qRT-PCR reference genes suitable for Rehmannia chingii Li and application of qRT-PCR reference genes

A technology of Tianmu Dihuang and internal reference gene, which is applied to qRT-PCR internal reference gene and its application field, can solve the problems that cannot be used as Tianmu Huang internal reference gene, internal reference gene has no universality, lack of Tianmu Huang internal reference gene, etc., achieves high sensitivity, Good specificity and good stability

Inactive Publication Date: 2021-01-12
HENAN AGRICULTURAL UNIVERSITY +1
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Problems solved by technology

However, internal reference genes are not constant in different plants and under different experimental conditions, and internal reference genes are not universal in different plants
In addition, the research on internal reference genes of Tianmu Dihuang is still relatively scarce. Although the screening and utilization of internal reference genes of Tianmu Dihuang's close relative species Rehmannia glutinosa have been reported, the gene sequences of Tianmu Dihuang and Dihuang are quite different, and the expression characteristics are also different. Therefore, the internal reference gene of Rehmannia glutinosa cannot be used as the internal reference gene of Tianmu Rehmannia qRT-PCR
[0004] At present, there are no reports on internal reference genes suitable for qRT-PCR research of Rehmannia glutinosa

Method used

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  • qRT-PCR reference genes suitable for Rehmannia chingii Li and application of qRT-PCR reference genes
  • qRT-PCR reference genes suitable for Rehmannia chingii Li and application of qRT-PCR reference genes
  • qRT-PCR reference genes suitable for Rehmannia chingii Li and application of qRT-PCR reference genes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Extraction of plant total RNA and synthesis of cDNA

[0032] Total RNA was extracted from all test samples according to the instructions of the RNA extraction kit (TaKaRa, Dalian), and the contamination of genomic DNA was removed by DNase treatment. The concentration and quality of the total RNA were measured by an ultra-micro nucleic acid protein analyzer, and the integrity of the total RNA was detected by 1% agarose gel electrophoresis.

[0033] The OD260 / OD280 and OD260 / OD230 of each test sample RNA met the requirements, and the agarose gel electrophoresis test showed that the 28S and 18S bands were clear without degradation, which met the requirements.

[0034] cDNA was synthesized by reverse transcription using reverse transcription kit type 6210A (TaKaRa, Dalian). About 1 μg of total RNA, 1 μL of oligo dT primer (concentration: 50 μmol L -1 ), 1 μL dNTP, 0.5 μL RNase inhibitor (40U·μL -1 ), 1 μL PrimeScript II reverse transcriptase (200U·μL -1 ),...

Embodiment 2

[0036] Example 2: Selection of candidate internal reference genes and primer design

[0037]According to the transcriptome sequencing data of Tianmudi Huang flowers and leaves, six different candidate internal reference genes were screened, namely RcTIP41, RcUBQ, RcActin, Rc18S, RcGAPDH and RcEF-1α, in all tested samples (flowers at different developmental stages: The FPKM values ​​of these six genes were relatively stable in the small bud stage (C1), budding stage (C2), big bud stage (C3), early flowering stage (C4), full flowering stage (C5) and leaf (L) ( Table 1). Among them, FPKM refers to the fragments of transcription per thousand bases per million mapped reads. This value can reflect the expression level of the gene. The larger the FPKM value, the higher the expression level of the gene. NCBI online analysis software ORFFinder was used to predict the open reading frame of the cDNA sequence of each gene. Then, according to the sequence of the gene, quantitative specif...

Embodiment 3

[0043] Example 3: Stability analysis of candidate internal reference genes

[0044] Using the 10-fold diluted cDNA as a template, according to the Premix Ex TaqTM II (TliRNaseH Plus) (TaKaRa, Dalian) kit instructions prepared qRT-PCR reaction system, each reaction was repeated 3 times, and carried out on the 96-well plate of BIO-RAD IQ5 quantitative instrument (Bio-Rad, Shanghai) Real-time fluorescence quantitative PCR.

[0045] The reaction system is: 12.5 μL of Premix Ex enzyme, 1 μL forward primer (10 μmol L-1), 1 μL reverse primer (10 μmol L-1), 1.0 μL cDNA template, plus 9.5 μL deionized water, the total volume is 25 μL.

[0046] The qRT-PCR reaction program was: 95°C for 30s; 95°C for 5s, 60°C for 30s, 40 cycles.

[0047] The expression level of candidate internal reference genes can be initially evaluated by the original Cq value obtained by qRT-PCR. The larger the Cq value of the gene, the lower the expression level of the gene, and vice versa. The stability of c...

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Abstract

The invention discloses qRT-PCR reference genes suitable for Rehmannia chingii Li and application of the qRT-PCR reference genes. According to the invention, six candidate reference genes with relatively stable expression quantities are screened out based on five different development stages and leaf transcriptome data of flowers of the Rehmannia chingii Li, and the stability of the candidate reference genes is analyzed through three different algorithms including GeNorm, NormFinder and Bestkeeper by utilizing a qRT-PCR technology. Results show that the number of the optimal reference genes for the flowers and leaf tissues of the Rehmannia chingii Li is two, and the optimal reference genes are RcTIP41 and Rc18S, wherein a nucleotide sequence of the RcTIP41 gene is as shown in SEQ ID NO.1,and a nucleotide sequence of the Rc18S gene is as shown in SEQ ID NO.2. According to the invention, the optimal reference genes for qRT-PCR of the flowers and leaves of the Rehmannia chingii Li are determined, and specific primers of the reference genes are provided, so that an important reference is provided for accurate and quantitative analysis of expression of related genes in the Rehmannia chingii Li.

Description

technical field [0001] The invention relates to a qRT-PCR internal reference gene suitable for Tianmu Dihuang and its application, and belongs to the technical field of molecular biology. Background technique [0002] Tianmu Dihuang (Rehmannia chingii Li.) belongs to the perennial herbaceous plant of Scrophulariaceae Rehmannia genus, mainly distributed in Zhejiang, Anhui and other places. The wild relative species of Chinese herbal medicine Rehmannia glutinosa Libosch. The yellow flowers and leaves of Tianmudi are rich in various chemical components such as iridoid glycosides, phenylethanol glycosides, anthocyanins, and polysaccharides, and have various pharmacological activities such as liver protection, anti-inflammation, neuroprotection, anti-oxidation, and hypoglycemia. Not only has important value in the development and utilization of traditional Chinese medicine, but also can be used for food, feed and natural product extraction. To study the development, synthesis a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11C12N15/29
CPCC12Q1/6895C07K14/415C12Q2600/158C12Q2600/166
Inventor 王丰青左鑫李铭铭李欣容苗春妍杨旭孙瑞斌杜家方周艳洪利亚张重义
Owner HENAN AGRICULTURAL UNIVERSITY
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