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Analysis and application of salvia miltiorrhiza acetyl-CoA acyltryansferase (SmAACT) gene

A technology of gene and Salvia miltiorrhiza, applied in the field of genetic engineering of medicinal plants, can solve the problems such as no relevant reports, achieve good application prospects and improve the effect of material yield

Inactive Publication Date: 2009-12-30
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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1, the making of Danshen cDNA chip

[0018] 1. Isolation and detection of total RNA from Salvia miltiorrhiza

[0019] 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 , 2% PVP40, 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

[0029] Example 2: Cloning of Salvia miltiorrhiza acetyl-CoA acyltransferase gene

[0030] 1. Preparation of experimental materials

[0031] 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 6-7V 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, cultured to 18 days later as the test material. The culture conditions are 25°C, 110-120r·min-1, and cultured in the dark.

[0032] 2. Preparation and processing of elicitors

[0033] 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 precipitate Dissolve in 125mL distilled water, ...

Embodiment 3

[0053] Embodiment 3, the bioinformatics analysis of SmAACT gene

[0054] The full-length cDNA of the salvia miltiorrhiza acetyl-CoA acyltransferase gene involved in the present invention is 1623bp in length, and the detailed sequence is shown in sequence 1 in the sequence list, wherein the open reading frame is located at 126-1325bp. 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 databases with BLAST program, the results showed that SmAACT and The homology of acetyl-CoA acyltransferases in other plants ranged from 70.6% (rice, NP-001041797.1) to 84.0% (Huhuanglian, ABC74567), showing a high degree of homology. The homology of wood (AAL18924) etc. is also high. According to the results of multiple alignments of protein amino acid sequences, it can be seen that the amino acid sequences of different species have multiple conserve...

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PUM

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Abstract

The invention discloses a salvia miltiorrhiza acetyl-CoA acyltryansferase (SmAACT) gene, prolease encoded by the gene and application. The gene is obtained by cloning from salvia miltiorrhiza by utilizing cDNA chip technology for the first time, and therefore fills the gap of separating and cloning an acetyl-CoA acyltryansferase gene from the salvia miltiorrhiza, a traditional rare medicinal material in our country. The SmAACT gene has a nucleotide sequence shown in SEQ IDNO.1 or a homologous sequence which adds, substitutes, inserts or deletes one or more nucleotides or nucleotide sequences of an allelic gene and a derivative thereof. Protein encoded by the gene has a nucleotide sequence shown in SEQ ID No.2 or a homologous sequence which adds, substitutes, inserts or deletes one or more nucleotides. The salvia miltiorrhiza acetyl-CoA acyltryansferase catalyzes a first enzymatic reaction on an MVA pathway, ensures that acetyl-CoA with two molecules is condensed into acetyl acetyl-CoA, and is first key enzyme on a biosynthetic pathway of diterpenoid tanshinone compounds from the salvia miltiorrhiza; and the expression level of the gene is estimated to be closely related to the content of tanshinone components. The SmAACT gene can improve the content of diterpene active components, namely tanshinones in the salvia miltiorrhiza through biotechnology, can be used for the research and industrialization on improving the content of tanshinone substances by utilizing the biotechnology, is favorable for the quality improvement and breeding of salvia miltiorrhiza medicinal materials, 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 gene of Salvia miltiorrhiza acetyl-CoA acyltransferase (acetyl-CoA acyltryansferase, AACT), and transform the gene to increase the concentration of diterpenoids in Salvia miltiorrhiza. Methods for the content of 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 mater...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/63C12N5/10C12Q1/68C12N15/84
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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