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QRT-PCR reference genes of grapes and application of qRT-PCR reference genes

An internal reference gene, grape technology, applied in the field of molecular biology, can solve problems such as lack of internal reference gene research

Pending Publication Date: 2021-01-01
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in grapes, the research on internal reference genes is still relatively scarce, and there are many varieties of grapes, and a large number of new varieties have been bred in recent years, all of which lead to a certain degree of blindness in the selection of internal reference genes in grapes. Absolutely ideal internal reference genes to choose from

Method used

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  • QRT-PCR reference genes of grapes and application of qRT-PCR reference genes
  • QRT-PCR reference genes of grapes and application of qRT-PCR reference genes
  • QRT-PCR reference genes of grapes and application of qRT-PCR reference genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Candidate internal reference genes

[0035]According to the previous transcriptome data, ten candidate reference genes were analyzed and identified in this study (Table 1), namely: Actin, 18s-rRNA, GAPDH, VvEF1-α, VvEF1-γ, EF1-α, Ubiquitin and three The nucleotide sequences of the newly discovered candidate internal reference genes RRM1 (VIT_07s0005g03980), PPR2 (VIT_11s0065g00380) and MRE11 (VIT_19s0014g03680) are shown in the sequence listing. RRM1 contains an RNA recognition motif and encodes a heterogeneous ribonucleoprotein; PPR2 encodes a protein containing repeat triangular pentapeptide; MRE11 encodes a double-strand break repair protein. The functions of RRM1 and PPR2 are unknown and neither has been identified in Vitis vinifera nor in other species. MRE11 orthologs in Arabidopsis are involved in cell cycle activation and double-strand DNA repair.

[0036] Wherein, the nucleotide sequence of the VvEF1-γ gene is shown in SEQ ID NO.28; the nucleotide ...

Embodiment 2

[0037] Example 2: Design of primers for candidate internal reference genes

[0038] qRT-PCR primers were designed with Primer 5 software, and the nucleotide sequences of the primers are shown in Table 1.

[0039] Wherein, the primers for specifically amplifying the VvEF1-γ gene are as follows:

[0040] F: CAAGAGAAACCATCCCTAGCTG (SEQ ID NO. 16)

[0041] R: TCAATCTGTCTAGGAAAGGAAG (SEQ ID NO. 17)

[0042] The primers used to specifically amplify the 18s-rRNA gene are as follows:

[0043] F: CATAAACGATGCCGACCAG (SEQ ID NO. 12)

[0044] R: TTCAGCCTTGCGACCATACT (SEQ ID NO. 13)

[0045] The primers used to specifically amplify the EF1-α gene are as follows:

[0046] F: GAACTGGGTGCTTGATAGGC (SEQ ID NO. 20)

[0047] R: AACCAAAATATCCGGAGTAAAAAGA (SEQ ID NO. 21)

[0048] The primers for specific amplification of the Actin gene are as follows:

[0049] F: CATTGTGAGCAACTGGGATG (SEQ ID NO. 10)

[0050] R: GATTAGCCTTCGGGTTGAGA (SEQ ID NO. 11)

[0051] The primers used to specifical...

Embodiment 3

[0056] Example 3: Stability analysis of candidate internal reference genes

[0057] 1. Total RNA extraction and cDNA synthesis

[0058] The collected samples (fruits, leaves, tendrils of grapes) were ground into powder form with liquid nitrogen. The total RNA was extracted using a polysaccharide and polyphenol total RNA extraction kit (TIANGEN, Beijing). For specific methods, refer to the instruction manual. The quality and integrity of the extracted RNA were checked by 1% agarose gel electrophoresis. Using the extracted total RNA as a template, use First-strand cDNA synthesis kit (Vazyme, Nanjing) was used to synthesize cDNA, and the specific method was referred to the instruction manual, and the synthesized cDNA was stored in a -20°C refrigerator.

[0059] 2. Real-time fluorescent quantitative PCR (qRT-PCR)

[0060] The total qRT-PCR reaction system was 10 μL, including 5 μL 2×TransStart Top Green qPCR SuperMix (Quanshi Jinbiology, Beijing), 1 μL cDNA, 0.3 μL forward an...

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Abstract

The invention discloses qRT-PCR reference genes of grapes and an application of the qRT-PCR reference genes. The stability of ten candidate reference genes (Actin, 18s-rRNA, GAPDH, VvEF1-gamma, VvEF1-alpha, EF1-alpha, Ubiquitin, RRM1, PPR2 and MRE11) are analyzed in fruits, leaves and tendrils of seven different grape varieties and nine Jufeng fruit samples in different development stages throughthree pieces of analysis software including geNorm, NormFinder and BestKeeper according to early transcriptome data. Results show that the optimal reference gene number of the grape samples is two, the selection of the optimal reference genes of different samples is different, the optimal reference gene combination in the fruits comprises the VvEF1-gamma and the 18s-rRNA, the reference genes of the tendrils are the EF1-alpha and the Actin, and the reference genes in the fruit development stage are the EF1-alpha and the VvEF1-alpha. The qRT-PCR reference genes can provide important reference for related researchers to carry out qRT-PCR work in the grapes.

Description

technical field [0001] The invention relates to a grape qRT-PCR internal reference gene and application thereof, and belongs to the technical field of molecular biology. Background technique [0002] Grapes are one of the most important fruit trees widely grown worldwide. The fruit of grapes can be eaten fresh or used to make wine and raisins, so it has high nutritional and economic value. At present, relevant researchers have discovered and cultivated a large number of new grape varieties, and have carried out a lot of scientific research work using these germplasm resources. Research on the molecular mechanism of important agronomic traits of grapes and related functional genes has greatly promoted the development of the grape industry. [0003] In the process of carrying out basic research on grapes, real-time fluorescent quantitative PCR (qRT-PCR) technology has played an important role, which has many advantages such as short reaction time, good repeatability and high...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/166C12Q2600/13C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 郭大龙韦同路余义和裴茂松刘海楠郑玉萍陈苏丹
Owner HENAN UNIV OF SCI & TECH
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