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A kind of lycoris lldfrb gene and its expressed protein and application

A long tube lycoris and gene technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of less analysis and achieve the effect of improving ornamental characters

Active Publication Date: 2020-08-25
南京嘉德生态环境科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The function and expression regulation mode of DFR gene in plants are changeable, but there is little analysis of the expression regulation mechanism of this gene in monocotyledonous plants. Studying the function of DFR gene in Lycoris longifolia in the regulation of flower color can provide Using genetic engineering to improve ornamental traits in plants provides useful molecular tools

Method used

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  • A kind of lycoris lldfrb gene and its expressed protein and application
  • A kind of lycoris lldfrb gene and its expressed protein and application
  • A kind of lycoris lldfrb gene and its expressed protein and application

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1: Cloning and expression analysis of L1DFRb gene

[0024] (1) Clone LlDFRb gene

[0025] In this example, TIANGEN Plant RNA Extraction Kit (DP432) was used to extract total plant RNA. Using TaKaRa PrimeScript TM The RT Master Mix (Perfect Real Time) reverse transcription kit reverse-transcribed the extracted RNA into cDNA, and the final cDNA was diluted 10 times with water and stored in a -20°C refrigerator.

[0026] According to the Lycoris petal transcriptome database obtained by the previous research group, a Unigene fragment with high homology of DFR protein was screened, and compared with the NCBI database, it was preliminarily confirmed that the full-length integrity of these screened genes was good. And use Primer Premier5.0 software to design a pair of specific primers (F: 5'-GAGAGAGAGAGATGAAGGGGC-3'; R: 5'-GTCATAAGCTTGTATTGTGGGTG-3') to clone the coding region sequence.

[0027] Using the 1st Strand cDNA as a template, use the EasyPfu Mix high-f...

Embodiment 2

[0043] Embodiment 2: Vector construction and functional verification of LlDFRb

[0044] I. Observation of subcellular localization of LlDFRb gene expression

[0045] (1) Target fragment clone recovery and sequencing

[0046] The complete ORF of the L1DFRb gene was analyzed for restriction sites in the BioXM software, and the available restriction endonucleases Kpn I and Sma I were finally selected. Primers designed to contain enzyme cleavage sites are:

[0047] F: 5'-GAGAGAGAGAGATGAAGGGGC-3';

[0048] R: 5'-GTCATAAGCTTGTATTGTGGGTG-3'.

[0049] Using the correctly sequenced pEASY-Blunt-LlDFRb plasmid as a template, use PrimerStar Max (TaKaRa) high-fidelity enzyme for PCR amplification. The reaction system is 50 μL: 1 μL 1st Strand cDNA, 2 μL ORF Forward Primer (10 mM), 2 μL ORF Reverse Primer ( 10mM), 25μL 2×PrimerStar Max (Takara), 20μL ddH 2 O. Reaction conditions: pre-denaturation at 98°C for 1min; denaturation at 98°C for 10s, annealing for 5s, extension at 72°C for 1...

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Abstract

The invention discloses a Lycoris lycoris L1DFRb gene and its expressed protein and application, belonging to the field of plant molecular biology. The nucleotide sequence of the L1DFRb gene is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.1. Shown in ID NO.2. The LlDFRb gene is an important regulatory gene in the downstream pathway of anthocyanin synthesis. It is actively expressed at the early bud stage of Lycoris, and then decreases with the opening of flowers, which can deepen the flower color of genetically transformed tobacco, from pink to light red. is a DFR gene that can change flower color. Applying the LlDFRb gene to transgenic plants can improve the flower color of the plants so as to improve their ornamental value, which has practical application value.

Description

technical field [0001] The invention belongs to the field of plant molecular biology, and in particular relates to a Lycoris lycoris L1DFRb gene and its expressed protein and application. Background technique [0002] Lycoris longituba (Lycoris longituba) belongs to the genus Lycoris of the family Amaryllidaceae. It is a unique species in my country. It is mainly produced in Jiangsu and Anhui. Its flower color variation is extremely rich, including pink, yellow, orange, purple Red, red-blue and rare emerald green, etc., and the flower type is also very variable, and some can emit a delicate fragrance, which has great research value and application potential. Dihydroflavonol 4-reductase (DFR) is an important regulatory gene in the downstream pathway of anthocyanin synthesis, which catalyzes dihydroflavonol to produce colorless geranium, colorless delphinidin and colorless cyanidin, respectively , is an important node for anthocyanins to change from colorless to colored. As a ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/84A01H5/02A01H6/82
CPCC12N9/0006C12N15/825C12Y101/01219
Inventor 岳远征史俐莎贾瑞瑞陈敏杨秀莲施婷婷王良桂
Owner 南京嘉德生态环境科技有限公司
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