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Liriodendron transcription factor LcbHLH02399 gene as well as expression protein and application thereof

A lcbhlh02399, transcription factor technology, applied in the field of plant genetic engineering, to achieve the effect of enhancing tolerance

Active Publication Date: 2022-07-29
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 LcbHLH02399 gene as well as expression protein and application thereof
  • Liriodendron transcription factor LcbHLH02399 gene as well as expression protein and application thereof
  • Liriodendron transcription factor LcbHLH02399 gene as well as expression protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Analysis of Liriodendron bHLH family and cloning of LcbHLH02399 gene

[0027] 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, the protein domain alignment software HMMER3.0 was used to build a model to find out the potential bHLH family sequences of Liriodendron, and then use BLASTP to align the candidate bHLH family sequences of Liriodendron. Through sequencing and homology comparison, a sequenced sequence homologous to bHLH transcription factor was cloned and named as LcbHLH02399 transcription factor.

[0028] 1. Extraction and quality inspection of total RNA

[0029] Before extracting total RNA, all containers used in the extraction process need to be RNase-free to remove interference from genomic DNA. Soak plastic utensils such as centrifuge tubes, grinding tools such as mortars and pestles, and tweezers in 0.1% DEPC water overnight. ...

Embodiment 2

[0061] Example 2: Construction of Liriodendron LcbHLH02399 gene expression vector

[0062] 1. Design of enzyme digestion primers

[0063] According to the pRI-101-6Flag plasmid map ( figure 2 ) and the characteristics of the coding region of the LcbHLH02399 gene, using the software Primer premier 5.0 to design primers containing the restriction enzyme cleavage site for the ORF of the LcbHLH02399 gene. Through the software search for the ORF restriction site of LcbHLH02399 gene, it is found that there is no XbaI and BamHI restriction sites in the ORF of LcbHLH02399 gene. The designed Nde I and Cla I double restriction enzyme primers are as follows:

[0064] LchICE1-Nde I-F: 5′-GGGAATTCCATATGCTGTCGAGGGTGAACGG-3′

[0065] LchICE1-Cla I-R: 5′-CCCATCGATGAATTCGGCGTCGAGGAATTCAT-3′

[0066] 2. PCR amplification of the target gene restriction fragment

[0067] Same as "4. PCR amplification of target gene sequence fragment" in Example 1

[0068] 3. Electrophoresis detection and re...

Embodiment 3

[0100] Example 3: Agrobacterium-mediated genetic transformation of LcbHLH02399 gene to hybrid Liriodendron

[0101] 1. Freeze-thaw method into Agrobacterium

[0102] (1) Thaw 100 μL of Agrobacterium EHA105 competent cells on ice, add 2 μL of the extracted plant expression vector plasmid, and mix by pipetting with a pipette tip;

[0103] (2) Ice bath for 5 minutes, liquid nitrogen freezing for 5 minutes, water bath at 37°C for 5 minutes, and ice bath for 5 minutes;

[0104] (3) Add 700 μL of LB liquid medium, 28 ° C, 200 rpm, shaking culture for 3 hours;

[0105] (4) Centrifuge at 4000rpm for 3min, leaving a little supernatant;

[0106] (5) the remaining bacterial liquid after the transformation is mixed and coated on the LB solid medium containing Kan;

[0107] (6) Inverted culture at 28°C for 3 days

[0108] (7) Identification of positive clones.

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Abstract

The invention discloses a liriodendron transcription factor LcbHLH02399 gene as well as an expression protein and application thereof, and belongs to the technical field of plant genetic engineering. The bHLH family sequence of liriodendron is analyzed through a bioinformatics tool, and then the LcbHLH02399 gene for coding the bHLH transcription factor is cloned through sequencing and homologous comparison. The method comprises the following steps: constructing an overexpression vector for an LcbHLH02399 gene, transforming hybrid liriodendron calluses, and carrying out liquid culture to obtain transgenic hybrid liriodendron; compared with phenotypes of LcbHLH02399 transgenic hybrid liriodendron plants and wild plants which are subjected to low-temperature treatment in an illumination incubator at 4 DEG C for 3 days, the influence of low-temperature stress on the growth of the transgenic plants is small, the growth of the wild plants is inhibited, and leaves are withered. The result shows that the liriodendron tulipifera LcbHLH02399 gene can enhance the tolerance of a liriodendron tulipifera plant to low-temperature stress, and has an important application value in molecular breeding for improving the low-temperature stress of the plant.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and more particularly relates to a Liriodendron tulipifera transcription factor LcbHLH02399 gene and its expression protein and application. Background technique [0002] Liriodendron Chinese (Liriodendron Chinese), as an important rare tree species in my country (national second-class protected plant), has scientific research, economic, ornamental, medicinal and ecological value. Mainly reflected in: 1. It has important scientific research value for paleobotany research; 2. The trunk is straight, the crown is umbrella-shaped, the leaves are peculiar, and the flowers are large and beautiful, and it is an excellent ornamental tree species; 3. The wood texture is straight, dry and less cracked , Soft and moderate, easy to process, less deformation, no moth-eaten, is a high-quality material for interior decoration and furniture production (Liu Jianping, 2020). Liriodendron tulipif...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00A01H6/00
CPCC07K14/415C12N15/8273
Inventor 陆叶陈金慧杨立明施季森杨蕤西郝兆东
Owner NANJING FORESTRY UNIV
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