Liparis nervosa lectin gene and protein as well as application thereof

A technology of lectin and lectin, which is applied in the field of lectin, can solve the problems that have not been reported, and achieve the effect of expanding the scope of application

Inactive Publication Date: 2011-09-07
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There has been no report about the separation and extraction of the protein contained in the ear orchid and the gene encoding the protein of the ear orchid

Method used

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  • Liparis nervosa lectin gene and protein as well as application thereof
  • Liparis nervosa lectin gene and protein as well as application thereof
  • Liparis nervosa lectin gene and protein as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Preparation of lectin protein

[0016] In the present embodiment, the preparation method of the lectin protein of sageria spp. is as follows

[0017] 1. Crude product preparation

[0018] Wash the rhizomes and leaves of Fenugreek, homogenize it with a mixer until it becomes a paste, then add 2 times the volume of 0.2M sodium chloride solution of Fenugreek paste, and place it at 4°C for 12 hours, 4 Filter through a layer of gauze, centrifuge (4°C, 5,000g, 30min), collect the supernatant, and add solid ammonium sulfate to the obtained supernatant to reach 50% saturation, place it at 4°C for 6 hours, centrifuge (4°C, 10,000g , 20min) to remove the precipitate, continue to add ammonium sulfate to the obtained supernatant to 80% saturation, place it at 4°C for 12 hours, centrifuge (4°C, 10,000g, 20min) to collect the precipitate, dissolve the precipitate in distilled water, and dialyze to remove salt, and freeze-dried to obtain the crude lamb ear lectin protein....

Embodiment 2

[0026] Example 2: Gene cloning of the lectin of Elephantia spp. (using the method of rapid amplification of cDNA ends, ie 3' / 5'RACE method)

[0027] 1. Materials and methods

[0028] 1.1 Materials

[0029] A lectin protein sample of sageria: prepared by the method described in Example 1.

[0030] Plant material: The rhizomes and leaves of Fructus L. were collected from Pengzhou City, Sichuan Province, China, and were used directly or quickly frozen and stored at -70°C.

[0031] Bacterial strain: Escherichia coli Top10 (purchased from Invitrogen).

[0032] Plasmid vector: PUC18-T vector (purchased from TaKaRabao Biological Engineering Co., Ltd.).

[0033]Enzymes and kits: Taq DNA polymerase, MLV reverse transcriptase, TdT (nucleotide terminal transferase) RNase Inhibitor, RNase A (nucleotidase), RNA extraction kit, 3' / 5'RACE kit etc. were purchased from TaKaRa Bao Biological Engineering Co., Ltd. (Dalian, China), and the column gel recovery kit was purchased from Omiga Comp...

Embodiment 3

[0102] Example 3: Inhibitory effect of the lectin protein of Elephant spp. on agricultural harmful fungi

[0103] 1. Materials and Methods

[0104] 1.1 Materials

[0105] Pleurotus agglutinin protein: prepared by the method described in Example 1.

[0106] Harmful agricultural fungi: P. oryzae Cav., B. maydis, and F. oxysporum vasinfectum were purchased from Shanghai Fuzhong Biotechnology Development Co., Ltd.

[0107] 1.2 Method

[0108] paper method

[0109] 1.2.1 Preparation of filter paper

[0110] Choose filter paper with strong water absorption and uniform texture, use a puncher to punch into discs with a diameter of 6.25 mm, autoclave at 121 °C for 40 minutes, and dry them for later use.

[0111] 1.2.2 Preparation of bacterial suspension and potato agar culture plate

[0112] Add normal saline and glass beads after autoclaving to three sterile Erlenmeyer flasks respectively, and then take the activated rice blast fungus P.oryzae Cav. The slant strains of Fusarium...

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Abstract

The invention relates to a liparis nervosa lectin protein obtained by separating and purifying rootstock and leaves of orchidaceae liparis nervosa. The liparis nervosa lectin protein has an amino acid sequence shown as SEQ ID NO:3 in a sequence table. In the invention, the liparis nervosa lectin protein obtained through separation and purification is subjected to determination on an N-end amino acid sequence to judge that the liparis nervosa lectin contains 29 N-end amino acid sequences, a degenerate primer is designed accordingly, a liparis nervosa lectin gene is cloned with 3'RACE and 5'RACE methods, and the nucleotide sequence of the liparis nervosa lectin gene is shown in SEQ ID NO:2. Experiments prove that the liparis nervosa lectin protein has an obvious inhibition effect on agricultural harmful fungi, and can be applied to preparation of pesticides for resisting agricultural harmful fungi and improvement of resistance of plants to the harmful fungi.

Description

technical field [0001] The invention belongs to the field of lectins, and in particular relates to the application of the orchid orchid agglutinin gene, the orchid orchid agglutinin protein and the anti-agricultural harmful fungus. Background technique [0002] Liparis nervosa belongs to Angiospermae, Monocotyledoneae, Liliidae, Orchidales, Orchidaceae, Liparis , is a kind of precious Chinese herbal medicine, mainly for: hematemesis, hemoptysis, intestinal wind bleeding, metrorrhagia, surgical bleeding, convulsions in children, pyretic sores, snake bites, etc. The researches on mutton's fungus mainly focus on the hemostatic function (see Zhou Jiming, Liu Changwu, Ran Chongxing, etc. Preliminary research on the compound hemostatic drug of goat's fungus for external use. Chinese Journal of Hospital Pharmacy, 1983, 3(3): 5 -7), it has been proved by animal experiments and clinical experiments that Muscaria can shorten the coagulation time, reduce the amount of bleeding and has...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/42A01N65/40A01P3/00C12N15/82A01H5/00
Inventor 鲍锦库吴传芳罗萍何雪梅张潇月徐怀龙
Owner SICHUAN UNIV
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