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Liriodendron transcription factor LcbHLH52 gene and application thereof

A technology of transcription factor, Liriodendron, applied in application, genetic engineering, plant genetic improvement and other directions to achieve the effect of enhancing tolerance

Active Publication Date: 2021-05-04
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the low temperature limits the large-scale promotion of Liriodendron tulipifolia in northern my country.

Method used

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  • Liriodendron transcription factor LcbHLH52 gene and application thereof
  • Liriodendron transcription factor LcbHLH52 gene and application thereof
  • Liriodendron transcription factor LcbHLH52 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Analysis of the bHLH family of Liriodendron tulipifera and the cloning of the LcbHLH52 gene

[0030] Download the model plant Arabidopsis (https: / / www, arabidopsis.org) and rice (http: / / rapdb.dna.affrc.go.jp) databases to obtain bHLH family sequences, 177 and 135. Then use the protein domain alignment software HMMER3.0 to build a model to find out the potential bHLH family sequences of Liriodendron tulipifera, and then use BLASTP to compare the candidate bHLH family sequences of Liriodendron tulipifera. Finally, LcbHLH52 transcription factor was selected for cloning and sequencing.

[0031] 1. Extraction and quality detection of total RNA

[0032] Before extracting total RNA, all containers used in the extraction process need to be treated without RNase, in order to remove the interference of genomic DNA. Soak plastic utensils such as centrifuge tubes, grinding tools such as mortar and pestle, and tweezers in 0.1% DEPC water overnight, drain the water, wrap...

Embodiment 2

[0086] Embodiment 2: Construction of Liriodendron LcbHLH52 gene expression vector

[0087] 1. Design of enzyme digestion primers

[0088] According to the Pcambia2301 plasmid map ( image 3 ) and the characteristics of the LcbHLH52 gene coding region, using the software Primerpremier 5.0 to design the primers containing the restriction site for the LcbHLH52 gene ORF. Searching the restriction site of LcbHLH52 gene ORF through software, it is found that there is no restriction site of KpnI and BamHI in the ORF of LcbHLH52 gene, and the double restriction primers of KpnI and BamHI can be designed as follows:

[0089] LcbHLH52-KpnI-F: 5′-GGggtaccATGGAAATAGATGAACATGG-3′,

[0090] LcbHLH52-BamHI-R: 5'-CGggatccCTACATGCATCTCCCTCC-3'.

[0091] 2. PCR amplification of the enzyme-cut fragment of the target gene

[0092] Same as "4, PCR amplification target gene sequence fragment" in Example 1

[0093] 3. Electrophoresis detection and recovery of amplified products

[0094] Same as...

Embodiment 3

[0121] Example 3: Genetic transformation of Arabidopsis thaliana by the LcbHLH52 gene mediated by Agrobacterium

[0122] 1. Freezing and thawing into Agrobacterium

[0123] (1) Melt Agrobacterium EHA105 competent cells on ice, add 2 μL of the extracted plant expression vector plasmid, and pipette the tip to mix;

[0124] (2) Ice bath for 30 minutes, freeze in liquid nitrogen for 1 minute, and then bathe in water at 37°C for 5 minutes;

[0125] (3) Add 700 μL LB liquid medium, shake at 28°C, 200 rpm for 3 hours;

[0126] (4) Centrifuge at 4000rpm for 3min, leaving a little supernatant;

[0127] (5) Mix the remaining bacterial liquid after transformation, and apply it on the LB solid medium containing Kan;

[0128] (6) Inverted culture at 28°C for 3 days

[0129] (7) Identification of positive clones.

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Abstract

The invention discloses a liriodendron transcription factor LcbHLH52 gene and an application thereof, and belongs to the technical field of plant genetic engineering. Sequences of liriodendron bHLH family are analyzed through a bioinformatics tool, and then the LcbHLH52 transcription factor gene is successfully cloned through sequencing and homologous comparison. An overexpression vector is constructed for the cloned LcbHLH52 transcription factor gene, and used to transform arabidopsis thaliana to obtain T1 generation of seeds. T2 generation of LcbHLH52 transgenic arabidopsis thaliana plants and wild type arabidopsis thaliana plants are processed at the low temperature of 4 DEG C for 3 days simultaneously. Phenotypic observation shows that the growth of the transgenic plants is unlikely to be affected by low-temperature stress, while the growth of the wild type plants is inhibited with wilted leaves. The result shows that the liriodendron LcbHLH52 gene can enhance the tolerance of arabidopsis thaliana plants to low-temperature stress, and has important application value in improving molecular breeding of plants against low-temperature stress.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a gene of transcription factor LcbHLH52 of Liriodendron tulipifera and its application. Background technique [0002] Liriodendron Chinese, as an important rare tree species in my country (a national second-class protected plant), has many aspects such as scientific research, economy, ornamental, medicinal and ecological values. Mainly reflected in: 1. It has important scientific research value for paleobotany research; 2. The trunk is straight, the crown is umbrella-shaped, the leaf shape is strange, the flowers are large and beautiful, and it is an excellent ornamental tree species; 3. The wood texture is straight, dry and less cracked , moderate softness, easy processing, less deformation, and no insects, it is a high-quality material for interior decoration and furniture production (Liu Jianping, 2020). Liriodendron chinensis is mainly distr...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00A01H6/20
CPCC07K14/415C12N15/8205C12N15/8273
Inventor 郝兆东杨立明王占军高敏朱礼明施季森陈金慧
Owner NANJING FORESTRY UNIV
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