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Cytokinin oxidase osckx 3 surface display system and its construction method and application

A surface display carrier and oxidase technology, applied in the field of genetic engineering, can solve the problems of detecting cytokinin content, etc., and achieve the effect of reducing extraction and purification steps, rapid detection, and good specificity

Active Publication Date: 2020-06-09
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, there are currently no relevant reports in the art that use the above mechanism to detect cytokinin content

Method used

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  • Cytokinin oxidase osckx 3 surface display system and its construction method and application
  • Cytokinin oxidase osckx 3 surface display system and its construction method and application
  • Cytokinin oxidase osckx 3 surface display system and its construction method and application

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Embodiment Construction

[0023] The present invention will be described in detail below in conjunction with specific examples, but the protection scope of the present invention is not limited to the following examples.

[0024] 1. PCR amplification of CKX

[0025] According to the open reading frame sequence of rice Nipponbare OsCKX3 gene retrieved from NCBI, specific primers F / R (see Table 1) were designed, and restriction sites Xba I and Sal I were inserted into F and R respectively. Take several young Nipponbare leaves, grind the tissue with liquid nitrogen into powder and extract its RNA. The electrophoresis of total RNA is as follows: figure 1 As shown, there are three bands of 28S, 18S and 5S. Using RNA as a template to reverse transcribe into cDNA, the program of the PCR machine is: 45°C, 60min; 70°C, 5min. The quality of the product cDNA was tested with Actin primers, and the test results were as follows: figure 2 As shown, the detection result is correct and can be used as an amplificatio...

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Abstract

Firstly, the invention, in accordance with a published OsCKX4 gene sequence (GeneBank, Accession NO.NC029256), extracts RNA of Nipponbare and conducts reverse transcription so as to obtain cDNA, and with the cDNA as a template, a polymerase chain reaction (PCR) is implemented so as to amplify the OsCKX4. A PCR amplification primer pair comprises an upstream primer and a downstream primer. Subsequently, the invention provides a surface display vector pMAL-p2x-INP-CKX of the cytokinin oxidase OsCKX4 gene. The rice Nipponbare cytokinin oxidase OsCKX4 surface display system provided by the invention comprises host escherichia coli and the surface display vector pMAL-p2x-INP-CKX of directional interpolation. On the basis of the system, a cytokinin electrochemical biosensor is constructed, so that an isopentenyladenine content of the cytokinin can be detected.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the cloning of rice Nipponbare cytokinin oxidase OsCKX 3 gene, the surface display of bacteria and its application to the electrochemical detection of cytokinin. Background technique [0002] Cytokinins, as plant growth regulator hormones, participate in the regulation of plant growth and development. Accurate analysis of the content and distribution of plant hormones in plants is of great significance for the study of plant life activities, crop genetics and breeding. [0003] Rice Nipponbare cytokinin oxidase OsCKX 3 can irreversibly catalyze the degradation of isopentenyl cytokinin. The activity of CKX is closely related to its coenzyme FAD. The reaction mechanism is: cytokinin binds to OsCKX 3 and catalyzes the reaction , so that cytokinin produces an intermediate product of imine compound, and at the same time, the internal FAD of OsCKX 3 is reduced to FADH 2 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/66A01H5/00A01H6/46C12Q1/26
CPCC12N9/0032C12N15/66C12Q1/005
Inventor 吴云华李勇龚斌
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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