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Gene for generating related diterpene synthase together with tanshinone type compound as well as encoding product and application thereof

A technology of diterpene synthase and compound, which is applied in the field of diterpene synthase gene and its coded product and application in the biosynthesis of tanshinone compounds, and can solve the problems of no related reports

Inactive Publication Date: 2009-09-23
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

cDNA chips have been applied in many plants such as Arabidopsis thaliana, rice, tomato, cotton, etc., but there is no relevant report in the study of functional genes of medicinal plants

Method used

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  • Gene for generating related diterpene synthase together with tanshinone type compound as well as encoding product and application thereof
  • Gene for generating related diterpene synthase together with tanshinone type compound as well as encoding product and application thereof
  • Gene for generating related diterpene synthase together with tanshinone type compound as well as encoding product and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1. Preparation of Salvia miltiorrhiza cDNA chip:

[0025] 1. Isolation and detection of total RNA of Salvia miltiorrhiza

[0026] Take 2 g of Shaanxi Shangluo salvia (Salvia Miltiorrhiza Bge) roots, quickly grind into powder with liquid nitrogen in a mortar, and quickly transfer to 10 mL of extraction buffer preheated at 65 °C (CTAB (W / V) 2%, Tris-HCl (pH8.0)100mmol·L -1 , EDTA 25m mol·L -1 , NaCl 2.0mol·L -1 , PVP402%, spermidine 0.5g / L, mercaptoethanol 2%), fully shaken and mixed; extracted twice with an equal volume of chloroform, and centrifuged at 7500g for 15 minutes. Add 1 / 4 volume of 10M LiCl to the supernatant, mix well and place it at 4°C to precipitate overnight; centrifuge at 7500g for 20 minutes, use 500μL SSTE (SDS 0.5%, NaCl 1mol·L for the precipitation) -1 , Tris-HCl (pH8.0) 10mmol·L -1 , EDTA 1mmol·L -1 , dissolve at 65°C for 5 minutes. Extracted with an equal volume of chloroform, centrifuged at 13,000g for 5 minutes; added 2 volumes of ...

Embodiment 2

[0036] Example 2: Cloning of diterpene synthase gene in Salvia miltiorrhiza:

[0037] 1. Preparation of experimental materials

[0038] The hairy roots of Salvia miltiorrhiza were induced by Ri-plasmid transformation by direct infection with Agrobacterium rhizogenes 15834. Take the hairy roots of Salvia miltiorrhiza preserved in 6-7V solid medium (without hormones), inoculate 2g wet roots in a 500mL conical flask containing 200ml of hormone-free 6-7V liquid medium under aseptic conditions for the following steps. Subculture, cultured to 18d as the test material. The culture conditions were 25°C, 110-120 r·min-1, and cultured in the dark.

[0039] 2. Preparation and processing of elicitors

[0040] Preparation of yeast extract (ycast extract, YE) biological elicitor: Dissolve 25g of yeast extract in 125mL of distilled water, add 100mL of absolute ethanol, put it in a refrigerator at 4°C for 4 days, pour off the supernatant, and colloidally precipitate Dissolve in 125mL dist...

Embodiment 3

[0060] Embodiment 3, the bioinformatics analysis of SmKSL gene sequence:

[0061] The length of the full-length cDNA of the Salvia diterpene synthase gene SmKSL involved in the present invention is 2110 bp, and the detailed sequence is shown in Sequence 1 in the sequence table, wherein the complete open reading frame is located at 192-1979 bp. The full-length cDNA sequence of Salvia miltiorrhiza was searched for nucleotide homology in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translation+PDB+Swissprot+Superdate+PIR database with BLAST program, the result nucleotide No homologous sequence was found at the level, it can be seen that it is very difficult to clone the gene using conventional degenerate primers. The comparison analysis of this gene at the amino acid level showed that the amino acid sequence of the protein encoded by the SmKSL gene of Salvia miltiorrhiza had low homology with other species, and had the highest homology with the amino acid seq...

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Abstract

The invention discloses a gene for generating related diterpene synthase together with a tanshinone compound as well as an encoding product and an application thereof. The gene is named as the tanshin ent-kaurene synthase gene (SmKSL) and is obtained from tanshin by cloning and adopting a gene chip technology, and as for the sequence, see to SEQ ID No.1. The protein encoded by the SmKSL is the protein provided with an aminoacid residue sequence with SEQ ID No.2 or is the protein derived via the SEQ ID No. 2 by replacement, deletion or addition of one or more aminoacid residues for the aminoacid residue sequence with SEQ ID No.2 and provided with the same activity with the aminoacid residue sequence with SEQ ID No.2. The SmKSL is a key enzyme gene in a tanshin diterpene secondary metabolic pathway, the genetic expression is closely related with the generation of the tanshinone compounds, for example, tanshinone IIA, and the invention has important theoretical and practical significances for adjusting and producing plant diterpenoid compounds and cultivating the excellent medical new plant variety.

Description

technical field [0001] The present invention relates to cloning of plant diterpene synthase gene by cDNA chip technology, its encoded product and application, in particular to a diterpene synthase gene for biosynthesis of tanshinone compounds, the main active components of Salvia miltiorrhiza, and its encoded product and application, belonging to medicinal plant genes engineering field. Background technique [0002] The formation of active ingredients (secondary metabolites) of medicinal plants is the product of specific genes in the secondary metabolic pathways of plants. With the extensive and in-depth study of plant functional genomes, the research on functional genes related to secondary metabolism and synthesis of medicinal plants with unique characteristics and broad application prospects has gradually become a research hotspot (Wei Xiaoyong, Fang Hua, Li Jiefen. Research on Functional Genes of Plants Traditional Chinese Medicine . Chinese Herbal Medicine, 2005, 36(8)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H1/00
Inventor 黄璐琦高伟崔光红王学勇戴住波
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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