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Hibiscus esculentus reference gene EF-1 alpha and application thereof

An internal reference gene, EF-1 technology, applied in the field of molecular biology, to achieve the effect of improving detection efficiency, improving reliability and strong specificity

Pending Publication Date: 2019-07-16
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

But there is no report about the cloning of the okra elongation growth factor gene (EF-1α) gene and the research on it as an internal reference gene of okra

Method used

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  • Hibiscus esculentus reference gene EF-1 alpha and application thereof
  • Hibiscus esculentus reference gene EF-1 alpha and application thereof
  • Hibiscus esculentus reference gene EF-1 alpha and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Obtaining of internal reference gene EF-1α

[0024] (1) Design a pair of primers according to the EF-1α gene of upland cotton (Gossypium raimondii) and Asian cotton (Gossypium arboreum L.) on GenBank, and use the clustalx software to perform sequence alignment on the designed primer pair, and the platinum Shang Biotechnology (Shanghai) Co., Ltd. synthesized the primer pair, specifically, the primer pair (as shown in SEQ ID NO.1, 2):

[0025] Forward primer 5'-TTCCGAGTTCTATAATGGGTAA-3',

[0026] Reverse primer 5'-ACGCCACAGCTTATTTCTTC-3'.

[0027] (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 65 2×CTAB extraction buffer at ℃, add 7 μL of 2-mercaptoethanol at the same time, mix thoroughly, put in a water bath at 65℃ for 30 min, and invert several times; take out the centrifuge tube, cool to ...

Embodiment 2

[0032] Example 2 Real-time fluorescent quantitative PCR design and routine PCR detection

[0033] 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 196 bp. The pair of fluorescent quantitative specific primers are the real-time fluorescent quantitative PCR primers (as shown in SEQ ID NO: 4, 5):

[0034] Forward primer 5'-GCTGCCAAGAAGAAATAAGC-3',

[0035] Reverse primer 5'-ATGAAGCAAACCTCGACACT-3'.

[0036] 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 as primer pai...

Embodiment 3

[0040] Example 3 Real-time fluorescent quantitative PCR primer verification

[0041] 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:

[0042] 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 particularly relates to a hibiscus esculentus reference gene EF-1 alpha and an application thereof. The invention discloses a sequence of a transcriptional elongation factor gene (EF-1 alpha) of the hibiscus esculentus reference gene, based on the sequence, a quantitative specific primer is designed, and the transcriptional elongation factor gene (EF-1 alpha) can be stably expressed at the development stage of hibiscus esculentus fruits and under high temperature stress, and can be used as a reference gene during expressionresearch of the hibiscus esculentus gene at the fruit development stage and under the high temperature stress. According to the hibiscus esculentus reference gene EF-1 alpha disclosed by the invention, the transcriptional elongation factor gene (EF-1 alpha) is used as the reference gene for expression analysis of the hibiscus esculentus gene at the fruit development stage and under high temperature stress for the first time, and stability, reliability and repeatability of the expression analysis research of the hibiscus esculentus gene at the fruit development stage and under the high temperature stress are facilitated. The detection primer provided by the invention has specificity, the detection efficiency is greatly improved, and the credibility of the detection result is increased.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to an okra internal reference gene EF-1α 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.), the genus Okra, originates in 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 the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/29C12N15/11
CPCC07K14/415C12Q1/6895C12Q2600/166
Inventor 李永平朱海生叶新如王彬陈敏氡
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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