Nanjing bass 3-hydroxyl-3-methyl glutaryl coenzyme A reductase protein encoding sequence

Abstract

The invention provides a Nanjing Linden tm-Hmgr protein encoding sequence, wherein the separated DNA molecules comprise, nucleic acid sequence encoding polypeptides having Nanjing Linden tm-Hmgr protein activity, the nucleic acid sequence has at least 70% homology with position No:131-1888 nucleic acid sequence in the SEQ ID No:3 in the sequence table, the nucleic acid sequence can be hybridized with position No:131-1888 nucleic acid sequence in the SEQ ID No:3 at 40-55 deg C.

Description

technical field

[0001] The present invention relates to the technical fields of molecular biology and genetic engineering. Specifically, the present invention relates to a tm-Hmgr protein (Nanjing linden 3-hydroxy-3-methylglutaryl-CoA reductase protein, Tilia miqucliana 3-hydroxy-3-methylglutaryl-CoA Reductase , TMHMGR) and their nucleic acid sequences. Background technique

[0002] Nanjing linden (Tilia miqueliana Maxim), commonly known as linden tree, belongs to the tilia family Tilia genus. It is produced in the provinces of East China, eastern Hunan, and Guangdong, and is born in hillsides and ravine forests. Nanjing linden is a deciduous tree with straight trunk, good material, slow growth and long lifespan. Its leaves are beautiful, the tree posture is quiet, the summer yellow flowers are full of trees, and the fragrance is fragrant. It is a famous shade tree species and an excellent nectar source plant. The linden plant is a famous herbal medicine in the West. It i...

Examples

Embodiment 1

[0176] Cloning of tm-Hmgr protein gene from Tilia nanjing

[0177] 1. Tissue separation (isolation)

[0178] The young leaves of Tilia Nanjing come from the Key Laboratory of Biotechnology of Medicinal Plants in Jiangsu Province. After the materials are collected, they are immediately frozen in liquid nitrogen.

[0179] 2. RNA isolation (RNA isolation)

[0180] 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, GIBCO BRL, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0181] 3. Cloning of Full-length cDNA

[0182] 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 Sm...

Embodiment 2

[0190] Sequence Information and Homology Analysis of the tm-Hmgr Protein Gene of Tilia Nanjing

[0191] The full-length cDNA of the new Nanjing Tilia tm-Hmgr protein of the present invention is 2160bp in length, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at nucleotides 131-1888. The amino acid sequence of Nanjing Tilia tm-Hmgr protein was deduced according to the full-length cDNA, which has a total of 585 amino acid residues, a molecular weight of 62956.49, and a pI of 6.11. See SEQ ID NO.4 for the detailed sequence.

[0192] The full-length cDNA sequence of Nanjing Tilia tm-Hmgr protein and its encoded protein were analyzed by BLAST program in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redunoant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR databases And protein homology search, it was found that it has high homology with upland cotton 3-hydroxy-3-methylglutaryl-CoA reductase 1 gene (AF038045.1) at the nucleotide l...

Embodiment 3

[0194] Prokaryotic Expression and Purification of Nanjing Tilia tm-Hmgr Protein in Escherichia coli

[0195] In this example, the full-length Nanjing Tilia tm-Hmgr protein coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0196] The Nanjing Tilia tm-Hmgr protein polypeptide was prokaryotically expressed in Escherichia coli in the form of fusion protein.

[0197] Construction of prokaryotic expression vector and transformation of Escherichia coli

[0198] According to the amino acid sequence of the Nanjing Tilia tm-Hmgr protein, design primers for the protein coding region, and respectively introduce restriction endonuclease sites on the forward and reverse primers (this depends on the pET32a(+) vector selected), so as to construct the expression carrier. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Nanjing Linden tm-Hmgr p...

technical field

[0001] The present invention relates to the technical fields of molecular biology and genetic engineering. Specifically, the present invention relates to a tm-Hmgr protein (Nanjing linden 3-hydroxy-3-methylglutaryl-CoA reductase protein, Tilia miqucliana 3-hydroxy-3-methylglutaryl-CoA Reductase , TMHMGR) and their nucleic acid sequences. Background technique

[0002] Nanjing linden (Tilia miqueliana Maxim), commonly known as linden tree, belongs to the tilia family Tilia genus. It is produced in the provinces of East China, eastern Hunan, and Guangdong, and is born in hillsides and ravine forests. Nanjing linden is a deciduous tree with straight trunk, good material, slow growth and long lifespan. Its leaves are beautiful, the tree posture is quiet, the summer yellow flowers are full of trees, and the fragrance is fragrant. It is a famous shade tree species and an excellent nectar source plant. The linden plant is a famous herbal medicine in the West. It i...

Examples

Embodiment 1

[0176] Cloning of tm-Hmgr protein gene from Tilia nanjing

[0177] 1. Tissue separation (isolation)

[0178] The young leaves of Tilia Nanjing come from the Key Laboratory of Biotechnology of Medicinal Plants in Jiangsu Province. After the materials are collected, they are immediately frozen in liquid nitrogen.

[0179] 2. RNA isolation (RNA isolation)

[0180] 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, GIBCO BRL, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0181] 3. Cloning of Full-length cDNA

[0182] 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 Sm...

Embodiment 2

[0190] Sequence Information and Homology Analysis of the tm-Hmgr Protein Gene of Tilia Nanjing

[0191] The full-length cDNA of the new Nanjing Tilia tm-Hmgr protein of the present invention is 2160bp in length, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at nucleotides 131-1888. The amino acid sequence of Nanjing Tilia tm-Hmgr protein was deduced according to the full-length cDNA, which has a total of 585 amino acid residues, a molecular weight of 62956.49, and a pI of 6.11. See SEQ ID NO.4 for the detailed sequence.

[0192] The full-length cDNA sequence of Nanjing Tilia tm-Hmgr protein and its encoded protein were analyzed by BLAST program in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redunoant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR databases And protein homology search, it was found that it has high homology with upland cotton 3-hydroxy-3-methylglutaryl-CoA reductase 1 gene (AF038045.1) at the nucleotide l...

Embodiment 3

[0194] Prokaryotic Expression and Purification of Nanjing Tilia tm-Hmgr Protein in Escherichia coli

[0195] In this example, the full-length Nanjing Tilia tm-Hmgr protein coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0196] The Nanjing Tilia tm-Hmgr protein polypeptide was prokaryotically expressed in Escherichia coli in the form of fusion protein.

[0197] Construction of prokaryotic expression vector and transformation of Escherichia coli

[0198] According to the amino acid sequence of the Nanjing Tilia tm-Hmgr protein, design primers for the protein coding region, and respectively introduce restriction endonuclease sites on the forward and reverse primers (this depends on the pET32a(+) vector selected), so as to construct the expression carrier. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Nanjing Linden tm-Hmgr p...