In th...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/11C12Q1/6851C12Q1/6895
CPCC12Q1/6851C12Q1/6895C12Q2600/166C12Q2545/101C12Q2531/101C12Q2563/107
Inventor 李永平朱海生刘建汀陈敏氡张前荣王彬温庆放
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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