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Fluorescent quantification of internal reference genes for different insect states of Cinnamomum camphorata and their primers and applications

A fluorescence quantitative and gene technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as quantitative research on relative expression of functional genes of pests, etc., to improve repeatability and amplification. Efficient and specific effects

Active Publication Date: 2020-09-18
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is necessary to screen the stability of internal reference genes in different insect states 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 quantification of internal reference genes for different insect states of Cinnamomum camphorata and their primers and applications
  • Fluorescent quantification of internal reference genes for different insect states of Cinnamomum camphorata and their primers and applications
  • Fluorescent quantification of internal reference genes for different insect states of Cinnamomum camphorata and their primers and applications

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Embodiment 1

[0025] 1. Extraction of total RNA and synthesis of cDNA in different stages of C.

[0026] Eggs, larvae, pupae, and adults were collected in different stages, and each treatment had three biological replicates, which were frozen in liquid nitrogen. After the treatment, total RNA was extracted by Trizol method, and 5 mL of RNase-free DNase I (TakaraBio Inc., Kusatsu, Shiga Prefecture, Japan) for 15 minutes to remove DNA. The concentration and quality of RNA were detected using a ThermoScientific NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific 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 μL reaction system containing 4.0 μL 5XPrimeScript IV cDNA Synthesis Mix, 1.0 μL random primer (50 μM), 4.0 μL total RNA,...

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Abstract

The invention discloses a fluorescent quantitative internal reference gene of different insect states of Cinnamomum camphorensis, primers and application thereof, and belongs to the field of forestry insect molecular biology. The fluorescent quantitative internal reference genes are RPL28 and RPS15 genes, the nucleotide sequences of which are shown in SEQ ID NO.6 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 the candidate internal reference genes through 5 algorithms, and obtains the different stages of Cinnamomum syringus. Fluorescent quantitative internal reference genes, and real-time fluorescent quantitative PCR primers for internal reference genes were designed, which have strong specificity and high amplification efficiency. This application fills in the current situation that there is no internal reference gene in different stages of C. camphorensis, and provides a reliable analysis basis for the quantification of the expression of related functional genes in different stages of C. camphorensis.

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 insect states 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 t...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/166C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 陈聪李慧郝德君李寿银苏鹏陈亮樊彬琦
Owner NANJING FORESTRY UNIV