Internal reference gene of Fengdan and its special primers and application under drought stress

An internal reference gene and drought stress technology, applied to Fengdan internal reference gene and its special primers and application fields under drought stress, can solve problems such as inconsistencies, unstable expression levels, errors, etc., and achieve the effect of improving stability and reliability

Active Publication Date: 2021-02-09
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In daily experiments, the expression levels of traditionally used internal reference genes may be unstable under different conditions, which may easily lead to wrong results or even contradictory conclusions

Method used

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  • Internal reference gene of Fengdan and its special primers and application under drought stress
  • Internal reference gene of Fengdan and its special primers and application under drought stress
  • Internal reference gene of Fengdan and its special primers and application under drought stress

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054]Example 1: Screening of Fengdan real-time fluorescent quantitative PCR internal reference genes under drought stress

[0055]Based on the RNA-seq database obtained from the leaves of Fengdan normal growth and drought stress for 12 days in the previous period, 78392 Unigene RPKM values ​​were obtained, and 10 stable expression Unigenes were found as candidate internal reference genes (see Table 1).

[0056]Table 1 RPKM value representing the expression level of each Unigene in RNA-seq

[0057] Sequence numbering in the transcriptome Normal growth Drought stress for 12 days Serial number Unigene0040750 9.539.69SEQ ID NO.1 Unigene0029456 13.5814.29SEQ ID NO.2 Unigene0043536 19.0518.33SEQ ID NO.3 Unigene0041281 56.9854.97SEQ ID NO.4 Unigene0043455 9.088.90SEQ ID NO.5 Unigene0041583 144.36136.35SEQ ID NO.6 Unigene0030732 5.345.62SEQ ID NO.7 Unigene0032356 16.1715.81SEQ ID NO.8 Unigene0006653 9.999.43SEQ ID NO.9 Unigene0043820 10.8510.95SEQ ID NO.10

[0058]Aiming at the 10 candid...

Embodiment 2

[0061]Example 2: Analysis of the expression stability of candidate internal reference genes

[0062]Analysis of the stability of the expression of candidate internal reference genes in Fengdan leaves under different drought stress times: Using Fengdan three-year-old potted seedlings as plant material, watering normally at 17:00 every day for 3 consecutive days before natural drought treatment, and then Carry out natural drought treatments (0 days, 4 days, 8 days and 12 days), each treatment has three biological repetitions. After harvest, liquid nitrogen is used for quick freezing and stored at -80°C.

[0063]The samples were extracted using MiniBEST Plant RNA Extraction Kit (Takara, Japan), and the RNA concentration and OD value were detected by a nucleic acid protein analyzer, and the integrity of total RNA was detected by 1.5% agarose gel electrophoresis; reverse transcription was used 5×M-MLV Buffer reverse transcriptase (TaKaRa, Japan), Oligo dT 18 primer was used for cDNA synthesis,...

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Abstract

The present invention relates to an internal reference gene of Fengdan under drought stress, its special primers and its application. The internal reference genes are TATA box binding protein TBP gene, actin ACT1 gene, actin ACT2 gene, glyceraldehyde-3-phosphate dehydrogenase GAPDH gene , eukaryotic translation initiation factor eIF1 gene, eukaryotic translation initiation factor eIF2 gene, tubulin α-TUB gene, tubulin β-TUB gene, RNA polymerase II RNA Pol II gene or RNA polymerase II Transcription factor RP II gene; the nucleotide sequence of the above gene is shown in the sequence list. The purpose of the present invention is to provide a gene stably expressed in the gene expression profile of Fengdan under drought stress, which is used as an internal reference gene for drought stress of Fengdan.

Description

Technical field[0001]The invention belongs to the field of plant biotechnology, and specifically relates to the Fengdan internal reference gene and its special primers and applications under drought stress.Background technique[0002]The internal reference gene is a gene whose expression level is not affected by the research conditions and is constantly expressed, and is often used for the calibration of real-time fluorescent quantitative PCR detection. In daily experiments, the expression levels of traditionally used internal reference genes under different conditions may be unstable, which may easily lead to wrong results or even contradictory conclusions. Therefore, it is particularly important to select and screen out suitable genes as internal reference genes for reference and correction of gene expression.[0003]Fengdan (Paeonia ostii) is a perennial woody plant of the genus Paeonia in the family Paeoniaceae, and a variety of Yangshan Peony. Because of its high medicinal value, o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/53C12Q1/6895C12Q1/6851C12N15/11
CPCC07K14/415C12N9/0008C12Q1/6851C12Q1/6895C12Q2600/166C12Q2545/101C12Q2563/107C12Q2531/113
Inventor 赵大球陶俊许聪栾雨婷
Owner YANGZHOU UNIV
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