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Application of phalaenopsis aphrodite PP2A gene as internal reference gene

An internal reference gene, Phalaenopsis technology, applied in the field of Phalaenopsis genetic engineering, can solve problems that have not been seen yet, and achieve good practical value and scientific research application significance

Active Publication Date: 2019-04-12
ZHENGZHOU NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As far as the Phalaenopsis cold-tolerance gene screening is concerned, there are not yet good relevant reports that can be used as internal reference genes in the cold-tolerance gene screening.

Method used

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  • Application of phalaenopsis aphrodite PP2A gene as internal reference gene
  • Application of phalaenopsis aphrodite PP2A gene as internal reference gene
  • Application of phalaenopsis aphrodite PP2A gene as internal reference gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This embodiment is mainly to Phalaenopsis internal reference gene PP2A A brief introduction to the acquisition process and sequence structure characteristics of is as follows.

[0041] (1) Low temperature stress treatment and cDNA template preparation

[0042] In the previous experimental design, in order to screen and obtain genes with relatively stable expression under low temperature stress conditions and normal growth, the inventors carried out low temperature stress treatment on related Phalaenopsis materials. The specific treatment methods are as follows:

[0043] In the greenhouse, two biennial Phalaenopsis varieties "Big Pepper" (represented by D below) and "Fule Xiyang" (represented by F below) in the same growth period were selected, and the normal temperature was 26°C in a plant artificial climate box. Pre-culture at ℃ / 22℃ for 15 days (cultivation conditions: light 60 μmol·m -2 ·s -1, light-to-dark ratio 12 h / 12 ​​h, relative humidity 70-90%), and then low...

Embodiment 2

[0055] On the basis of embodiment 1, present embodiment mainly just Phalaenopsis PP2A The stability of gene transcription and expression under normal growth temperature and low temperature stress conditions has been studied and analyzed. The specific experimental conditions are briefly introduced as follows.

[0056] (1) Primer design for real-time fluorescent quantitative PCR

[0057] Based on the sequencing results of Example 1, the design PP2A Gene real-time fluorescent quantitative PCR primer sequences are as follows:

[0058] qPP2A-F: 5'-TCTGTTGGCTGTGGAAGGAT-3',

[0059] qPP2A-R: 5'-AAATCCGACCTGGTAGTTTCTG-3';

[0060] It should be noted that the length of the amplified product based on the primer sequence is 186 bp.

[0061] (2) Real-time fluorescent quantitative PCR reaction

[0062] Get 21 cDNA samples of the leaves of Phalaenopsis different species, different low temperature treatment conditions and time prepared in the embodiment 1 of equal amount, mix homogeneou...

Embodiment 3

[0073] Based on the results of Example 2, it can be judged that Phalaenopsis PP2A The gene has the potential to be used as an internal reference gene. Therefore, the inventor uses Phalaenopsis PP2A The gene was used as an internal reference gene, and the NAC domain protein gene PhNAC1 As an example, the expression characteristics of this gene in the leaves of two different varieties of Phalaenopsis at different treatment times under the condition of low temperature stress were studied and analyzed. The relevant experimental process is briefly introduced as follows.

[0074] (1) Primer design

[0075] Real-time fluorescent quantitative PCR detection of NAC domain protein gene ( PhNAC1 ), the primers were designed as follows:

[0076] qPhNAC1-F: 5'-ATCTGAACAAGTGCGAGCCT-3',

[0077] qPhNAC1-R: 5'-ATCCTTACCAGTTGCCTTCC-3';

[0078] It should be noted that when the primer sequence is used for amplification, the length of the amplified product is 155 bp.

[0079] (2) Real-ti...

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Abstract

The present invention belongs to the technical field of phalaenopsis aphrodite gene engineering and particularly relates to a patent application for a phalaenopsis aphrodite PP2A gene applied as an internal reference gene. A PP2A gene cDNA sequence is 2,207 bp in length, contains a complete 1,764 bp ORF, and encodes phosphoprotease 2A (PP2A). A real-time fluorescent quantitation PCR is further utilized to study an expression condition of the PP2A gene of phalaenopsis aphrodite under normal growth temperature and low temperature stress, the gene expression amount is relatively stable and has anapplication potential as the internal reference gene. Further, when the phalaenopsis aphrodite PP2A gene is used as the internal reference gene to be used to analyze expression of PhNAC1 gene, a moreaccurate analysis result is shown. The phalaenopsis aphrodite PP2A gene can lay a good foundation for analysis and application of cold tolerance gene screening and cold tolerance germplasm resource screening, thus the phalaenopsis aphrodite PP2A gene has relatively good practical value and scientific research application significance.

Description

technical field [0001] The invention belongs to the technical field of Phalaenopsis genetic engineering, in particular to a Phalaenopsis PP2A Gene as an internal reference gene application patent application matters. Background technique [0002] Protein phosphatase 2A (protein phosphatase 2A, PP2A) is a major serine / threonine protein phosphatase in eukaryotes, composed of structural subunit A, catalytic subunit C and regulatory subunit B, mainly in plants Controls signal transduction and cellular metabolism. In animals and yeast, PP2A plays a role in multiple signaling pathways. However, its role in plant signaling pathways is poorly understood, and it is currently found to be mainly involved in the signal transduction of hormones such as abscisic acid, phytogenin and ethylene. [0003] Phalaenopsis( Phalaenopsis spp.) is the general name of single-stem epiphytic orchids of the genus Phalaenopsis in the family Orchidaceae. Because of its beautiful flower shape, rich an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12Q1/6895C12N15/11C12Q1/686
CPCC12Q1/686C12Q1/6895C07K14/415C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 梁芳张燕牛苏燕郝平安蒋素华许申平袁秀云马杰崔波王墨霏
Owner ZHENGZHOU NORMAL UNIV
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