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Process for producing koda using lemna paucicostata

A technology of Ximaiping and compounds, which is applied in the field of plant hormones and 15-octadecadienoic acid, can solve the problems of low cost, insufficient KODA, suicide substrates, etc.

Inactive Publication Date: 2012-12-19
SHISEIDO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, allene oxide synthase usually has the property of a suicide substrate, and when the concentration of the substrate is increased, the production amount will be reduced instead.
Due to these shortcomings of lipoxygenase, allene oxide synthase, enzymatic methods are not suitable for mass production
On the other hand, it is difficult to achieve the low cost required in the agricultural field by chemical synthesis
[0009] On the other hand, in the existing extraction method, the extraction method was carried out by cultivating the 441 strain of Lemaipina known to produce flower bud-inducing substances with high efficiency, but even in the case of using this strain, the amount of KODA produced can't say enough

Method used

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  • Process for producing koda using lemna paucicostata
  • Process for producing koda using lemna paucicostata
  • Process for producing koda using lemna paucicostata

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: the screening of KODA high production Ximaiping strain

[0044] Sixty-two species of Lenima spp. collected from various places were prepared and subcultured in 1 / 2-fold diluted Hunter's medium under continuous light irradiation from daylight-colored fluorescent lamps at 24 to 25°C. Hunter's Medium diluted 1 / 2 contains the following components:

[0045]

[0046] Use KOH (50%) to adjust to pH 6.2-6.5.

[0047] Spread the proliferated Lemipina on filter paper, let it stand for 2 hours, and then soak it in water for 1 hour. Use this water with high-speed liquid chromatography (HPLC; Column: TYPE UG120 5 μm SIZE 4.6mm I.D × 250mm; Protective filter: INERTSTL 4.6mm × 50mm; Eluent: 50% acetonitrile+0.1% trifluoroacetic acid; Condition: absorbance The wavelength of 210λ (nm), flow rate 1.ml / min, column temperature 40 ℃) to analyze the concentration of KODA. The average amount of KODA production of all Lemipina strains was 4.97 μM. Among them, the Ximaipin...

Embodiment 2

[0048] Example 2: Cloning and activity determination of lipoxygenase from Ximaiping SH strain

[0049] After extracting total RNA from Ximaiping (SH strain) using RNeasy Plant Mini Kit (QIAGEN), 1.8 μg of total RNA was used as a template to synthesize cDNA by LongRange 2Step RT-PCR Kit (QIAGEN).

[0050] Thereafter, using the cDNA as a template, degenerate PCR was carried out using the following degenerate primers (LpDPf, LpDPr) (PCR conditions: initial denaturation at 94°C for 3 minutes; The cycle was carried out 39 times), and the partial sequence of the target 9-lipoxygenase was obtained.

[0051] LpDPf: 5'-GCITGGMGIACIGAYGARGARTTY-3' (sequence number 5)

[0052] LpDPr: 5'-GCRTAIGGRTAYTGICCRAARTT-3' (SEQ ID NO: 6)

[0053] Here, I represents inosine.

[0054] The nucleotide sequence of this partial sequence was determined, and a BLAST search was performed based on the obtained sequence information. As a result, the sequence obtained showed high homology with respect to...

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Abstract

The genes of a novel lipoxygenase and a novel allene oxide synthase derived from Lemna paucicostata strain SH are identified, and a plant growth regulator (KODA) is produced with high yield by using a Lemna paucicostata strain that expresses said lipoxygenase and said allene oxide synthase.

Description

technical field [0001] The present invention relates to the plant hormone (common name: 9-hydroxyl-10-oxo-cis-12(Z), 15(Z)-octadecadienoic acid, Hereinafter referred to as the production method of KODA). [0002] Background technique [0003] KODA is a phytohormone that has plant flower bud formation promoting action, plant activation action, and plant growth regulation action including these actions (Japanese Patent Application Laid-Open No. 9-295908, Japanese Patent Laid-Open No. 11-29410, Japanese Patent Laid-Open No. 2001-131006 Gazette, Japanese Patent Application Laid-Open No. 2009-17829) known. KODA is known to be present in various plant species, and the stressed Lemna paucicostata is known to release extremely high levels (hundreds of times) of KODA compared to other plants. Utilizing this property, KODA can be produced using an extraction method obtained by extracting Lemna spp., which is a kind of duckweed plant. As another production method, KODA can be pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/40C07C59/76A01H5/00C12N15/29
CPCC12N9/0069C12N15/8247C12N9/88A01H3/00C07C59/76C12P7/42A01H5/12
Inventor 横山峰幸别府敏夫
Owner SHISEIDO CO LTD