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.
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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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