Luffa cylindrica.L reference gene EF-1 alpha and primer and application thereof

An internal reference gene, EF-1 technology, applied in the field of molecular biology, can solve problems such as the lack of internal reference genes

Active Publication Date: 2020-09-18
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

[0004] At present, the internal reference gene of loofah is only 18S rRNA gene, the cloning of other internal reference genes and the stability screening of internal ...

Method used

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  • Luffa cylindrica.L reference gene EF-1 alpha and primer and application thereof
  • Luffa cylindrica.L reference gene EF-1 alpha and primer and application thereof
  • Luffa cylindrica.L reference gene EF-1 alpha and primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 internal reference gene of loofah EF-1α get

[0023] (1) Obtain internal reference genes from loofah transcriptome data EF-1α , using Primer Premier 5.0 to design a pair of primers and synthesize the pair of primers by Platinum Biotechnology (Shanghai) Co., Ltd., the pair of primers are: forward primer 5'-ATGGGTAAGGAAAAGATTCACATC-3', reverse primer 5'-TTATTTCTTCTTGACAGCGGACTT- 3'.

[0024] (2) Extraction of total RNA and synthesis of the first strand of cDNA: the total RNA of loofah leaves was extracted using the Universal Plant RNA Extraction Kit (Beijing Biotech Biotechnology Co., Ltd.). The concentration and purity of total RNA were measured using Thermo NanoDrop2000C (ThermoScientific), and RNA samples with an absorbance ratio of about 1.8-2.0 at 260 / 280 nm were selected. Total RNA integrity was assessed by 1% agarose gel electrophoresis. The first strand of cDNA was synthesized according to the instructions of PrimeScript II 1st Strand cDNA Synthesis ...

Embodiment 2

[0029] Example 2 Primer Design and Specificity Detection

[0030] (1) Based on the nucleotide sequence of the internal reference gene obtained in Example 1, use Primer Premier5.0 software and follow the principles of real-time fluorescent quantitative PCR primer design to design a pair of fluorescent quantitative specific primers. The amplified fragment is 223bp, the pair of fluorescent quantitative specific primers are the real-time fluorescent quantitative PCR primers (as shown in SEQ ID No.2 and SEQ ID No.3): forward primer 5'-TCAAGAAGGTCGGATACA-3', reverse primer 5' -ACAGGGACAGTTCCCAATAC-3'.

[0031] (2) The general plant RNA extraction kit (Beijing Biotech Biotechnology Co., Ltd.) was used to extract the total RNA of loofah leaves. The first strand of cDNA was synthesized according to the instructions of PrimeScript II 1st Strand cDNA Synthesis Kit (Takara), and stored in a -20°C refrigerator for later use.

[0032] (3) Conventional PCR detection: Use the cDNA processed...

Embodiment 3

[0040] Example 3 internal reference gene EF-1α Expression stability analysis under high temperature treatment

[0041] Robust loofah seedlings with the same growth were selected and subjected to high temperature treatment at 42 °C for 0, 2, 6, 12 and 24 h, and the treated seedling leaves were picked and immediately frozen with liquid nitrogen and placed in a -80 °C ultra-low temperature refrigerator. The total RNA of the samples was extracted, and the first strand of cDNA was synthesized according to the instructions of the PrimeScript II 1st strand cDNA synthesis kit (Takara).

[0042] Using the synthesized sample cDNA as a template, 7 internal reference genes (actin gene ( ACTIN ), α-tubulin gene ( TUA ), β-tubulin gene ( TUB ), elongation transcription factors ( EF-1α ), glyceraldehyde-3-phosphate dehydrogenase gene ( GAPDH ), ubiquitin gene ( UBQ ) and 18S ribosomal RNA gene ( 18S rRNA )) fluorescent quantitative primer pair (primer pair described in Example 2...

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Abstract

The invention belongs to the field of molecular biology, and particularly relates to a luffa cylindrica.L reference gene EF-1 alpha and a primer and application thereof. The nucleotide sequence of theluffa cylindrica.L reference gene EF-1 alpha is shown as SEQID No.1. A luffa cylindrica.L real-time fluorescent quantitation PCR primer designed with the gene sequence is shown as SEQID No.2-3. BestKeeper, GeNorm, NormFinder and RefFinder analysis software and a Delta Ct method are adopted for evaluating the stability of the EF-1 alpha gene, and the reference gene EF-1 alpha is the most stable under high temperature and low temperature and ABA coercion condition. The designed real-time fluorescent quantitation PCR primer is high in specificity, and has high stability, reliability and repeatability, a strong support is provided for precise quantitation of relevant functional groups under adversity stress of temperature stress, ABA coercion and the like of luffa cylindrica.L, and research stability, repeatability and reliability are improved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a loofah internal reference gene EF-1α and its primers and applications. Background technique [0002] Loofah ( Luffa cylindrica.L ) is one of the important summer vegetables in China. It has the advantages of high nutritional value, wide adaptability, heat resistance, moisture resistance, strong stress resistance, etc., and also has medicinal functions. Therefore, planting loofah has great commercial value and huge market potential. However, loofah plants are often affected by various adversity stresses during cultivation. Therefore, improving the resistance of the plant itself to adversity stress is one of the important goals of the loofah breeding work. [0003] With the continuous development of molecular biology research, functional gene research has become an important means of plant stress resistance breeding, and gene expression analysis is the ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/11C12Q1/6895
CPCC12Q1/6895C12Q2600/158C12Q2600/166
Inventor 陈敏氡王彬朱海生李永平温庆放刘建汀叶新如曾美娟裘波音
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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