Davilpepper tryptophan decarboxylase protein coded sequence

A technology of tryptophan decarboxylase and coding sequence, which is applied in the direction of lyase, biochemical equipment and methods, applications, etc., and can solve the problems of undiscovered and unseen tryptophan decarboxylase gene cloning, etc.

Inactive Publication Date: 2007-08-08
SOUTHWEST UNIV
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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 cloning of the tryptophan decarboxylase gene on the biosynthetic pathway of Rauwolfia terpene-indole alkaloids, 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Cloning of Tryptophan Decarboxylase Gene from Rauwolfia

[0067] 1. Isolation

[0068] Rauvolwolfia (species "Yunnan Rauvolwolfia") was collected from Nanshan, Chongqing, and the seeds were sown in the greenhouse. When the Rauvolwolfia seedlings grew to a height of 10cm, DNA or RNA was prepared to be extracted.

[0069] 2. RNA isolation (RNA isolation)

[0070] 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.

[0071] 3. Cloning of Full-length cDNA

[0072] According to the nucleotide conservative sequence of the tryptophan decarboxylase gene of various plants, using the principle of homologous gene clonin...

Embodiment 2

[0084] Sequence Information and Homology Analysis of Tryptophan Decarboxylase Gene of Rauwolfia

[0085] The length of the full-length cDNA of the new tryptophan decarboxylase gene sequence of the present invention is 1824bp (including the 5-terminal non-coding region of 153bp, the 1500bp coding region and the 3-terminal non-coding region of 171bp), and the detailed sequence is shown in SEQ ID NO.1, wherein The open reading frame is located at nucleotides 154-1653 (1500 nucleotides). The amino acid sequence of Rauwolfia tryptophan decarboxylase was deduced based on the full-length cDNA, with a total of 499 amino acid residues, a molecular weight of 55.54 kDa, and an isoelectric point (pI) of 5.35. See SEQ ID NO.1 for the detailed sequence.

[0086] The full-length cDNA sequence related to Rauwolfia tryptophan decarboxylase gene and its encoded protein were stored in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR datab...

Embodiment 3

[0088] Expression of Rauwolfia tryptophan decarboxylase protein or polypeptide in eukaryotic cells and detection of TIAs content in transgenic plants

[0089] The construction of the expression vector containing the target gene, according to the Rauwolfia tryptophan decarboxylase gene coding sequence (SEQ ID NO.1), design primers to amplify the complete coding reading frame, and introduce restrictions on the upstream and downstream primers respectively Sexual endonuclease sites (this may depend on the carrier selected), in order to construct expression vectors. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Rauwolfia tryptophan decarboxylase 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 was identified under the premise of ensuring the correct reading frame, and then it was transferred into A...

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Abstract

The invention discloses a davilpepper tryptophan decarboxylase protein coding sequence in the gene engineering domain, which is characterized by the following: coding the ribonucleotide sequence with 70% consanguinity with 154-1653rd in the SEQ ID NO.1; crossing 154-1653rd in the SEQ ID NO.1 under 45-55 deg.c.

Description

technical field [0001] The invention relates to a protein coding sequence, particularly a protein coding sequence of Rauwolfia tryptophan decarboxylase, which belongs to the field of genetic engineering. Background technique [0002] Hypertension is a disease that seriously endangers human health, and my country has become the country with the most serious hazards of hypertension in the world. According to the latest nutritional and health survey results of residents released by the Health Education Institute of the Chinese Center for Disease Control and Prevention, the number of people with high blood pressure in mainland China has reached 160 million, so there is a demand for antihypertensive drugs, especially natural antihypertensive drugs Very huge. Rauvolfia verticillata is an important medicinal plant belonging to the genus Rauvolfia in the family Apocynaceae, endemic to China. Its root contains various medicinal terpene indole alkaloids (TIAs) such as reserpine, ajm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/79
Inventor 廖志华陈敏谌容
Owner SOUTHWEST UNIV
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