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Tanshinone biosynthesis inhibiting factor gene SmJAZ3 and encoding protein and application thereof

A technology for biosynthesis and inhibitory factors, applied in the field of genetic engineering, which can solve the problems that have not yet been discovered and that there are few researches on the signal transduction of Salvia miltiorrhiza

Active Publication Date: 2016-03-09
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As a traditional Chinese medicine with extensive medicinal value, Salvia miltiorrhiza has become a research hotspot of scholars at home and abroad, but the current research on Danshen is mainly based on its tanshinone (MEP or MVA pathway) or salvianolic acid key enzyme genes. Research, less research on Danshen signaling
The present invention mainly obtains a JAZ3 protein in the JA signaling pathway of Salvia miltiorrhiza by cloning, constructs an overexpression and antisense suppression carrier by means of genetic engineering, genetically transforms Salvia miltiorrhiza to obtain hairy roots, and finds that the expression of the JAZ3 gene in Salvia miltiorrhiza significantly affects the metabolic synthesis of tanshinone. In particular, by inhibiting the expression of the Danshen JAZ3 gene, the accumulation and synthesis of tanshinone can be significantly promoted, and the hairy root of Salvia miltiorrhiza with high yield of tanshinone can be obtained, which can provide new drug source raw materials for commercially obtaining tanshinone. No inhibitor of JAZ3 mentioned in this topic has been found yet. Related reports of gene expression strategies to increase tanshinone content

Method used

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  • Tanshinone biosynthesis inhibiting factor gene SmJAZ3 and encoding protein and application thereof
  • Tanshinone biosynthesis inhibiting factor gene SmJAZ3 and encoding protein and application thereof
  • Tanshinone biosynthesis inhibiting factor gene SmJAZ3 and encoding protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Cloning of Example 1 Danshen SmJAZ3 Gene

[0022] 1. Tissue separation (isolation)

[0023] The tender tissues of Salvia miltiorrhiza plants were taken and stored in -80°C refrigerator

[0024] 2. RNA isolation (RNAisolation)

[0025] Take part of the tissue and grind it with a mortar, add the lysate to homogenize it, transfer it to a 1.5mLEP tube, and extract the total RNA. Ordinary agarose gel electrophoresis was used to identify the quality of total RNA, and then the RNA content was determined on NanoDrop.

[0026] 3. Cloning of Full-length cDNA

[0027] The hairy roots of Salvia miltiorrhiza in shake flasks cultured for two months were induced by methyljasmonic acid, RNA was extracted, reverse transcribed into cDNA, and transcriptome analysis was performed by high-throughput sequencing.

[0028] 3.1. Transcriptome sequencing

[0029] Through transcriptome sequencing of the hairy root material of Salvia miltiorrhiza, a highly homologous sequence to Arabidopsis J...

Embodiment 2

[0035] Example 2 (Sequence information and homology analysis of Danshen SmJAZ3 gene)

[0036] The length of the full-length cDNA of the SmJAZ3 gene of the present invention is 1011bp, and the detailed sequence is shown in SEQ ID NO.1. The amino acid sequence of the Danshen SmJAZ3 gene was deduced according to the full-length cDNA, with a total of 336 amino acid residues. See SEQ ID NO.2 for the detailed sequence.

[0037] The homologous comparison of the amino acid sequences of Salvia miltiorrhiza SmJAZ3 and tobacco (Nicotianatabacum) jasmonateZIM-domainprotein7b is shown in Table 1. The gene was compared with the Arabidopsis JAZ gene by software such as ClustalW and Boxshade. This gene has the conserved ZIM structure of AtJAZs and the Jas domain (SLX2FX2KRX2RX5PY). The result is as Figure 10 , Figure 11 shown. The phylogenetic tree was established by Mega6.0, and the local Blast found that the obtained JAZ gene had the highest homology with Arabidopsis JAZ3, and named i...

Embodiment 3

[0056] Example 3 Construction of overexpression and antisense suppression vectors, genetic transformation of Salvia miltiorrhiza hairy roots

[0057] 1. Construction of SmJAZ3 overexpression and antisense suppression vector

[0058] SmJAZ3 forward and reverse full-length clones into pCAMBIA2300 + On, the overexpression vector pCAMBIA2300 was obtained + -SmJAZ3 and antisense suppression vector pCAMBIA2300 + -Anti-SmJAZ3.

[0059] 2. Agrobacterium rhizogenes C58C1 mediates pCAMBIA2300 + , pCAMBIA2300 + -SmJAZ3 and pCAMBIA2300 + -Anti-SmJAZ3 plasmid genetic transformation of Salvia miltiorrhiza to obtain hairy roots of Salvia miltiorrhiza.

[0060] (1) Pre-cultivation of explants

[0061] Cut the leaves and petioles of sterile salvia miltiorrhiza seedlings, inoculate them on the preculture medium (1 / 2MS), and culture them in the dark at 25°C for 2 days.

[0062] (2) Co-cultivation of Agrobacterium and explants

[0063] Put the above-mentioned explants of pre-cultivated l...

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Abstract

The invention discloses an inhibiting factor JAZ (Jasmonate ZIM domain) gene-SmJAZ3 and corresponding protein sequence thereof in salviae miltiorrhizae jasmonic acid signal pathway. The SmJAZ3 gene has a nucleotide sequence shown in SEQ ID NO.1. Gene encoded protein has an amino acid sequence shown in SEQ ID No.2. An over-expression and antisense inhibitory carrier is constructed, genetic transformation is conducted on salvia miltiorrhiza hairy roots, and tanshinone content in salvia miltiorrhiza is obviously affected. The SmJAZ3 is closely related to tanshinone, quality improvement of radix salviae miltiorrhizae is facilitated, and a new action target and research idea are provided for solving the problem of medicine source shortage of the tanshinone.

Description

technical field [0001] The invention relates to genetic engineering technology, in particular to a tanshinone biosynthesis inhibitor gene SmJAZ3 and its encoded protein and application. Background technique [0002] Salviamiltiorrhiza Bunge, also known as red ginseng, purple danshen, red root, etc., is a commonly used traditional Chinese medicine of Labiatae (Labiatae), and its roots and rhizomes are used as medicine. effect. There are two main types of pharmacological components of tanshinone, one is fat-soluble diterpenoids, and the other is water-soluble phenolic acids. Tanshinone compounds with high content include tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone, which have various pharmacological activities such as anti-tumor, anti-bacterial, anti-inflammatory, anti-oxidation, and anti-atherosclerosis, so they have a huge market demand. The main components of phenolic acids include protocatechualdehyde, rosmarinic acid, danshensu, caffeic acid, s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N5/10
Inventor 开国银时敏耿丽媛张建林
Owner SHANGHAI NORMAL UNIVERSITY
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