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Peony ACC synthase gene Ps-ACS1 specific probe

A synthetase, specific technology, applied in the direction of DNA/RNA fragment, recombinant DNA technology, microbial assay/inspection, etc., to avoid the effect of expression interference

Inactive Publication Date: 2009-12-16
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But in peony, even in the whole Paeoniaceae, the cloning of ACS gene and the research on its regulation mechanism have not been reported yet.

Method used

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  • Peony ACC synthase gene Ps-ACS1 specific probe
  • Peony ACC synthase gene Ps-ACS1 specific probe
  • Peony ACC synthase gene Ps-ACS1 specific probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The cloning of embodiment 1Ps-ACS1 gene

[0017] 1. Extraction of total RNA from peony petals

[0018] (1) The plastics, glassware and reagents used for RNA extraction were prepared to remove RNase according to the "Molecular Cloning Experiment Guide" (third edition).

[0019] (2) Take 1.0 g of the petal sample of the peony 'Luoyang Red' in full bloom, grind it to powder in liquid nitrogen, quickly transfer it to a 1.5 mL centrifuge tube, add 600 μL of CTAB extract preheated at 65 ° C (2% CTAB, 2% PVP, 100mM TrisCl (pH8.0), 10mMNaCl, 25mM EDTA (pH8.0), 2% β-mercaptoethanol (add before use)), close the lid tightly, and immediately vortex vigorously for 30s to completely disperse the powder In the solution, incubate at 65°C for 2-5min, vortex vigorously 3-5 times during the period, after cooling to room temperature, add an equal volume of chloroform / isoamyl alcohol (24:1) to the above mixture for extraction twice, After vortexing for 5 minutes each time, centrifuge at r...

Embodiment 2

[0051] Preparation and hybridization detection of embodiment 2 probes

[0052] 1. Probe Preparation

[0053] According to the instructions of the PCR DIG probe synthesis Kit (Roche, Cat. No. 1636090), the Ps-ACS1 intermediate fragment obtained by RT-PCR was labeled with digoxigenin to prepare probe A. In addition, according to the sequence near the 3′ end of Ps-ACS1, a pair of specific primers were designed, Ps-ACS1F: 5′-CAGGTCTCT TCTTATGGATGG-3′, Ps-ACS1R: 5′-GTAATTAAGCTCGAAGAAGGG-3′, and Ps-ACS13′ RACE The product is used as a template, and the amplified fragment is labeled with digoxigenin to prepare specific probe B. After the reaction, 3 μl of the PCR product was taken to detect the labeling status, and the remaining probes were stored in a -20°C refrigerator for future use.

[0054] 2. Southern hybridization

[0055] (1) Main reagents and their preparation

[0056] A. Denaturing solution: 0.5M NaOH, 1.5M NaCl.

[0057] B. Neutralizing solution: 0.5M Tris base, 1.5M ...

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Abstract

The invention provides a peony ACC synthase gene Ps-ACS1 specific probe with a nucleotide sequence presented by a sequence table SEQ ID NO.1 or useful length segments thereof. Proved by experiments, the probe can be used for the specificity detection of a Ps-ACS1 gene, avoids the expression interference brought by other gene families because of higher homology in the gene expression analysis research, is beneficial to researching the adjusting and controlling mechanisms and the action modes of different ACS members in a peony ethane reaction on the basis and provides the basis for thoroughly investigating the relations between ethane and the flower-cutting, the blossoming and the wilting of peonies and disclosing the action mode and the adjusting-controlling mechanism of the ethane in the deflowered blossoming and wilting processes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a specific probe for ACC synthase gene Ps-ACS1 of tree peony. Background technique [0002] Peony (Paeonia suffruticosa) is a fine product among traditional Chinese famous flowers. It is known as "National Beauty and Heavenly Fragrance" and "King of Flowers". Therefore, it has gradually become one of the most popular flowers and trees in the world. In recent years, with the growing market demand for fresh cut flowers, in addition to garden seedlings and potted flowers, the potential international market for peony cut flowers is also expanding. However, due to the concentrated natural flowering period of peony, the short flowering period of a single flower, and the unqualified post-harvest storage and fresh-keeping technology, it is difficult to guarantee the quality of fresh cut flowers, and it is impossible to achieve long-term and long-distance supply. This not only restricts the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 董丽周琳贾培义王玮然
Owner BEIJING FORESTRY UNIVERSITY
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