Analysis and application of salvia miltiorrhiza bunge isopentenylpyrophosphate isomerase (SmIPPI) gene

A technology of isopentenyl pyrophosphate and isomerase, applied in the direction of isomerase, application, genetic engineering, etc., to achieve good application prospects and increase yield

Inactive Publication Date: 2010-02-24
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

Before the present invention is published, there is no publication or report of the Danshen prenyltransferase gene and its amino acid sequence mentioned in this patent application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the making of Danshen cDNA chip

[0020] 1. Isolation and detection of total RNA from Salvia miltiorrhiza

[0021] Take 2 g of Shaanxi Shangluo Salvia (Salvia Miltiorrhiza Bge) roots, quickly grind them into powder with liquid nitrogen in a mortar, and quickly transfer them to 10 mL of extraction buffer (CTAB (W / V) 2%, Tris-HCl (pH8.0)100mmol·L -1 , EDTA 25mmol·L -1 , NaCl 2.0mol·L -1 , PVP402%, spermidine 0.5g / L, mercaptoethanol 2%), fully shake and mix; extract twice with equal volume of chloroform, and centrifuge 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, and use 500 μL SSTE (SDS 0.5%, NaCl 1mol L -1 , Tris-HCl (pH8.0) 10mmol L -1 , EDTA 1mmol·L -1 , dissolved at 65°C for 5 minutes. Extract with an equal volume of chloroform, centrifuge at 13,000g for 5 minutes; add 2 times the volume of absolute ethanol to the supernatant, and ...

Embodiment 2

[0031] Embodiment 2, the cloning of Danshen isopentenyl pyrophosphate isomerase gene

[0032] 1. Preparation of experimental materials

[0033] The hairy roots of Salvia miltiorrhiza were induced by Ri-plasmid transformation by direct infection with Agrobacterium rhizogenes 15834. Get the hairy roots of Salvia miltiorrhiza in 6,7-V solid medium (without hormones), and inoculate 2g of wet roots in a 500mL Erlenmeyer flask containing 200ml of hormone-free 6-7V liquid medium under aseptic conditions. Subculture was carried out, and it was used as the test material after 18 days of cultivation. The culture condition is 25℃, 110~120r·min -1 , cultivated under dark conditions.

[0034] 2. Preparation and processing of elicitors

[0035] Preparation of yeast extract (yeast 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 a colloidal ...

Embodiment 3

[0055] Embodiment 3, the bioinformatics analysis of SmIPPI gene

[0056] The full-length cDNA of the salvia miltiorrhiza isopentenyl pyrophosphate isomerase gene involved in the present invention has a length of 1320 bp, and encodes a protein sequence SEQ ID No. 2 consisting of 305 amino acid residues in the sequence list.

[0057] See sequence 1 in the sequence listing for the detailed sequence, wherein the open reading frame is located at 127-1044bp. The full-length cDNA sequence of Salvia miltiorrhiza was searched for nucleotide homology in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBankCDS translation+PDB+Swissprot+Superdate+PIR databases with BLAST program, the results showed that Salvia miltiorrhiza SmIPPI and The IPPI homology is between 70.4% (pueraria, AAQ84167) and 99.1% (tobacco, BAB40974), and has a high degree of homology. are also higher, at 89.8% and 88.9%, respectively. There are many conserved amino acid sequences in the IPPI gene, which can be...

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PUM

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Abstract

The invention discloses a salvia miltiorrhiza bunge isopentenylpyrophosphate isomerase (IPPI) gene, a protease coded by the IPPI genes and an application thereof. The gene is obtained by cloning fromsalvia miltiorrhiza bunge through utilizing a cDNA chip technology for the first time, thus the invention fills the blank of separating and cloning the SmIPPI gene from the salvia miltiorrhiza bunge which is a conventional famous and precious Chinese medicinal plant. The SmIPPI gene has a nucleotide sequence represented by SEQ ID NO.1 or adds, substitutes, inserts or deletes one or more nucleotidehomologous sequences or an allele thereof and a nucleotide sequence derived from the nucleotide homologous sequence. A protein coded by the SmIPPI genes has an amino acid sequence represented by SEQID NO.2 or adds, substitutes, inserts or deletes one or more amino acid homologous sequences. The SmIPPI gene can be applied to researches and industrialization for improving the content of diterpeneactive constituents in the salvia miltiorrhiza bunge by a biotechnology method and improving the content of tanshinone substances by utilizing a transgenic technology, is helpful to the quality improvement, the selective breeding, and the like of medicinal materials of the salvia miltiorrhiza bunge and has good application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering of medicinal plants, in particular, it relates to the use of cDNA chip technology to clone a new Danshen isopentenyl pyrophosphate isomerase (isopentenyl diphosphate isomerase, IPPI) gene, transforming the gene to improve the secondary Methods for content of terpene secondary metabolites. Background technique [0002] The formation of active ingredients (secondary metabolites) of medicinal plants is the product of specific genes in plant secondary metabolic pathways. With the extensive and in-depth study of plant functional genomes, the study of functional genes related to secondary metabolism synthesis of medicinal plants with unique characteristics and broad application prospects has gradually become a research hotspot. The biosynthetic pathway and its regulatory mechanism provide a theoretical basis for the formation of the quality of medicinal materials, and at the same time bring a broad a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N9/90C12N15/63C12N1/21C12N5/10C12Q1/68C12N15/84C12R1/19
Inventor 黄璐琦张夏楠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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