Tea tree miRNA fluorescent quantitative PCR reference gene under low temperature stress as well as screening method and application of reference gene

A fluorescence quantitative, internal reference gene technology, applied in the field of molecular biology, can solve problems such as unstable expression

Active Publication Date: 2015-12-09
ANHUI QIMEN COUNTY QIHONG TEA
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Problems solved by technology

For example, U6 is the most commonly used internal reference gene for studying the relative expression of miRNA. It has been reported that the expression of U6 i

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  • Tea tree miRNA fluorescent quantitative PCR reference gene under low temperature stress as well as screening method and application of reference gene
  • Tea tree miRNA fluorescent quantitative PCR reference gene under low temperature stress as well as screening method and application of reference gene
  • Tea tree miRNA fluorescent quantitative PCR reference gene under low temperature stress as well as screening method and application of reference gene

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Embodiment

[0044] Selection of miRNAqRT-PCR internal reference candidate genes and primer design

[0045] Six internal reference genes commonly used in plant miRNAqRT-PCR (U6, 18SrRNA, 26SrRNA, 5.8SrRNA, 5SrRNA and GAPDH) and three tea tree miRNAs with relatively stable expression (cs-miR396b, Pc-222-3p and Pc-45545- 5p), using these 9 miRNAs as internal reference candidate genes. The sequences of miRNAU6, 18SrRNA, 26SrRNA, 5.8SrRNA, 5SrRNA and GAPDH were obtained from NCBI, and the sequences of tea tree miRNAcs-miR396b, Pc-222-3p and Pc-45545-5p were obtained from tea tree genome sequencing data and miRNA low-temperature sequencing library. The nucleotide sequences are respectively shown in SEQ ID NO: 3, SEQ ID NO: 1 and SEQ ID NO: 4.

[0046] Primer Premier 5.0 software was used to design upstream and downstream primers of internal reference genes. The upstream and downstream primers of U6, 18SrRNA, 26SrRNA, 5.8SrRNA, 5SrRNA and GAPDH are designed according to the specific sequence, ...

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Abstract

The invention relates to a tea tree miRNA fluorescent quantitative PCR reference gene Pc-222-3p, which has a nucleotide sequence as shown in SEQ ID NO: 1, as well as a screening method of the reference gene. According to the method, 6 common plant miRNA reference genes and 3 tea tree miRNAs are selected as candidate reference genes, and the expression stabilities of the candidate reference genes under different low temperature stresses and in different tea tree tissues are evaluated by virtue of qRT-PCR technology through two software, namely BestKeeper and geNorm, so as to provide a reference for the selection of reference genes for tea tree miRNA differential expression analysis under low temperature stress. The obtained tea tree miRNA fluorescent quantitative PCR reference gene Pc-222-3p is stable in gene expression under different temperatures of treatment and in different tea tree tissues; therefore, the reference gene Pc-222-3p can be used as a reference gene for tea tree miRNA fluorescent quantitative PCR under low temperature stress.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to an internal reference gene of miRNA fluorescent quantitative PCR under low temperature stress of tea trees, a screening method and application thereof. Background technique [0002] Tea tree (Camelliasinensis) originates in the tropics and subtropics of my country, and is a perennial economic crop that likes temperature and chills. With the continuous development of the tea industry, the tea tree planting area has expanded northward to 38° north latitude. Most tea areas in my country, especially alpine tea gardens, often suffer from low temperature freezing damage during winter or "cold spring" in spring. Freeze injury has become a more prominent and urgent problem to be solved in the high-quality and high-yield cultivation and production of tea in my country. For a long time, the cold resistance of tea gardens has generally been improved by optimizing cultivation me...

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 李叶云班秋艳添先凤谢小芳江昌俊
Owner ANHUI QIMEN COUNTY QIHONG TEA
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