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Chimonanthus praecox CpSRG1 gene, promoter and application of Chimonanthus praecox CpSRG1 gene

A wintersweet and gene technology, applied in the field of molecular biology, can solve problems such as discovering dioxygenase superfamily genes

Pending Publication Date: 2021-05-04
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the discovery of dioxygenase superfamily genes in Wintersweet

Method used

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  • Chimonanthus praecox CpSRG1 gene, promoter and application of Chimonanthus praecox CpSRG1 gene
  • Chimonanthus praecox CpSRG1 gene, promoter and application of Chimonanthus praecox CpSRG1 gene
  • Chimonanthus praecox CpSRG1 gene, promoter and application of Chimonanthus praecox CpSRG1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Isolation of Wintersweet CpSRG1 Gene

[0026] Randomly selected clones were sequenced from the Wintersweet cDNA library (provided by the Flower Research Office of the School of Horticulture and Landscape Architecture, Southwest University) to obtain EST sequences, which were clustered and analyzed to preliminarily confirm the functional properties. According to the annotation results, the target clones were selected for bidirectional sequencing confirmation. Obtain the gene sequence of Wintersweet senescence-related gene (Senescence Related Gene 1) SRG1, use the software Primer primer 5.0 to design specific primers and carry out PCR amplification, and the primer sequences are as follows:

[0027] CpSRG1-F: 5'-ccgagccgcgtgagctgctac-3' (SEQ ID No.4)

[0028] CpSRG1-R: 5'-ccgcatgcataagcttgctcgag-3' (SEQ ID No.5)

[0029] Using Wintersweet cDNA as a template to amplify the CpSRG1 gene of Wintersweet, the PCR reaction system and reaction conditions are as follows:...

Embodiment 2

[0032] Example 2 Analysis of the expression characteristics of Wintersweet CpSRG1 gene

[0033] 1. RNA extraction

[0034] (1) The equipment used for RNA extraction, mortar, mortar stick, spoon, scissors, etc., were wrapped in tin foil, placed in an oven at 180°C for 4 hours, and cooled for later use.

[0035] (2) Take wintersweet leaves, grind them fully with liquid nitrogen in a mortar, quickly pour them into a RNase-removing centrifuge tube, add 600 μL Trizol (plant RNA extraction reagent), vortex to mix, and place at room temperature for 10 minutes.

[0036] (3) Add 120 μL of chloroform, vortex and mix well, and let stand at room temperature for 10 min.

[0037] (4) Centrifuge at 12,000 rpm for 10 min at 4°C, and pipette 200 μL of the supernatant into another centrifuge tube.

[0038] (5) Add 2 times the volume of absolute ethanol and mix well, then centrifuge at 12000rpm at 4°C for 30min, discard the supernatant, and keep the precipitate.

[0039] (6) Wash the precipit...

Embodiment 3

[0055] Cloning of Example 3 Wintersweet CpSRG1 Promoter

[0056] 1 Acquisition of CpSRG1 Gene Promoter Fragment in Wintersweet

[0057] The promoter of CpSRG1 gene was amplified by hiTAIL-PCR. Based on the cloned CpSRG1 gene 5' end sequence, three specific nested primers RB0, RB1, and RB2 were designed, combined with degenerate primers LAD and AC0 / AC1, and three rounds of amplification were performed respectively. The primer sequences used in the PCR process are shown below. Wherein n, v, b, d are degenerate bases, n represents a, g, c or t, v represents g, a or c, b represents g, t or c, and d represents g, a or t.

[0058] LAD1: 5'-acgatggactccagagcggccgcvnvnnnggaa-3' (SEQ ID No. 12);

[0059] LAD2: 5'-acgatggactccagagcggccgcbnbnnnnggtt-3' (SEQ ID No. 13)

[0060] LAD3: 5'-acgatggactccagagcggccgcvvnvnnnccac-3' (SEQ ID No. 14)

[0061] LAD4: 5'-acgatggactccagagcggccgcbdnbnnnccaa-3' (SEQ ID No. 15)

[0062] LAD5:5'-acgatggactccagagcggccgcbdnbnnncggt-3' (SEQ ID No. 16)

...

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PUM

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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a chimonanthus praecox CpSRG1 gene, a promoter and application of the Chimonanthus praecox CpSRG1 gene. The invention provides a chimonanthus praecox CpSRG1 protein, a gene for coding the protein, cloning of the promoter and application of the chimonanthus praecox CpSRG1 protein. The chimonanthus praecox CpSRG1 gene is obtained through cloning for the first time, the expression characteristic of the CpSRG1 gene is detected through a real-time fluorescent quantitative PCR technology, and a function of the promoter of the gene is verified in a plant (arabidopsis thaliana) through a transgenic technology. Reference and basis are provided for research on senescence, stress response (hormone) and regulation mechanism of chimonanthus praecox and leaves, and possibility is provided for further improvement of ornamental plants, such as prolonging of the flowering phase of chimonanthus praecox.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to the wintersweet CpSRG1 gene, promoter and application thereof. Background technique [0002] Wintersweet (Chimonanthus praecox) is a deciduous shrub of the family Chimonanthus genus Chimonanthus praecox, native to China and widely distributed in south-central and southwest China. Wintersweet is a rare winter flowering plant, and the flowering period is from December to February of the following year. With its unique flower color and pleasant fragrance, wintersweet can be used as potted plants, ornamental cut flowers and landscaping plants. At present, some progress has been made in the research of wintersweet on abiotic stress and flower development. [0003] The dioxygenase superfamily 2-OGD is the second largest enzyme family in plants, and its members are widely involved in various oxidation / hydroxylation reactions. Recent studies have also found that ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/113C12N15/82C07K14/415A01H5/02A01H5/12A01H6/20A01H6/00
CPCC07K14/415C12N15/8266C12N15/827C12N15/8291C12N15/8297
Inventor 马婧李桂香刘道凤罗江会门维婷李名扬眭顺照
Owner SOUTHWEST UNIVERSITY
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