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The cDNA of the gene of fenugreek dihydroflavonol-4 reductase

A technology of dihydroflavonol and reductase of Fragrantia japonica, which can be applied in the field of cDNA and can solve the problems of limited DHK activity and the like

Inactive Publication Date: 2018-10-09
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alfalfa ( Medicago truncatula ) DFR1 belongs to the Asn type, which can effectively reduce DHK; DFR2 belongs to the Asp type, which has very limited activity on DHK

Method used

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  • The cDNA of the gene of fenugreek dihydroflavonol-4 reductase
  • The cDNA of the gene of fenugreek dihydroflavonol-4 reductase
  • The cDNA of the gene of fenugreek dihydroflavonol-4 reductase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: FhDFRs Full-length gene cloning

Embodiment 1-1

[0039] Example 1-1: FhDFRs Obtaining the 3'end: According to the intermediate fragments obtained from the screening, primers are designed for 3'RACE-PCR amplification. The primer combinations are:

[0040]

[0041] The reaction system is as follows:

[0042]

[0043] The PCR reaction program is as follows: 95 ℃ 5 min; 94 ℃ 30 s, X ℃ 30 s, 72 ℃ 1 min, 30 Cycle.

[0044] Where X ℃ represents the annealing temperature, FhDFR2 (58℃) , FhDFR3 (58℃) , FhDFR6 (58°C).

[0045] After the PCR product gel is recovered, it is connected to the T vector and transformed into E. coli JM109 Among the competent cells, use Amp-resistant LB plate medium for selection. Randomly pick a single clone, shake the bacteria and perform PCR verification of the bacterial solution, and sequence the identified positive clones.

Embodiment 1-2

[0046] Example 1-2: FhDFRs Obtaining the 3'end: Assemble the intermediate fragments obtained from screening and the amplified 3'end fragments, and then design primers based on this, and use Clontech's RACE kit (SMART RACEcDNA Amplification Kit) to perform 5'RACE experiments.

[0047] The primer combinations are as follows:

[0048]

[0049] The reaction system is as follows:

[0050] ① FhDFR2 Landing PCR reaction

[0051] The reaction system is as follows:

[0052]

[0053] The response procedure is as follows:

[0054]

[0055] ② FhDFR3 Nested PCR

[0056] A. Using DFR3-GSP2 as downstream primer 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] Amplification. The reaction system is as follows:

[0062]

[0063] The reaction procedure is as follows:

[0064]

[0065] ③ FhDFR6 Normal PCR reaction

[0066] The re...

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PUM

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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, and specifically relates to the separation of three encoding chalcone isomerases from the petals of safflower vanilla FhDFR ) The cDNA of the gene and its encoding. Background technique [0002] Since people first isolated the DFR gene from snapdragon and corn in 1985, DFR genes of many species have been cloned, 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 are from dicots (592). There are only 52 monocots, including 17 in Gramineae, and only 1 in Iridaceae, which has a large number of ornamental plants. The function has not been verified. [0003] DFR belongs to the NADPH-dependent short-chain reductase (SDR) family. The gene is generally a single copy in the plant genome, and exists as a small gene family in a few species, such as petunia, popla...

Claims

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Application Information

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