Application of Phalaenopsis pp2a Gene as Internal Reference Gene

An internal reference gene, phalaenopsis technology, applied in the field of Phalaenopsis genetic engineering, can solve the problems that have not yet been seen.

Active Publication Date: 2022-06-07
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 pp2a Gene as Internal Reference Gene
  • Application of Phalaenopsis pp2a Gene as Internal Reference Gene
  • Application of Phalaenopsis pp2a Gene as Internal Reference Gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This example mainly focuses on the internal reference gene of Phalaenopsis PP2A A brief introduction to the acquisition process and sequence structure characteristics of .

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

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

[0043] In the greenhouse, two biennial Phalaenopsis varieties of the same growth period, "Big Pepper" (represented by D in the following) and "Fu Le Sunset" (represented by F in the following), were selected in the same growth period, and the normal temperature in the plant artificial climate box was 26 ℃ / 22℃ pre-incubated for 15 d (culture conditions: light 60 μmol m -2 ·s -1, light-dark ratio of 12 h / 12 ​​h...

Embodiment 2

[0055] On the basis of Embodiment 1, this embodiment mainly focuses on Phalaenopsis PP2A The stability of gene transcription and expression under normal growth temperature and low temperature stress was studied and analyzed. The specific experimental situation is briefly described as follows.

[0056] (1) Real-time fluorescent quantitative PCR primer design

[0057] Based on the sequencing results of Example 1, the design PP2A Gene real-time 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 fluorescence quantitative PCR reaction

[0062] Take equal amount of 21 cDNA samples of leaves of different varieties of Phalaenopsis prepared in Example 1, different low temperature treatment conditions and times, mix them evenly as a standard, and dilute 10 time...

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 inventors used Phalaenopsis PP2A gene as an internal reference gene, with NAC domain protein gene PhNAC1 As an example, the expression characteristics of this gene in leaves of two different cultivars with different treatment times under the condition of low temperature stress of Phalaenopsis were studied and analyzed. The relevant experimental process is briefly introduced as follows.

[0074] (1) Primer design

[0075] Real-time quantitative PCR detection of NAC domain protein genes ( PhNAC1 ), 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-time fluorescence quantitative PCR...

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Abstract

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. said PP2A The cDNA sequence of the gene is 2207 bp in length, including a complete ORF of 1764 bp in length, encoding phosphoprotease 2A (PP2A). Further use of real-time fluorescent quantitative PCR technology for the PP2A The study on the expression of the gene under the normal growth temperature and low temperature stress conditions of Phalaenopsis showed that the expression of the gene is relatively stable, and it has the potential to be used as an internal reference gene. It was further used as an internal reference gene for analysis PhNAC1 When it comes to gene expression, it shows more accurate analysis results. It can lay a good analysis and application foundation for the screening of cold tolerance genes of Phalaenopsis and the selection of cold tolerance germplasm resources, so it has 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. Background technique [0002] Protein phosphatase 2A (PP2A) is a major serine / threonine protein phosphatase in eukaryotes, consisting of structural subunit A, catalytic subunit C and regulatory subunit B. Controls signal transduction and cellular metabolism. In animals and yeast, PP2A functions 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 collective name for the single stem epiphytic orchid of the Orchidaceae Phalaenopsis genus. It has become a popular product among Chinese New Year flowers for many years because of its beautiful...

Claims

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Application Information

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Patent Type & Authority Patents(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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