Sequence of the glo/pi MADS-box gene and its coded amino acid sequence

An amino acid and nucleotide sequence technology, applied in genetic engineering, plant genetic improvement, DNA preparation, etc.

Inactive Publication Date: 2011-11-30
广东省农业科学院花卉研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the relevant research reports on the molecular mechanism of the development of Paphiopedilum are rare at home and abroad.

Method used

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  • Sequence of the glo/pi MADS-box gene and its coded amino acid sequence
  • Sequence of the glo/pi MADS-box gene and its coded amino acid sequence
  • Sequence of the glo/pi MADS-box gene and its coded amino acid sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Extraction of total RNA

[0055] Take the flowers of Paphiopedilum of the same color, and use the following extraction steps:

[0056] Grind the sample into powder in liquid nitrogen, quickly add 500 μl of Solution D, 500 μl of water-saturated phenol, 200 μl of 3 M sodium acetate (pH 5.2), 50 μl of 4 M β-mercaptoethanol at the rate of 10 ml / g sample, Shake and mix thoroughly, and place at room temperature for 1 min; add 200 μl chloroform, shake vigorously for 1 min; centrifuge at 12 000 g for 10 min at 4 °C, carefully transfer the supernatant to another RNase-free centrifuge tube; add silica to suspend Solution 100 μl, absolute ethanol 200 μl, 3 M sodium acetate (pH 5.2) 200 μl, mix slowly with a micropipette, room temperature, centrifuge at 12 000 g for 1 min, discard the supernatant; add 1 ml of 70% ethanol, Mix with a pipette, centrifuge at 12 000 g for 1 min at room temperature, discard the supernatant, and repeat twice; dry at room temperature for 8-10 m...

Embodiment 2

[0057] Example 2 Synthesis of the first strand of cDNA and amplification of full-length double-stranded cDNA by LD-PCR

[0058] cDNA first-strand synthesis: follow SMART TM The operation steps of PCR cDNA Synthesis Kit (CLONTECH, U.S.A.), take 1.0 μg of total RNA of Phyllostachys chinensis, mix with 1.0 μl of reverse transcription primer 3' SMART CDS Primer ⅡA (12 μM), and put it on the PCR machine at 72°C for 3min , then 42°C, 2min, quickly take out, put at room temperature, then add 5×First Strand Buffer 2μl, DTT (100 mM) 0.25μl, dNTP Mix (10 mM of each dNTP) 1μl, SMART ⅡA Oligonucleotide (12 μM) 1μl , Rnase inhibitor 0.25μl, Power Script Reverse Transcriptase (100U) 1μl, the reaction system is 10μl. The reaction program was 60 minutes at 42°C, 7 minutes at 72°C, and finally stored at -20°C for future use.

[0059] LD-PCR method to amplify full-length double-stranded cDNA: Preheat the PCR instrument to 95°C, take 2 μl of diluted ss cDNA as the reaction template, Deionized...

Embodiment 3

[0060] Example 3 Design primers and synthetic primers

[0061] Download related species from the GenBank database (such as Paphiopedilum longifolia, Cymbidium arvensis, Claw orchid, Chunlan, Cymbidium, etc.) GLO / PI The amino acid sequence encoded by -like MADS-box gene was compared with CLUSTAL X (1.8) to determine the conserved region of the amino acid sequence, and the primers were designed according to the conserved region sequence RQVTFSK GLO / PI F1 (5'→3'): CGGCAAGTGACCTTCTCGAAG; Design degenerate primers based on the conserved region sequence MLEEENKR GLO / PI R1 (5'→3'): CTTTTATTTTCCTCTTCCAGCAT. The PCR amplified fragment size is about 430bp. The primers were designed and synthesized by Shanghai Handsome Biotechnology Company.

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Abstract

The invention provides a Paphiopedilum GLO / PIMADS-box gene sequence and a polypeptide encoded by the gene sequence. In addition, the present invention also discloses a method for obtaining the clone of P. paphiopedilum GLO / PI gene. The method is to design a degenerate primer for the amino acid conservation region of the closely related species GLO / PIMADS-box gene, extract total RNA from the flower of P. -PCR amplification, combined with RACE technology to clone to obtain the full-length cDNA sequence of the GLO / PIMADS-box gene of Paphiopediphyllum. The P. paphiopedilum GLO / PIMADS-box gene of the present invention is a new gene related to the development of P. paphiopedilum. The P. paphiopedilum GLO / PI gene provided by the present invention not only lays a foundation for further research on the function of the P. paphiopedilum GLO / PI protein, but also provides a certain basis for the molecular mechanism of the paphiopedilum organ development.

Description

technical field [0001] The invention belongs to the field of genes in molecular biology, in particular to Paphiopediphyllum GLO / PI MADS-box gene sequence and its encoded amino acid sequence. Background technique [0002] Paphiopedilum ( Paphiopedilum spp.), also known as slipper orchid, belongs to Orchidaceae (Orchidaceae) Paphiopedilum ( Paphiopedilum Pfitz.) plants, distributed in tropical Asia to some islands in the Pacific Ocean; they are terrestrial orchids, and a few are epiphytic orchids; the leaves are basal, banded, and dichotomous. The orchid has a peculiar shape, large and colorful flowers, and has high potted ornamental value and economic value. At present, Paphiopedilum is one of the important commercial Cayenne orchids, and occupies a pivotal position in the Cayenne orchid flower market. Therefore, it is of great theoretical and practical significance to explore the molecular mechanism of Paphiopedilum development. However, there are few research repor...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/10C07K14/415C12Q1/68
Inventor 李冬梅朱根发吕复兵操君喜
Owner 广东省农业科学院花卉研究所
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