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Reference gene TUB of Luffa cylindrica. L, primers of reference gene TUB and application of primers

An internal reference gene and loofah technology, which is applied in the field of molecular biology and can solve problems such as lack of internal reference genes

Pending Publication Date: 2020-09-18
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the internal reference gene of loofah is only 18Sr RNA gene, the cloning of other internal reference genes and the stability screening of internal reference genes have not been reported. Therefore, there is still a lack of suitable internal reference genes in the study of gene expression under adversity stress in loofah

Method used

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  • Reference gene TUB of Luffa cylindrica. L, primers of reference gene TUB and application of primers
  • Reference gene TUB of Luffa cylindrica. L, primers of reference gene TUB and application of primers
  • Reference gene TUB of Luffa cylindrica. L, primers of reference gene TUB and application of primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 internal reference gene of loofah TUB get

[0021] (1) Obtain internal reference genes from loofah transcriptome data TUB , using Primer Premier 5.0 to design a pair of primers, and synthesize the pair of primers by Platinum Biotechnology (Shanghai) Co., Ltd., specifically, the pair of primers: forward primer 5'-ATGAGGGAAATTCTTCACGTGC-3', reverse primer 5'- CTAATTGTCGAGGTCCTTCTTCTTC-3'.

[0022] (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 Synt...

Embodiment 2

[0027] Example 2 Primer Design and Specificity Detection

[0028] (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 226bp, 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'-GTGCTGGTAATAACTGGG-3', reverse primer 5' -GGGAAGACGGAGAAAGTA-3'.

[0029] (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 the PrimeScript II 1st strand cDNA synthesis kit (Takara), and stored in a -20°C refrigerator for later use.

[0030] (3) Conventional PCR detection: Use the cDNA process...

Embodiment 3

[0038] Example 3 internal reference gene TUB Analysis of expression stability under hydrogen peroxide treatment

[0039] Select robust loofah seedlings with the same growth, and use 100 μmmol·L -1 Hydrogen peroxide was sprayed 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).

[0040] 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) f...

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Abstract

The invention belongs to the field of molecular biology, and specifically relates to a reference gene TUB of Luffa cylindrica. L, primers of the reference gene TUB and application of the primers. Thenucleotide sequence of the reference gene TUB of Luffa cylindrica. L is shown in SEQ ID No. 1, and real-time fluorescent quantitative PCR primers of Luffa cylindrica. L are designed by using the genesequence, and are shown in SEQ ID No. 2-3. The stability of the TUB gene is evaluated by using analysis software of BestKeeper, GeNorm, NormFinder and RefFinder and a Delta Ct method, and the reference gene TUB is the most stable under the stress conditions of hydrogen peroxide and drought. The designed real-time fluorescent quantitative PCR primers have high specificity, high stability, high reliability and good reproducibility, vigorous support is provided for precise quantification of related functional genes of Luffa cylindrica. L under the conditions of hydrogen peroxide, drought and other adversity stress, and the stability, reproducibility and reliability of research 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 TUB 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 ba...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/11C12Q1/6895
CPCC12Q1/6895C12Q2600/158C12Q2600/166
Inventor 朱海生陈敏氡王彬温庆放李永平刘建汀叶新如曾美娟张前荣
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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