Two-dimensional electrophoresis method for separating protein from kenaf leaf efficiently and stably

A two-dimensional electrophoresis and protein technology, applied in the field of plant molecular biology, can solve the problem of late start of kenaf proteomics research, and achieve the effects of stable experimental results, large numbers and uniform distribution.

Inactive Publication Date: 2012-03-21
FUJIAN AGRI & FORESTRY UNIV
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  • Claims
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AI Technical Summary

Problems solved by technology

In order to further study the molecular mechanism of kenaf development, growth, stress resistance, etc., it is particularly important to explore its gene expression differences at the proteome level. However, kenaf proteomics research started relatively late, and there are few domestic reports. The present invention Firstly, the effects of TCA-acetone method, urea-thiourea method, and phenol extraction method on extracting protein from kenaf leaves were compared in several aspects, in order to find the most suitable protein extraction method for kenaf two-dimensional electrophoresis

Method used

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  • Two-dimensional electrophoresis method for separating protein from kenaf leaf efficiently and stably
  • Two-dimensional electrophoresis method for separating protein from kenaf leaf efficiently and stably
  • Two-dimensional electrophoresis method for separating protein from kenaf leaf efficiently and stably

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0018] (1) Preparation of protein samples

[0019] Extraction of kenaf leaf protein by TCA-acetone method: Take 1g of fresh kenaf leaves (the raw material of kenaf is kenaf variety Fuhong 952), add appropriate amount of PVP, grind with liquid nitrogen for 45min, add 10mL-20℃ pre-cooled Contain 0.07% β-mercaptoethanol, 10% TCA in acetone solution, grind at low temperature for 15 minutes, and stand overnight at -20°C. Centrifuge (15000r / min, 4°C) for 15min, discard the supernatant, add 10mL of cold acetone (containing 0.07% β-mercaptoethanol) to shake, and precipitate at -20°C for 2 hours. Wash twice with -20°C pre-cooled acetone containing 0.07% β-mercaptoethanol, and vacuum-dry the precipitate to a dry powder.

[0020] Extraction of kenaf leaf protein by urea-thiourea method: take 1g of fresh kenaf leaves, add appropriate amount of PVP, grind with liquid nitrogen for 45min, add 6ml extraction buffer (40mmol / LTris-base, 5mol / LUrea, 2mol / Lthiourea, 2mol / Lthiourea, % CHAPS, 5% ...

Embodiment 1

[0034] (1) Extract kenaf leaf protein by TCA-acetone method:.

[0035] (2) Using bovine serum albumin as the standard protein, the protein content in the prepared samples was determined by Brafford method.

[0036] (3) Two-dimensional electrophoresis was carried out using 24 cm IPG strips with a pH value of 4-7, an active loading method, a loading volume of 1.4 mg, and a separation gel with a concentration of 12%.

[0037] (4) Fix for 2 hours, stain with Coomassie Brilliant Blue G250 staining solution overnight, decolorize with distilled water, scan, and analyze with software.

Embodiment 2

[0039] (1) Extract kenaf leaf protein by urea-thiourea method: Take 1g of fresh kenaf leaves, add appropriate amount of PVP, grind with liquid nitrogen for 45min, add 6ml extraction buffer (40mmol / LTris-base, 5mol / LUrea, 2mol / Lthiourea, 2% CHAPS, 5% PVP, 2% β-mercaptoethanol) low temperature grinding for 15min. Centrifuge (15000r / min, 4°C) for 15min, take the supernatant, add pre-cooled acetone containing 0.07% β-mercaptoethanol, and let stand overnight at -20°C. Wash twice with 0.07% β-mercaptoethanol, and vacuum-dry the precipitate to dry powder.

[0040] (2) Using bovine serum albumin as the standard protein, the protein content in the prepared samples was determined by Brafford method.

[0041] (3) Two-dimensional electrophoresis was carried out using 24 cm IPG strips with a pH value of 4-7, an active loading method, a loading volume of 1.4 mg, and a separation gel with a concentration of 12%.

[0042] (4) Fix for 2 hours, stain with Coomassie Brilliant Blue G250 staini...

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Abstract

The invention discloses a two-dimensional electrophoresis method for separating protein from a kenaf leaf efficiently and stably. In combination of biomass chemical features of the kenaf leaf, on the basis of the conventional protein two-dimensional electrophoresis method, the method for separating protein from the kenaf leaf, the selection of the pH range of an IPG gel strip, the protein sample applying way, the protein sample applying amount, the concentration of a separation gel, the setting of parameters of protein spot analyzing software and other aspects are improved and optimized. By the method, a two-dimensional electrophoretogram of the protein of the kenaf leaf with more clear protein spots which are uniformly distributed and with clear background can be obtained; the experimental result is stable; and averagely, 1300 protein spots can be detected on the gel with the size of 24cm*16cm. The invention paves a working foundation for further proteomic and molecular biologic research on kenaf and other hemps.

Description

technical field [0001] The invention belongs to the field of plant molecular biology and relates to a kenaf proteomics technology, in particular to a stable and efficient two-dimensional electrophoresis method for separating proteins from kenaf leaves. Background technique [0002] Kenaf (Kenaf) is Malvaceae (Malvaceae) Hibiscus genus ( Hibiscus ) annual bast fiber crop, which was cultivated in African Sudan in 4000 BC. As a natural fiber crop, kenaf has the characteristics of fast growth, strong stress resistance, and wide adaptability. The huge biological yield is 3- 4 times, the extremely strong carbon dioxide absorption capacity is 4-5 times that of forests, and it is an advantageous crop for developing a low-carbon economy. In addition to being traditionally used as raw materials for processing hemp rope, sacks, carpets, etc., kenaf fiber is also a good material for processing automotive interiors, panels, and hemp carbon. Antibacterial and other biological functions....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/26
Inventor 祁建民陈涛李小珍徐建堂秦先超方平平林培清陶爱芬
Owner FUJIAN AGRI & FORESTRY UNIV
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