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Transcription factor ptomyb115 specifically regulating tannin synthesis and its use

A technology of transcription factor and expression vector, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of poplar susceptible to diseases and insect pests, difficult to consolidate engineering achievements, failure, etc., to improve the ability of canker disease and improve disease resistance. Effect

Inactive Publication Date: 2018-02-06
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because poplar is susceptible to pests and diseases, it is difficult to consolidate the project results, or even fail
In contrast to the extensive investigation of herbaceous plant-pathogen interactions using genetic and molecular approaches, our knowledge of the defense mechanisms of tree species against pathogens remains largely incomplete (Miranda et al. .,2007)

Method used

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  • Transcription factor ptomyb115 specifically regulating tannin synthesis and its use
  • Transcription factor ptomyb115 specifically regulating tannin synthesis and its use
  • Transcription factor ptomyb115 specifically regulating tannin synthesis and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Poplar total RNA extraction and reverse transcription synthesis of cDNA

[0032] 1. Poplar total RNA extraction

[0033]The young leaves of poplar were used as materials, and the medicine spoons, pestles and mortars used in RNA extraction were all burned with 95% alcohol until cooled before use. Take a spoonful of quartz sand and heat it in a heater for 10 minutes to cool it before use. 1.5mL centrifuge tubes, plastic products, etc. use RNase-treated (RNase Free) AXYGEN products.

[0034] Total RNA extraction was performed according to the operation steps of Huashun Server-SPIN DNA-free Plant RNA Isolation Kit:

[0035] 1) Add lysate (500 μL RL+100 μL 10% PVP+10 μL 2-ME) to a 1.5 mL centrifuge tube;

[0036] 2) Mix young poplar leaves (100mg) and quartz sand under liquid nitrogen freezing conditions and quickly grind them into powder. Before the liquid nitrogen is completely volatilized, transfer all the powder to the pre-prepared lysis solution, and pipett...

Embodiment 2

[0051] Example 2 Design of PCR primers and PCR amplification to obtain target fragments

[0052] 1. PCR primer design

[0053] Log on to PHYTOZOME (http: / / www.phytozome.com) to find the CDS sequence of the PtrMYB115 gene of P. tomentosa. According to the sequence, the specific primer of PtoMYB115CDS of Populus tomentosa was designed. The upstream primer PtoMYB115-F sequence of the PtoMYB115 fragment is: 5 '-ATGGGAAGGGCTCCTTGTTG-3' (SEQ ID No. 4), downstream primer PtoMYB115-R sequence: 5'-TTGTCATACCAGCAGTGACT-3' (SEQ ID No. 5); target fragment 858bp.

[0054] Primers were synthesized by BGI (Beijing). The reaction reagents used in PCR are all products of TaKaRa company: dNTP Mixture (2.5mM each), Taq DNA polymerase (5U / μL), with 10×PCR Buffer (0.1M Tris-HCl [pH8.3]), 0.5M KCl) and MgCl 2 (25mM).

[0055] 2. PCR amplification of the target fragment

[0056] The PtoMYB115 gene fragment was amplified using poplar cDNA as a template, and the system for amplifying the PtoMYB11...

Embodiment 335

[0068] Example 335S: PtoMYB115 vector construction and transformation of Agrobacterium EHA105

[0069] After the amplified fragment gel was recovered, the carrier pCXSN (provided by Professor Wang Guoliang of South China Agricultural University) was digested with XcmI and then connected with the recovered fragment to obtain 35S:PtoMYB115 carrier (sequence shown in SEQ ID No. 3). The map is shown in figure 1 . The ligation product was transformed into Escherichia coli DH5α competent cells, and the target gene fragment was amplified by PCR and verified by enzyme digestion to obtain transformants, and the strains were stored.

[0070] The 35S:PtoMYB115 plasmid was extracted from the positive transformant Escherichia coli respectively, and the steps were in accordance with the extraction steps of the PlasmidMini Kit. The specific operations are as follows:

[0071] ① Take the bacterial liquid cultured to the logarithmic phase in an EP tube, centrifuge at 13400 rpm for 1 min to co...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and in particular relates to a transcription factor PtoMYB115 for specific regulation and control of tannin synthesis and an application of the transcription factor PtoMYB115. The transcription factor PtoMYB115 disclosed by the invention has an amino acid sequence shown by SEQ ID No.2. The invention also provides an expression vector containing the transcription factor PtoMYB115. The invention also provides a host containing the expression vector. The transcription factor provided by the invention improves the disease resistance of a plant by regulating and controlling the tannin content of a Chinese white poplar at a transcription level, thereby providing a new method for obtaining a high-quality poplar variety with high disease resistance.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to a transcription factor PtoMYB115 that specifically regulates tannin synthesis and uses thereof. Background technique [0002] Poplar (Populus spp.) is one of the most widely planted fast-growing trees in the world. Under ideal conditions, poplar can become a material in about 4 years. It has the characteristics of strong adaptability, fast reproduction, easy hybridization, easy improvement of heredity, and easy asexual reproduction. It is an important building material and papermaking raw material. At the same time, it can also be used as a Raw materials for renewable energy. The genome sequencing of P. trichocarpa was completed in 2006 (Tuskan et al., 2006), which is regarded as a model plant of woody transgenic plants, and is widely used in genetic transformation research of forest tree species. Populus tomentosa (Populus tomentosa) belongs to the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N1/21A01H5/00A01H6/00
CPCC07K14/415C12N15/8282
Inventor 侯一粟罗克明王显强汪丽君
Owner SOUTHWEST UNIV
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