Application of salvia miltiorrhiza transcription factor SmbHLH124 in improvement of yield character of salvia miltiorrhiza hairy roots

A technology of transcription factor and hairy root, applied in the field of plant biology, can solve the problems of increasing the growth of hairy roots of Salvia miltiorrhiza, and achieve the effects of improving yield traits, biomass and tanshinone content, and good application prospect.

Pending Publication Date: 2021-11-12
UNIV OF ELECTRONIC SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are many members of the bHLH transcription factor family in Salvia miltiorrhiza, SmbHLH1, SmbHLH 3, SmbHLH 53, SmbHLH 10 and other members have been reported to have certain regulatory effects on the biosynthesis of secondary metabolites, but no effect on the growth of hairy roots in Salvia miltiorrhiza. reports of increased volume

Method used

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  • Application of salvia miltiorrhiza transcription factor SmbHLH124 in improvement of yield character of salvia miltiorrhiza hairy roots
  • Application of salvia miltiorrhiza transcription factor SmbHLH124 in improvement of yield character of salvia miltiorrhiza hairy roots
  • Application of salvia miltiorrhiza transcription factor SmbHLH124 in improvement of yield character of salvia miltiorrhiza hairy roots

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The construction of embodiment 1 Salvia miltiorrhiza SmbHLH124 overexpression vector

[0055] (1) SmbHLH124 cDNA fragment acquisition

[0056] Design specific primers SmbHLH124-F and SmbHLH124-R according to the sequence information of the SmbHLH124 gene in the published Salvia miltiorrhiza genome and transcriptome data. synthesis. Using the cDNA (20ng / μL) of the underground root of Salvia miltiorrhiza as a template, the SmbHLH124 gene fragment was amplified. The target fragment amplification system: 10×KOD Plus Buffer 5μL, dNTP (2.5mM) 5μL, MgSO4 (25mM) 3μL, KODPlus Neo (1U / μL) 1 μL, SmbHLH124-F (10 μM) 1 μL, SmbHLH124-R (10 μM) 1 μL, cDNA 1 μL, ddH 2 O 33 μL. The PCR amplification program is as follows: 95°C / 5min→(95°C / 30s→56°C / 30s→68°C / 30s)×40cycles→68°C / 5min→12°C / 10min. The PCR product was subjected to agarose electrophoresis, and the target fragment of about 300bp was recovered using the AxyPrep DNA Gel Recovery Kit. figure 1 , and sent the gel-recovered produ...

Embodiment 2

[0067] Example 2 Agrobacterium-mediated genetic transformation of Salvia miltiorrhiza hairy roots

[0068] Agrobacterium-mediated Salvia miltiorrhiza hairy root genetic transformation reference (Toki et al., Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. The Plant Journal, 2006, 47 (6): 969-976.) disclosed method . The steps of genetic transformation of Salvia miltiorrhiza are as follows, and the flowchart is shown in image 3 :

[0069] (1) Agrobacterium preparation

[0070]Pick up Agrobacterium tumefaciens C58C1 carrying the target vector pLXP003 (Shanghai Titan Technology Co., Ltd.) stored at -80°C with an inoculation loop, streak on LB+50mg / L Kan+50mg / L Rif plate and culture it upside down at 28°C Pick up a single colony on the above LB plate with a toothpick, inoculate it in a test tube (or 10 mL EP tube) containing 5 mL LB+50 mg / L Kan+50 mg / L Rif liquid medium, activate at 28°C and 180 rpm Cultivate for 48 hours; on t...

Embodiment 3

[0081] Example 3 Molecular identification of Salvia miltiorrhiza overexpressing SmbHLH hairy roots

[0082] (1) Genomic DNA extraction from the hairy root of Salvia miltiorrhiza

[0083] The DNA extraction of Salvia miltiorrhiza hairy root adopts CTAB method, and the specific operation steps are as follows:

[0084] The CTAB extract was preheated in a 65°C water bath. Take a single root 1-2cm away from the root tip and put it into a 2mL centrifuge tube with steel balls, put it into liquid nitrogen for quick freezing, and then shake it into powder. Add 500 μL of preheated CTAB extract, bathe in water at 65°C for 30-50 min, and mix well during this period. Add 500 μL of chloroform:isoamyl alcohol (24:1), mix thoroughly by inversion, and centrifuge at 12000 rpm for 10 min at room temperature. Take the supernatant, add an equal volume of isopropanol to precipitate, and precipitate at -20°C for 1 h. Centrifuge at 12000rpm for 10min at room temperature to collect the precipitate...

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Abstract

The invention belongs to the technical field of plant biology, and particularly relates to novel application of a salvia miltiorrhiza transcription factor SmbHLH124. The technical problem to be solved by the invention is to improve the yield character of salvia miltiorrhiza hairy roots. According to the technical scheme for solving the technical problem, the application of the salvia miltiorrhiza transcription factor SmbHLH124 or an encoding DNA molecule thereof in improvement of the yield character of the salvia miltiorrhiza hairy roots and a method for improving the yield character of the salvia miltiorrhiza hairy roots are provided. According to the technical scheme, the hairy roots with obviously increased biomass and tanshinone content can be obtained, and the yield character of the salvia miltiorrhiza hairy roots is effectively improved. The application can provide an ideal material for tanshinone medicine source production, and has a good application prospect in solving the problem of insufficient medicine sources of tanshinone compounds.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and specifically relates to the use of the Danshen transcription factor SmbHLH124 in improving hairy root yield traits. Background technique [0002] Salvia miltiorrhiza Bunge is a medicinal plant with a long history of use. It has been recorded in the "Chinese Pharmacopoeia" for a long time, and its roots and rhizomes are used as medicine. Its medicinal materials and preparations are widely used clinically. Tanshinone compounds are fat-soluble active ingredients in Danshen, and the representative ingredients include cryptotanshinone (CTS), dihydrotanshinone (DTS), tanshinone I (TSI) and tanshinone IIA (TSA). , has clear pharmacological effects in many aspects such as cardiovascular disease, anti-inflammatory and immune, anti-tumor, liver protection, and neuroprotection (Liu Huiying, 2016). [0003] The hairy root of Salvia miltiorrhiza, like other plant hairy roots, has the characteristics of...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/06A01H6/50
CPCC07K14/415C12N15/8243C12N15/8261Y02A40/146
Inventor 张勇邓科君郑雪莲仲昭辉刘晓佩方庆李梦珂
Owner UNIV OF ELECTRONIC SCI & TECH OF CHINA
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