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
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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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