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Fluorescent quantitative internal reference genes and their primers and applications in different tissues of Cinnamomum camphorensis

A fluorescence quantitative, internal reference gene technology, applied in the application, genetic engineering, plant gene improvement and other directions, can solve problems such as quantitative research on the relative expression of pest functional genes, and achieve the effect of high amplification efficiency and strong specificity

Active Publication Date: 2020-10-30
NANJING FORESTRY UNIV
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AI Technical Summary

Problems solved by technology

Therefore, it is necessary to screen the stability of internal reference genes in different tissues of the pest, otherwise it is impossible to quantitatively study the relative expression of the functional genes of the pest

Method used

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  • Fluorescent quantitative internal reference genes and their primers and applications in different tissues of Cinnamomum camphorensis
  • Fluorescent quantitative internal reference genes and their primers and applications in different tissues of Cinnamomum camphorensis
  • Fluorescent quantitative internal reference genes and their primers and applications in different tissues of Cinnamomum camphorensis

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Embodiment 1

[0026] 1. Extraction of total RNA and synthesis of cDNA from different tissues of Cinnamomum chinensis

[0027] Different tissues of 4th instar larvae and adults were collected (the head does not contain antennae, thorax, abdomen, wings, feet, and antennae). Each treatment had three biological replicates, and they were frozen in liquid nitrogen, and the Trizol method was used after treatment. Total RNA was extracted and treated with 5 mL of RNase-free DNase I (Takara Bio Inc., Kusatsu, Shiga Prefecture, Japan) for 15 minutes to remove DNA. The concentration and quality of RNA were detected using a Thermo Scientific NanoDrop 2000 UV-Vis spectrophotometer (Thermo FisherScientific Inc., Waltham, MA, USA). The integrity of total RNA was detected by 1.0% agarose gel electrophoresis. Using PrimeScript TM II first-strand cDNA synthesis kit (TaKaRa, Dalian, China), purify RNA and synthesize the first-strand cDNA according to the instructions. Perform reverse transcription in a 20.0...

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Abstract

The invention discloses a fluorescent quantitative internal reference gene of different tissues of Cinnamomum camphora and its primers and application, and belongs to the field of forestry insect molecular biology. The fluorescent quantitative internal reference genes are RPL18 and RPS15 genes, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.7. The present invention selects 8 candidate internal reference genes that are commonly used as internal reference genes through the transcriptome sequencing database of Cinnamomum chinensis, evaluates the stability of candidate internal reference genes through 5 algorithms, and obtains the fluorescence of different tissues of Cinnamomum syringus Quantify the internal reference gene, and design the real-time fluorescent quantitative PCR primers of the internal reference gene, which have strong specificity and high amplification efficiency. This application fills in the fact that there are no internal reference genes in different tissues of Cinnamomum chinensis, and provides a reliable analysis basis for the quantification of the expression of related functional genes in different tissue conditions of Cinnamomum syringus.

Description

technical field [0001] The invention belongs to the field of molecular biology of forestry insects, and in particular relates to fluorescent quantitative internal reference genes of different tissues of Cinnamomum camphorensis, primers and applications thereof. Background technique [0002] Pagiophloeus tsushimanus (Pagiophloeus tsushimanus) belongs to Coleptera of Coleptera, Curculionidae of Weevil family, and Pagiophloeus genus Pagiophloeus is a new record species in China. The adults mainly feed on the twig epidermis of 1-2 years old, and the larvae feed on the cambium between the xylem and the phloem, and the severely damaged camphor grows weak or dies. At present, Cinnamomum sinensis is only distributed in Shanghai, China. The insect has one generation per year in Shanghai, and overwinters as larvae and adults. At present, camphor forests in various ecological public welfare forests in Shanghai are affected by the insect in large areas, and there is a tendency to spre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/6888C12N15/12C12N15/11
CPCC07K14/43563C12Q1/6851C12Q1/6888C12Q2600/166C12Q2531/113C12Q2545/101C12Q2563/107
Inventor 郝德君李寿银陈聪李慧樊彬琦王焱张岳峰韩阳阳
Owner NANJING FORESTRY UNIV
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