TUA segment sequence for Phalaenopsis internal reference gene

An internal reference gene, Phalaenopsis technology, applied in the field of molecular biology, can solve the problems of blank, real-time quantitative PCR results, etc.

Inactive Publication Date: 2014-09-03
ZHENGZHOU NORMAL UNIV
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AI Technical Summary

Problems solved by technology

The high sensitivity of real-time quantitative PCR technology requires more stable expression of internal reference genes to meet the rigor of the experiment and ensure the reliability of the data. Blind selection of internal reference genes will cause real-time quantitative PCR results to vary due to changes in their own expression. The accuracy is reduced, and even the opposite or wrong conclusions are obtained. In real-time quantitative PCR, the selection of internal reference genes with stable expression is the core of the technology
In the study of gene quantitative analysis of Phalaenopsis using real-time quantitative PCR, there is only a report using the actin gene (ACTIN) as an internal reference gene, and the study of internal reference genes stably expressed in different developmental stages and tissues of Phalaenopsis is blank

Method used

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  • TUA segment sequence for Phalaenopsis internal reference gene
  • TUA segment sequence for Phalaenopsis internal reference gene
  • TUA segment sequence for Phalaenopsis internal reference gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0044] Step 1. Design of degenerate primers.

[0045] According to the homologous sequence of tubulin in the GenBank database, DNAMAN software was used to perform multiple sequence alignments, and then primer5.0 software was used to design a pair of degenerate primers in the conserved region of multiple sequences for the cloning of internal reference gene fragment sequences. and primer see

[0046] TUA-F: AACTTYGCCCGYGGTCAYT;

[0047] TUA-R: GGYTGGTAGTTGATRCCRCAC.

[0048] Step 2, PCR amplification and sequencing

[0049] Amplification of TUA fragment sequence

[0050] Using the leaves of the common Phalaenopsis cultivar 'Treasure Red Rose' as the experimental material, take 1 g of the leaves at the full flowering stage, grind them fully in liquid nitrogen, put the powder into 1.5 mL centrifuge tubes, add 1 mL of Trizol from Invitrogen Reagent, shake and mix well, place at room temperature for 10 m, centrifuge at 12000 rpm for 15 m at 4°C, pipette the supernatant into anot...

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Abstract

The invention discloses a TUA segment sequence for a Phalaenopsis internal reference gene. Multisequence comparison degenerate primer design, PCR (polymerase chain reaction) amplification and sequencing are performed to obtain the alpha-tubulin gene (TUA) segment sequence; and according to the designed fluorescence quantitative specific primers, real-time fluorescence quantitative PCR detection is performed to determine that the TUA segment sequence is an internal reference gene which can be stably expressed in different tissues in different Phalaenopsis development stages.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a TUA fragment sequence used for an internal reference gene of Phalaenopsis. Background technique [0002] Phalaenopsis spp., also known as Phalaenopsis orchid, has beautiful flower shape, bright colors, neatly arranged inflorescences, and a flowering period of 3 to 5 months. It is known as the "Queen of Orchids" and has high ornamental value. The first flagship product of Chinese New Year Flowers in the region occupies an increasingly important position in the flower industry. With the wide application of biotechnology in flower breeding, the molecular mechanism of Phalaenopsis development regulation has become a research hotspot. The expression of related genes Analysis is the main content of its molecular regulation mechanism research. Real-time quantitative PCR is an important means of gene quantitative expression analysis research. The key to implementing real-time quantitat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/10C12Q1/68
Inventor 袁秀云蒋素华梁芳王林青王默霏
Owner ZHENGZHOU NORMAL UNIV
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