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Fluorescent quantitative internal reference genes and their special primers and applications under waterlogging stress in Changshan, Haizhou

A fluorescence quantitative, internal reference gene technology, applied in the field of plant molecular biology, to achieve the effect of improving detection efficiency, strong primer specificity, and improving reliability

Inactive Publication Date: 2019-11-29
NANJING FORESTRY UNIV
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  • Application Information

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Problems solved by technology

So far, there has been no report on the research on genes under waterlogging stress in Changshan, Haizhou

Method used

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  • Fluorescent quantitative internal reference genes and their special primers and applications under waterlogging stress in Changshan, Haizhou
  • Fluorescent quantitative internal reference genes and their special primers and applications under waterlogging stress in Changshan, Haizhou
  • Fluorescent quantitative internal reference genes and their special primers and applications under waterlogging stress in Changshan, Haizhou

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Embodiment 1

[0030] 1. Plant tissue total RNA extraction and cDNA synthesis

[0031] Extract total RNA with EASYspin Plus Plant RNA Extraction Kit (Adley Biotechnology Co., Ltd.), use a spectrophotometer (METTLER TOLEDO, Switzerland) to detect RNA concentration and quality, and utilize 1.5% agarose gel electrophoresis to detect the total complete RNA. sex. Using Beijing Quanshijin Reverse Transcription Kit (Quanshijin Biotechnology Co., Ltd.), RNA was purified and the first strand of cDNA was synthesized according to the instructions.

[0032] 2. Selection of internal reference genes and design of primers

[0033] Query the main internal reference genes in other plants according to the literature and finally design 17 internal reference genes required by this embodiment, which are Actin (SEQ ID NO.1), PP2A (SEQ ID NO.2), RPL (SEQ ID NO. .3), PK (SEQ ID NO.4), 18S (SEQ ID NO.5), RAN (SEQ ID NO.6), APT (SEQ ID NO.7), SAND (SEQ ID NO.8), PROF (SEQ ID NO. NO.9), MDH (SEQ ID NO.10), EF-1A (S...

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Abstract

The invention discloses a fluorescent quantitative internal reference gene and its special primers and applications under waterlogging stress in Changshan, Haizhou. The fluorescent quantitative internal reference genes are MDH, UBC‑E2 and UBQ, wherein the gene sequence of the MDH gene is as shown in SEQ ID NO.10 The gene sequence of the UBC-E2 gene is shown in SEQ ID NO.12, and the gene sequence of the UBQ gene is shown in SEQ ID NO.16. The present invention screens candidate genes with relatively stable expression based on the existing published genes, evaluates their stability through software, and obtains three internal reference genes suitable for the most stable expression under the waterlogging stress in Changshan, Haizhou, and uses these genes as a basis to design The real-time fluorescent quantitative PCR primers have filled the current situation that there is no internal reference gene under the waterlogging stress of Changshan in Haizhou, and provided strong support for the accurate quantification of related functional genes under the waterlogging stress of Changshan in Haizhou, which can improve the stability and repeatability of the research and reliability.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and specifically relates to a fluorescent quantitative internal reference gene, a special primer and application thereof under waterlogging stress in Changshan, Haizhou. [0002] technical background [0003] Haizhou Changshan (Clerodendrum trichotomum Thunb.), also known as stinky phoenix tree, is a deciduous shrub or small tree under the genus Clerodendrum L. of the Verbenaceae family. It has certain cold resistance, few pests and diseases, and has strong adaptability. There has not been any report on the gene research under waterlogging stress in Changshan, Haizhou. In the process of exploring the differences in gene expression under waterlogging stress in Changshan, Haizhou, it is necessary to use real-time fluorescence quantitative technology to analyze and verify the expression level of genes, which requires stable and reliable internal reference genes. Stably expressed internal refe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/166
Inventor 杨秀莲刘华歧岳远征施婷婷王良桂华雅洁
Owner NANJING FORESTRY UNIV
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