GBSS1 specific enzyme activity determination method

A technology of specific enzyme activity and seeds, applied in the field of GBSS 1 specific enzyme activity determination

Inactive Publication Date: 2014-05-21
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The above two methods can obtain the enzyme activity of GBSS1 in the sample, but it is impossible to judge whether the difference in the enzyme activity in the sample is caused by the difference in the amount of the enzyme protein or the difference in the specific enzyme activity.

Method used

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  • GBSS1 specific enzyme activity determination method

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Embodiment 1

[0047] The preparation of embodiment 1, GBSS1 enzyme extract

[0048] Take 1 rice seed and freeze it in liquid nitrogen for 1 min, then manually remove the hull, seed coat and embryo, and then grind it (these steps are all carried out at 0-2°C). Three grains were taken from each rice variety as three repetitions. According to the method described by Nakamura et al. (Nakamura Y et al (1989). Plant Cell Physiol, 30:833-839), suspend the ground tissue with 1ml of extraction buffer, centrifuge at 12,000g at 4°C for 10min, and use the precipitate 1ml of extraction buffer was washed repeatedly for 2-3 times, and finally the precipitate was suspended in 100ul of extraction buffer to make GBSS1 enzyme extract.

Embodiment 2

[0049] Example 2, Quantitative Determination of GBSS1 Protein in Enzyme Extract

[0050] 2.1 Determination of total protein amount

[0051] The standard curve of bovine serum albumin was prepared according to the Bradford method (Bradford MM (1976). Anal Biochem, 72:248-254), and the rice GBSS 1 enzyme extract was diluted 1:100 to determine the amount of total protein.

[0052] 2.2 Determination of the protein content of GBSS1 in the enzyme extract by ELISA

[0053] 2.2.1 Establishment of standard curve for prokaryotically expressed GBSS 1 protein concentration

[0054] After the prokaryotic expressed GBSS1 was purified by Ni column and Superdex200 column, the samples with GBSS1 protein content above 99% were used as standard samples. Samples were assayed for protein concentration using the Brandford method and the standard curve prepared in 2.1. Then, standard samples were serially diluted so that the protein concentration was 1.0×10 -4 -1.0×10 -2 Between mg / ml, add an e...

Embodiment 3

[0060] Embodiment 3, the making of ATP quantitative standard curve

[0061] Accurately weigh the ATP powder purchased from SIGMA company and prepare it into 1.0×10 -8 to 1.0×10 -4 mol / l gradient solution, and then use the SIGMA company's kit (ATP assay kit for Luciferase / Luciferin) to react, measure the luminescence value on the Promega GloMax-20 / 20Luminometer, and obtain the standard curve of the luminescence value relative to the ATP concentration accordingly . Calculation formula 3 was obtained with Excel software: lg luminescence value=1.0114×(lgATP concentration+9)+1.052.

[0062] The lg (luminescence value) / lg (ATP) standard curve that bioluminescence meter counts obtains is as follows figure 2 .

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Abstract

The present invention relates to a GBSS1 specific enzyme activity determination method, and provides a granule-bound starch synthase (GBSS1 enzyme) specific enzyme activity determination method, wherein an ELISA technology is adopted to carry out quantitation on the enzyme extract in the GBSS1 protein level and the previous GBSS1 enzyme activity determination steps are simplified.

Description

technical field [0001] The invention relates to the field of biotechnology; more specifically, the invention relates to a method for measuring the specific enzyme activity of GBSS 1. Background technique [0002] GBSS1 (Granule-Bound Starch Synthase 1, 2.4.1.242) encoded by the rice waxy gene (Wx) is responsible for the synthesis of amylose in rice seed endosperm (Sano Y et al (1985). Gamma Field Symp, 24:63-77 ; Shapter et al (2009). J. Cereal Sci, 49:4-11). The expression level of rice waxy gene and the level of GBSS1 enzyme activity are significantly positively correlated with the amylose content in endosperm (Wang ZY et al(1995).The Plant Journal,7:613-622; Sano Y et al(2008 ). Theor Appl Genet, 116:979-989). Over the past 20 years, great progress has been made in the study of the expression and regulation of rice waxy genes and their application in rice quality improvement. In the above research process, the determination of GBSS1 enzyme activity is a routine experim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573
CPCC12Q1/48C12Q1/485G01N33/573
Inventor 蔡秀玲刘德瑞
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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