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A method for functional identification of carotenoid metabolism genes

A carotene and identification method technology, applied in the field of gene function identification, to achieve the effect of targeted identification

Active Publication Date: 2021-03-26
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the same strategy is difficult to achieve in the study of carotenoid synthesis pathways, because the carotenoid substrates and corresponding products utilized by most of the enzymes involved are fat-soluble, which is inconvenient for carotenoids in aqueous systems. Functional identification of hormone-metabolizing enzymes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] In this embodiment, a method for functional identification of carotenoid metabolism genes is proposed, including the following steps:

[0021] S1: Clone screening marker elements: cloned from pGBK-T7 vector by Amp r Promoter-driven kanamycin resistance gene Kan r , with this element (pAmp r ::Kan r ) as a subsequent screening marker;

[0022] S2: Clone the promoter: the promoter sequence of the RuBisCO large subunit gene (rbcL) in the cyanobacterium Synechocystis sp. PCC6803 was cloned, and the promoter was used to drive the enzyme gene to be detected;

[0023] S3: Cloning of the enzyme gene: the carotenoid metabolism-related enzyme gene Arabidopsis zeaxanthin epoxidase (AtZEP) is cloned downstream of the rbcL promoter (rbcL::AtZEP), driven by the promoter ;

[0024] S4: Construction of the expression cassette: a complete gene expression cassette includes a pAmp arranged in sequence r ::Kan r Screening marker elements and an rbcL::AtZEP element for expressing enz...

Embodiment 2

[0031] In this embodiment, a method for functional identification of carotenoid metabolism genes is proposed, including the following steps:

[0032] S1: Clone screening marker elements: cloned from pGBK-T7 vector by Amp r The promoter-driven kanamycin resistance gene Kanr, with this element (pAmp r ::Kan r ) as a subsequent screening marker;

[0033] S2: Clone the promoter: the promoter sequence of the RuBisCO large subunit gene (rbcL) in the cyanobacterium Synechocystis sp. PCC6803 was cloned, and the promoter was used to drive the enzyme gene to be detected;

[0034] S3: Cloning of the enzyme gene: the carotenoid metabolism-related enzyme gene Arabidopsis lycopene ε-cyclase (AtLUT2) was cloned downstream of the rbcL promoter (rbcL::AtLUT2). The promoter drives;

[0035]S4: Construction of the expression cassette: a complete gene expression cassette includes a pAmp arranged in sequence r ::Kan r Screening marker elements and a rbcL::AtLUT2 for expressing enzyme protein...

Embodiment 3

[0042] In this embodiment, a method for functional identification of carotenoid metabolism genes is proposed, including the following steps:

[0043] S1: Clone screening marker elements: cloned from pGBK-T7 vector by Amp r Promoter-driven kanamycin resistance gene Kan r , with this element (pAmp r ::Kan r ) as a subsequent screening marker;

[0044] S2: Clone the promoter: the promoter sequence of the RuBisCO large subunit gene (rbcL) in the cyanobacterium Synechocystis sp. PCC6803 was cloned, and the promoter was used to drive the enzyme gene to be detected;

[0045] S3: Cloning of enzyme gene: the carotene ε-hydroxylase (AtLUT1) of Arabidopsis thaliana, an enzyme gene related to carotenoid metabolism to be identified, is cloned downstream of rbcL::AtLUT2 in Example 3, through ribosome binding The site rbs sequence is concatenated to form (rbcL::AtLUT2::rbs::AtLUT1), and the expression of the two inserted enzyme genes is jointly driven by the rbcL promoter combined with t...

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Abstract

The invention discloses a carotenoid metabolism gene function identification method, which comprises the following steps: S1, cloning a selection marker element: cloning a kanamycin resistance gene Kanr driven by an Ampr promoter from a pGBK-T7 vector, and taking the element (pAmpr:: Kanr) as a subsequent selection marker; S2, cloning a promoter: cloning a promoter sequence of a RuBisCO large-subunit gene (rbcL) in the synechocystis sp. PCC6803 of the cyanobacteria, wherein the promoter is used for driving an enzyme gene to be detected; S3, cloning an enzyme gene: cloning a carotenoid metabolism-related enzyme gene X to be identified from different organisms to the downstream (rbcL:: X) of a rbcL promoter, and driving the carotenoid metabolism-related enzyme gene X by the promoter; and S4,constructing an expression cassette: a complete gene expression cassette comprises a pAmpr:: Kanr selection marker element and a rbcL:: X element for expressing zymoprotein which are arranged in sequence. Compared with a traditional identification method, the method is simpler and more convenient and more direct and can be adopted to clearly identify gene functions in a targeted mode.

Description

technical field [0001] The invention relates to the technical field of gene function identification, in particular to a method for identifying the function of carotenoid metabolism genes. Background technique [0002] Carotenoids are widely distributed in nature. They typically function as light harvesting and as photoprotective pigments, in addition to providing a vibrant color to the plant's pollen and aiding seed dispersal. For humans, these compounds are beneficial to human health and are essential phytonutrients in the daily diet. Almost all photosynthetic organisms are able to synthesize common carotenoids (lycopene and β-carotene), while more types of carotenoids appear in higher plants, including lutein and its oxidized derivatives, etc. . But some specific carotenoids are only found in certain species, such as astaxanthin in Haematococcus pluvialis and lettuce xanthin in lettuce. If the enzymes that catalyze the synthesis of these unique carotenoids can be ident...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/65C12N15/74G01N30/02G01N30/88C12R1/01
CPCC12N15/65C12N15/74G01N30/02G01N30/88G01N2030/884
Inventor 卢山曹天骏邓银银李朋富
Owner NANJING UNIV
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