Method for extracting and purifying wild rice stem total proteins

A purification method and a technology of protein extraction solution, which are applied in the field of extraction and purification of total protein of Zizania zizania plants, can solve the problems that the extraction and purification method has not been reported, and achieve the effect of less impurities, low nucleic acid content and high protein content

Inactive Publication Date: 2008-08-27
CHINA JILIANG UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there have been reports on the total protein two-dimensional electrophoresis of tomato and Arabidopsis, but the extraction and purification method of the total protein of the aquatic plant Zizania zizania by solid-phase two-dimensional electrophoresis has not been reported yet.

Method used

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  • Method for extracting and purifying wild rice stem total proteins

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: Mutual cultivation

[0033] (1) Isolation and cultivation of smut fungus:

[0034] Pick a small amount of teliospores of Ustilago smut from the freshly cut surface of Zizania japonica, spread them evenly on a PSA plate (preparation: 200g potato (peeled), 20g sucrose, 20g agar, add water to 1000mL, natural pH value), culture 4d. Pick the characteristic colonies and inoculate them on the PSA plate again for 18 days of purification and culture, then use a 4-5mm hole puncher to punch holes on the edge of the actively growing colonies, and inoculate the round bacterial blocks on the PSA plate (10ml per dish, the diameter of the dish is 90mm, pH6 .0) on. Other culture conditions are: 24-28°C, relative humidity 75-85%.

[0035] (2) Zizania zizania tissue culture:

[0036] The alternate lateral buds of male stems were used as culture material, inoculated on callus induction medium (preparation of callus induction medium: MS medium 1000mL (Qingdao Hi-Tech Park ...

Embodiment 2

[0039] Example 2: Extraction of Zizania total protein

[0040] (1) Weigh about 3 g of Zizania zizania callus after 12 days of interactive culture, cut it into pieces, add 0.3 g of tissue abrasive PVP-40 (Amresco) and a small amount of quartz sand, and grind it into a powder (gray green) with liquid nitrogen.

[0041] (2) Put the ground wild rice stem sample into a 50ml centrifuge tube with a stopper, add 10ml of acetone solution with a concentration of 10% trichloroacetic acid (w / w) and a concentration of β-mercaptoethanol of 0.07% (w / w), and shake slightly , placed at -20°C for 1 h.

[0042] (3) Place the stoppered centrifuge tube in an ultracentrifuge at 4° C. and 13,000 rpm, and centrifuge for 20 minutes (the supernatant is dark green, and the precipitate is light green).

[0043] (4) Pour off the supernatant, add 10ml of acetone solution containing β-mercaptoethanol concentration of 0.07% (w / w), shake, and place it at -20°C for precipitation for 1h. (The supernatant is li...

Embodiment 3

[0049] Example 3: Extraction of Zizania zizania total protein

[0050] (1) Weigh about 3 g of Zizania zizania callus that has been cultured for 15 days, cut it into pieces, add 0.3 g of tissue abrasive PVP-40 and a small amount of quartz sand, and grind it into a powder (gray green) with liquid nitrogen.

[0051] (2) Put the ground wild rice stem sample into a 50ml centrifuge tube with stopper, add 9.6ml of acetone solution with a concentration of 10% trichloroacetic acid and 0.07% concentration of β-mercaptoethanol, shake slightly, and place at -20°C for precipitation for 1.5h .

[0052] (3) Place the stoppered centrifuge tube in an ultracentrifuge at 4° C. and 13,000 rpm, and centrifuge for 20 minutes (the supernatant is dark green, and the precipitate is light green).

[0053] (4) Pour off the supernatant, add 9.8ml of 0.07% β-mercaptoethanol in acetone solution, shake, and place at -20°C for precipitation for 2 hours (the supernatant is light green or green, and the preci...

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Abstract

The invention provides an extraction method of zizania latifolia protein in the interaction of zizania latifolia and zizania smut, which simulates the interaction between zizania latifolia and zizania smut, to extract zizania latifolia gene groups and proteins which are not mixed. The invention can prepare the zizania total protein product without the protein of zizania smut, whose protein content is high, impurity content is low and nucleic acid content is low, thereby satisfying the demand of two-dimensional electrophoresis to protein purity.

Description

(1) Technical field [0001] The invention relates to a method for extracting and purifying the total protein of Zizania plant by simulating the interactive culture of Zizania and Ustilago smut, especially a kind of unmixed Zizania smut protein, which can be used for the extraction of total protein of Zizania plant by solid-phase two-dimensional electrophoresis Purification method. (2) Background technology [0002] Zizania latifolia Turcz (zizania latifolia Turcz), also known as bamboo shoots, is a perennial aquatic perennial herb of the family Poaceae, asexually propagated, native to China and Southeast Asia, and is an important aquatic vegetable in my country. my country's wild bamboo shoots are quite rich in variety resources, which are the second largest aquatic vegetable species after lotus root. , high nutritional value, deeply loved by consumers. Therefore, it is very important to study the molecular development and proteomics of the excellent germplasm resources of Z...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C07K1/30
Inventor 叶子弘邹克琴俞晓平刘倩尤文雨
Owner CHINA JILIANG UNIV
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