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Flavanone 3-hydroxylase protein coded sequence of scutellaria viscidula bge

A technology of hydroxylase protein and flavonoids, which is applied in the field of genetic engineering and can solve the problems such as undiscovered and undiscovered f3h gene cloning of Scutellaria baicalensis

Inactive Publication Date: 2009-09-30
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the analysis of existing documents, there is no clone of the f3h gene on the biosynthetic pathway of Scutellaria baicalensis flavonoids, and no reports of documents closely related to the subject of the present invention have been found so far

Method used

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  • Flavanone 3-hydroxylase protein coded sequence of scutellaria viscidula bge
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  • Flavanone 3-hydroxylase protein coded sequence of scutellaria viscidula bge

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Embodiment 1

[0058] Cloning of Flavanone 3-Hydroxylase from Scutellaria baicalensis

[0059] 1. Tissue separation (isolation)

[0060] Scutellaria baicalensis was purchased from Shanxi University, and the seeds were sown in the greenhouse. When the Scutellaria baicalensis seedlings grew to a height of 10 cm, DNA or RNA was prepared to be extracted.

[0061] 2. RNA isolation (RNA isolation)

[0062] 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.

[0063] 3. Cloning of Full-length cDNA

[0064] According to the nucleotide conservative sequence of the flavanone 3-hydroxylase gene of various plants, using the principle of homol...

Embodiment 2

[0076] Sequence information and homology analysis of the flavanone 3-hydroxylase gene of Scutellaria baicalensis

[0077] The full-length cDNA of the new flavanone 3-hydroxylase gene sequence of the present invention is 1317 bp in length, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at 74-1123 nucleotides (1050 nucleotides). According to the full-length cDNA, the amino acid sequence of flavanone 3-hydroxylase of Scutellaria baicalensis was deduced, with a total of 233 amino acid residues, a molecular weight of 39.38kDa, and an isoelectric point (PI) of 5.41. See SEQ ID NO.4 for the detailed sequence.

[0078] The full-length cDNA sequence related to the flavanone 3-hydroxylase gene gene of Scutellaria baicalensis and its encoded protein were published in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBankCDS translations+PDB+SwissProt+Superdate Nucleotide and protein homology searches were carried out in the +PIR datab...

Embodiment 3

[0080] Eukaryotic expression of flavanone 3-hydroxylase protein or polypeptide in Scutellaria baicalensis and detection of baicalin content in transgenic plants.

[0081] The construction of the expression vector containing the target gene, according to the Scutellaria baicalensis flavanone 3-hydroxylase gene coding sequence (SEQID NO.3), design the primers that amplify the complete coding reading frame, and respectively on the upstream and downstream primers Introduce restriction endonuclease sites (this can be determined by the vector selected), so as to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Scutellaria baicalensis 3-hydroxylase gene cDNA was cloned into an intermediate vector (such as pBluescript), and further cloned into a binary expression vector ( Such as pBI121 and improved pCAMBIA1304), the expression vector identified under the premise of ensuring the correct reading frame is then...

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Abstract

The invention discloses a flavanone 3-hydroxylase protein coded sequence of scutellaria viscidula bge, and belongs to the field of genetic engineering. A nucleotide sequence of polypeptide with flavanone 3-hydroxylase activity is coded, and consists of a nucleotide sequence from the 74th position to the 1,123th position of nucleotides in SEQ ID NO.3 or a degenerate sequence thereof; and the sequence codes the polypeptide with the nucleotide sequence shown in the SEQ ID NO.3. The flavanone 3-hydroxylase protein coded sequence has obvious effect of improving contents of flavonoid compounds such as baicalin and the like in scutellaria viscidula bge plants, and has great application value.

Description

technical field [0001] The invention relates to a protein coding sequence, particularly a protein coding sequence of Scutellaria baicalensis flavanone 3-hydroxylase, which belongs to the field of genetic engineering. Background technique [0002] Scutellaria baicalensis is a perennial medicinal herb belonging to the genus Scutellariae of the Lamiaceae. There are more than 300 species of plants in this genus, which are widely distributed in the world. There are more than 100 species in my country. There are about 7 species that can be used as Scutellaria baicalensis, and Scutellaria baicalensis is one of them. The main medicinal component of this plant is baicalin, which has been proved by modern pharmacology to have the functions of clearing heat and detoxifying, stopping bleeding and preventing miscarriage, antibacterial and anti-inflammatory, protecting liver and gallbladder, reducing blood pressure and desensitization, sun protection and whitening. With the continuous in...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N1/19C12N5/10
Inventor 孙敏雷桅
Owner SOUTHWEST UNIVERSITY
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