Jatropha curcas phytoene synthetase gene sequence and application thereof in plants
A technology of jatropha curcas and leprosy, applied in the fields of molecular biology and genetic engineering
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Embodiment 1
[0147] Cloning of PSY Protein Gene of Jatropha curcas
[0148] 1. Tissue separation (isolation)
[0149] The seeds of Jatropha curcas come from Panzhihua, Sichuan Province. After the seeds of Jatropha curcas are collected, they are stored in the laboratory.
[0150] 2. RNA isolation (RNA isolation)
[0151] Peel off the seed coat of Jatropha curcas seeds, take out the seed kernels, grind them with a mortar, add liquid nitrogen, grind them into powder, take 100mg and transfer them to 1.5mL EP tubes, and extract total RNA (two-step lysis method) . The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.
[0152] 3. Cloning of Full-length cDNA
[0153] According to the conserved amino acid sequence of PSY in some plants, design degenerate primers, using the principle of homologous gene cloning, SMART TM RACE cDNA amplification method (Clonetech kit) for full-length cDNA cloning is ...
Embodiment 2
[0164] Sequence information and homology analysis of PSY protein gene of Jatropha curcas
[0165] The full-length cDNA of the new Jatropha curcas PSY protein of the present invention is 1755bp in length, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at nucleotides 78-1364. The amino acid sequence of Jatropha curcas PSY protein was deduced according to the full-length cDNA, with a total of 429 amino acid residues, a molecular weight of 48068.52, and a pI of 8.96. See SEQ ID NO.2 for the detailed sequence.
[0166] The full-length cDNA sequence of the Jatropha curcas PSY protein and its encoded protein were nucleotide aligned in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR databases using the BLAST program. Protein homology search, the results found that the nucleotide sequence of Jatropha curcas PSY gene and other plant nucleotide sequences such as sweet orange (C.si...
Embodiment 3
[0167] The structural characteristic analysis of embodiment 3 Jatropha curcas PSY protein
[0168] 1. Domain analysis of Jatropha curcas PSY protein
[0169] The amino acid sequence of the Jatropha curcas PSY protein is retrieved in the NCBI database (the website is: http: / / www.ncbi.nlm.nih.gov / Blast.cgi), and the results are shown in image 3 .
[0170] In the amino acid sequence, there is a catalytic site of isopentenyl pyrophosphate synthase (Trans-Isoprenyl Diphosphate Synthases, Trans_IPPS) (i.e. amino acid residues 137-404 from the amino terminal (N terminal) of SEQ ID.1 in the sequence listing .
[0171] 2. Analysis of signal peptide of Jatropha curcas PSY protein
[0172] Plant carotenoid synthases are encoded by nuclear genes, and the codes are translated to form precursors (precursors) with transit peptide sequences at the N-terminal. Transit peptides have the function of transporting plant carotenoid synthases to plastids. After processing or translocation, they...
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