Coded sequence of reductase enzyme protein of eucommia 3-hydroxy-3-coenzyme of methyl glutaryl A
A technology of methylglutaryl coenzyme and reductase, applied in the fields of molecular biology and genetic engineering
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Embodiment 1
[0147] Cloning of Eucommia eu-Hmgr Protein Gene
[0148] 1. Tissue separation (isolation)
[0149] Young leaves of Eucommia ulmoides were obtained from the Key Laboratory of Medicinal Plant Biotechnology in Jiangsu Province. After the materials were collected, they were immediately frozen in liquid nitrogen.
[0150] 2. RNA isolation (RNA isolation)
[0151] Take part of the tissue, grind it with a mortar, add it to a 1.5mL EP tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to 1.5mL EP tube, and extract total RNA (TRIzol Reagents, GIBCOBRL, USA). 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 Hmgr in some plants, design annexation primers, use the principle of homologous gene cloning, and use the Smart-...
Embodiment 2
[0161] Sequence information and homology analysis of Eucommia eu-Hmgr protein gene
[0162] The novel eucommia eu-Hmgr protein full-length cDNA of the present invention has a length of 2281 bp, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at 170-1952 nucleotides. The amino acid sequence of eucommia eu-Hmgr protein was deduced according to the full-length cDNA, with a total of 590 amino acid residues, a molecular weight of 63313.88, and a pI of 6.83. See SEQ ID NO.4 for the detailed sequence.
[0163] The full-length cDNA sequence of Eucommia eu-Hmgr 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. The protein homology search found that it has a high homology with the 3-hydroxy-3-methylglutaryl-CoA reductase gene (AY352338.1) of Hevea parahevea at the nucleotide level (attached t...
Embodiment 3
[0165] Prokaryotic expression and purification of eucommia eu-Hmgr protein in Escherichia coli
[0166] In this example, the full-length eucommia eu-Hmgr protein coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.
[0167] The eucommia eu-Hmgr protein polypeptide was prokaryotically expressed in Escherichia coli in the form of fusion protein.
[0168] Construction of prokaryotic expression vector and transformation of Escherichia coli
[0169] According to the amino acid sequence of eucommia eu-Hmgr protein, design the primers of the protein coding region, and introduce restriction endonuclease sites (this depends on the pET32a (+) carrier selected) on the forward and reverse primers respectively, so as to construct the expression vector . Using the amplified product obtained in Example 1 as a template, after PCR amplification, the eucommia eu-Hmgr protein gene was cloned in...
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