Synthetase gene relative to carotene synthesis, coded protein and application thereof

A carotene, synthase technology, applied in application, genetic engineering, plant genetic improvement and other directions

Inactive Publication Date: 2010-03-17
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, GGPP is the first precursor substance that really enters the carotenoid synthesis pathway, and the relationship between its expression regulation and carotene synthesis in Salina salina has not been reported yet.

Method used

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  • Synthetase gene relative to carotene synthesis, coded protein and application thereof
  • Synthetase gene relative to carotene synthesis, coded protein and application thereof
  • Synthetase gene relative to carotene synthesis, coded protein and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1: Cloning and analysis of the full-length coding region of DvGGPS

[0028] Take advantage of SMART TM The cDNA construction kit constructed three cDNA libraries of Salina salina, and obtained the corresponding EST libraries by sequencing the cDNA sequences in the three libraries; through sequence comparison, a geranylgeranyl synthetase gene was isolated, Named DvGGPS. The corresponding clones from the cDNA library were selected for sequencing, and the cDNA sequence of DvGGPS was obtained. The longest cDNA fragment of DvGGPS is 15566bp, which contains the specific tailing signal TGTAA and poly(A) structure of the green algae gene. The analysis results of Vector NTI software showed that the longest reading frame of DvGGPS encoded a protein containing 345 amino acids, the molecular weight of the protein was 37.3kDa, and the isoelectric point was 6.44. ChloroP 1.1Server prediction results show that the protein may be localized in chloroplast. Homology compariso...

Embodiment 2

[0028] Take advantage of SMART TM The cDNA construction kit constructed three cDNA libraries of Salina salina, and obtained the corresponding EST libraries by sequencing the cDNA sequences in the three libraries; through sequence comparison, a geranylgeranyl synthetase gene was isolated, Named DvGGPS. The corresponding clones from the cDNA library were selected for sequencing, and the cDNA sequence of DvGGPS was obtained. The longest cDNA fragment of DvGGPS is 15566bp, which contains the specific tailing signal TGTAA and poly(A) structure of the green algae gene. The analysis results of Vector NTI software showed that the longest reading frame of DvGGPS encoded a protein containing 345 amino acids, the molecular weight of the protein was 37.3kDa, and the isoelectric point was 6.44. ChloroP 1.1Server prediction results show that the protein may be localized in chloroplast. Homology comparison shows that the protein has the conserved domain FARM (DDXXXXDXXDD) and domain SARM ...

Embodiment 3

[0033] Example three DvGGPS prokaryotic expression

[0034] In order to verify whether the DvGGPS gene plays a role in the carotene synthesis pathway of Salina salina, we combined the gene with the Erwinia erwinia carotene synthesis gene cluster CrtYIB, and determined the absorbance of the pigment synthesized by E. coli transformants Whether this gene can work together with downstream genes derived from Erwinia erwinia to synthesize β-carotene.

[0035] First, using the DvGGPS cDNA sequence and the Erwinia erwinia genome as templates, the target gene fragments DvGGPS, CrtE, and CrtYIB were amplified by specific primers, and specific restriction sites were introduced. Among them, CrtE is Erwinia erwinia geranylgeranyl synthetase gene, and this gene works together with the downstream gene cluster CrtYIB to synthesize β-carotene in this bacterium. The relevant primer sequences are as follows (the underlined part is the restriction endonuclease site introduced):

[0036] CrtYIB ...

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Abstract

The invention relates to a synthetase gene relative to carotene synthesis, a coded protein and an application thereof. The synthetase gene DvGGPS relative to carotene synthesis performs the function of geranyl synthetase in E.coli and provides a needed precursor GGPP for carotene synthesis for a downstream gene in a carotene synthesis way. Meanwhile, the pronucleus bacterial strain containing theDvGGPS gene of the invention has great potential on the aspect of using gene engineering to produce carotene.

Description

technical field [0001] The invention belongs to the field of biotechnology. The invention relates to a synthetase gene related to carotene synthesis, its encoded protein and its application. Background technique [0002] Plant carotenoids are mainly distributed in plant chloroplasts and chromoplast membranes, including carotene and xanthophylls. Plant carotenoids are important components of light-absorbing complexes, and play an important role in protecting photosynthetic organs and preventing photooxidative damage. Plant carotenoids are also important pigments in many flowers and fruits, giving plants flowers, fruits and other organs brilliant colors to attract insects, birds or other animals for pollination and seed dispersal. Plant carotenoids are also precursors for the biosynthesis of many physiologically active substances such as plant hormones (such as ABA), defense compounds and flavor aromas (such as β-indole). In addition, β-carotene and β-ring-containing carote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/10C12N15/63C12N1/19C12N1/21C12R1/89C12R1/19
Inventor 宋任涛许政暟宋婷常冲林钰靓孟祥宗
Owner SHANGHAI UNIV
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