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Fluorescence quantitation internal reference genes of leaf tissues of different parts of two pear trees different in three form and primer and application thereof

An internal reference gene and fluorescence quantitative technology, applied in the field of plant molecular biology, can solve the problem of low accuracy and achieve the effect of wide applicability

Active Publication Date: 2018-05-25
INST OF FRUIT & TEA HUBEI ACAD OF AGRI SCI
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  • Abstract
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  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to develop a method applicable to different tree shapes (scaffold tree shape and evacuation layered shape) and various tree body parts (outside, middle, inside) aiming at the deficiency that the accuracy of traditional single internal reference gene is not high. The dual internal reference genes and their specific primers for gene expression analysis in leaf tissue provide a reliable guarantee for the accurate quantification of genes under this experimental condition

Method used

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  • Fluorescence quantitation internal reference genes of leaf tissues of different parts of two pear trees different in three form and primer and application thereof
  • Fluorescence quantitation internal reference genes of leaf tissues of different parts of two pear trees different in three form and primer and application thereof
  • Fluorescence quantitation internal reference genes of leaf tissues of different parts of two pear trees different in three form and primer and application thereof

Examples

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Embodiment 1

[0026] Example 1: Screening out that Actin2 and ARM are the optimal double internal reference gene combinations for the fluorescence quantitative analysis of different tree parts of two pear trees

[0027] 1. Sampling of experimental materials: The adult leaf tissues of 'Yuanhuang' pears in two tree shapes (scaffold tree shape and evacuation layered shape) were used as experimental materials. 1m away from the trunk), middle (0.5-1.0m from the central trunk) and inner (0-0.5m from the central trunk) leaf tissue. Three pear trees of each tree shape were selected as three biological replicates. Leaf tissue samples were isolated and quickly frozen in liquid nitrogen, and then stored at -80°C.

[0028] 2. Extraction of total RNA from leaf materials: Use the RNAprep Pure Plant Kit (Tiangen) RNA extraction kit to extract total RNA from leaf tissue samples according to the instructions.

[0029] 3. Detection of total RNA integrity: 1.2% agarose gel electrophoresis, 1×TAE buffer, 100V...

example 2

[0043] Example 2: Calculation of the expression level changes of pear APX gene with different internal reference genes

[0044] The optimal internal reference gene combination (Actin2 and ARM), two unstable internal reference genes (HistoneH3 and TIP41-like), and two commonly used internal reference genes (UBQ10 and TUBβ) were used as normalization factors to normalize the APX gene. analyze( Figure 4 ). The results showed that when the two most stable internal reference genes Actin2, ARM and their combination were selected, the gene quantification was the most accurate. However, in the case of quantitative analysis using an unstablely expressed internal reference gene and two commonly used internal reference genes, the expression level of APX gene produced a large deviation in some samples.

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Abstract

The invention discloses fluorescence quantitation internal reference genes of leaf tissues of different parts of two pear trees different in three form and primer and application thereof. According tothe fluorescence quantitation internal reference genes of leaf tissues of different parts of the two pear trees different in three form and primer and application thereof, qRT-PCR (quantitative real-time polymerase chain reaction) is applied to analyzing the expression level of 13 candidate internal reference genes in pear leaf tissue samples of different parts of the two pear trees different intree form, geNorm software is applied to estimating the expression stable value of the candidate internal reference genes to determine that ARM and Actin2 are the optimal internal reference gene combination; different internal reference genes are applied to performing relative expression standardization analysis on APX to prove that the internal reference combination of ARM and Actin2 is high inreliability when serving as normalized factors.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and in particular relates to a fluorescent quantitative internal reference gene of leaf tissues in different tree body parts of two tree-shaped pears, primers and applications thereof. Background technique [0002] Pear is one of the most economically valuable fruit trees in the world and in my country. Different tree structures affected the distribution and penetration of photosynthetically active radiation in the canopy, and then had a regulating effect on fruit quality. The traditional pear tree shape is tall and the canopy is easy to close. In recent years, the trellis tree shape has been vigorously promoted in China. The tree structure is open and receives even light, which is conducive to improving the commercial performance of the fruit. Studying the expression level changes of related genes in various parts of different pear tree shapes will help to analyze the molecular mechanism ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/13C12Q2600/166C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 刘政秦仲麒伍涛李先明涂俊凡杨夫臣朱红艳
Owner INST OF FRUIT & TEA HUBEI ACAD OF AGRI SCI
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