CDNA of Freesia refracta Klatt flavanonol-4-reductase genes

A technology of vanilla dihydroflavonol and reductase, which is applied in the field of cDNA and can solve problems such as functions that have not been verified

Inactive Publication Date: 2015-12-02
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the number of DFR genes obtained has increased sharply, but most of them are from dicotyledonous plants (592), and there are only 52 from monocotyledonous plants, including 17 from Poaceae, and only 1 from Iridaceae, which gathers a large number of ornamental plants, and the other functionality has not been verified

Method used

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  • CDNA of Freesia refracta Klatt flavanonol-4-reductase genes
  • CDNA of Freesia refracta Klatt flavanonol-4-reductase genes
  • CDNA of Freesia refracta Klatt flavanonol-4-reductase genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: FhDFRs Cloning of full-length genes

Embodiment 1-1

[0039] Example 1-1: FhDFRs Acquisition of 3' end: According to the intermediate fragment obtained by screening, design primers for 3' RACE-PCR amplification. The primer combinations are:

[0040]

[0041] The reaction system is as follows:

[0042]

[0043] The PCR reaction procedure is as follows: 95°C for 5min; 94°C for 30s, X°C for 30s, 72°C for 1min, 30Cycle.

[0044] where X°C is the annealing temperature, FhDFR2 (58°C) ,FhDFR3 (58°C) , FhDFR6 (58°C).

[0045] After the gel recovery of the PCR product, it was connected to the T vector and transformed into Escherichia coli JM109Competent cells were screened with Amp-resistant LB plate medium. Randomly pick a single clone, shake the bacteria and carry out the PCR verification of the bacterial liquid, and the positive clones identified are sequenced.

Embodiment 1-2

[0046] Embodiment 1-2: FhDFRs Acquisition of the 3' end: splicing and screening the intermediate fragment and the amplified 3' end fragment, and then designing primers based on this, and using the RACE kit (SMARTRACE cDNA Amplification Kit) from Clontech to perform 5' RACE experiments.

[0047] Primer combinations are as follows:

[0048]

[0049] The reaction system is as follows:

[0050] ① FhDFR2 The touchdown PCR reaction

[0051] The reaction system is as follows:

[0052]

[0053] The reflection procedure is as follows:

[0054]

[0055] ② FhDFR3 nested PCR reaction

[0056] A. Using DFR3-GSP2 as the downstream primer for PCR, the reaction system is as follows:

[0057]

[0058] The reaction procedure is as follows:

[0059]

[0060] B. Dilute the above PCR product 50 times as a template, and use DFR3-GSP1 and NUP as nested primers for PCR

[0061] Amplify. The reaction system system is as follows:

[0062]

[0063] The reaction procedure ...

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Abstract

The new cDNA of three dihydroflavonol-4-reductase (DFR) genes is obtained through cloning from red flower Freesia refracta Klatt petals for the first time in the invention, and the nucleotide sequence of the cDNA and an amino acid sequence obtained from the nucleotide sequence are determined. Gene expression of the three genes has obvious correlation with anthocyanin synthesis in the coloring process of Freesia refracta Klatt flowers; and additionally, after eukaryotic expression vectors of the three FhDFRs are transferred to Arabidopis thaliana tt3 mutants, phenotypes of seedlings and seed coats are recovered.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, in particular to the separation of three coding chalcone isomerases ( wxya ) cDNA of the gene and its encoding. Background technique [0002] Since the DFR gene was first isolated from snapdragon and corn in 1985, DFR genes of many species have been cloned one after another, such as petunia, Arabidopsis, rice, purple sweet potato, apple, grape, peanut, rose, ginkgo and lily etc. In recent years, the number of DFR genes obtained has increased sharply, but most of them come from dicotyledonous plants (592), and there are only 52 from monocotyledonous plants, including 17 from Poaceae, and only 1 from Iridaceae, which gathers a large number of ornamental plants, and the other Functionality has not been verified. [0003] DFR belongs to the NADPH-dependent short-chain reductase (SDR) family. This gene is generally a single copy in the plant genome, and exists as a small gene family in a fe...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04
Inventor 高翔蔡馨荃刘兴雪王丽
Owner NORTHEAST NORMAL UNIVERSITY
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