A kind of gene of transcription factor lcbhlh16421 of Liriodendron tulipifera and its application

A technology of lcbhlh16421 and transcription factors, applied in the field of plant genetic engineering, to achieve the effect of enhancing tolerance

Active Publication Date: 2022-02-01
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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  • A kind of gene of transcription factor lcbhlh16421 of Liriodendron tulipifera and its application
  • A kind of gene of transcription factor lcbhlh16421 of Liriodendron tulipifera and its application
  • A kind of gene of transcription factor lcbhlh16421 of Liriodendron tulipifera and its application

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 LcbHLH16421 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. Through sequencing and homologous comparison, a sequence sequence homologous to bHLH transcription factor and highly similar to Lch16421 in Liriodendron tulipifera genome database was cloned, named LcbHLH16421 transcription factor.

[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. ...

Embodiment 2

[0085] Example 2: Construction of Liriodendron LcbHLH16421 gene expression vector

[0086] 1. Design of enzyme digestion primers

[0087] According to the Pcambia2301 plasmid map ( image 3 ) and the characteristics of the LcbHLH16421 gene coding region, using the software Primer premier 5.0 to design the primers containing the restriction site for the ORF of the LcbHLH16421 gene. Search the ORF restriction site of LcbHLH16421 gene by software and find that there is no XbaI and BamHI restriction site in the LcbHLH16421 gene ORF, and the XbaI and BamHI double restriction primer can be designed as follows: LcbHLH16421-XbaI-F: 5′-GCtctagaATGGAAGAAGGAGCAGATG-3′ , LcbHLH16421-BamHI-R: 5'-CGggatccCTACATTGTCCCTTGGAAGC-3'.

[0088] 2. PCR amplification of the enzyme-cut fragment of the target gene

[0089] Same as "4, PCR amplification target gene sequence fragment" in Example 1

[0090] 3. Electrophoresis detection and recovery of amplified products

[0091] Same as "5. Electrop...

Embodiment 3

[0117] Example 3: Genetic transformation of Arabidopsis thaliana by the LcbHLH16421 gene mediated by Agrobacterium

[0118] 1. Freezing and thawing into Agrobacterium

[0119] (1) Melt Agrobacterium EHA105 competent cells on ice, add 2 μL of the extracted plant expression vector plasmid, and pipette the tip to mix;

[0120] (2) Ice bath for 30 minutes, freeze in liquid nitrogen for 1 minute, and then bathe in water at 37°C for 5 minutes;

[0121] (3) Add 700 μL LB liquid medium, shake at 28°C, 200 rpm for 3 hours;

[0122] (4) Centrifuge at 4000rpm for 3min, leaving a little supernatant;

[0123] (5) Mix the remaining bacterial liquid after transformation, and apply it on the LB solid medium containing Kan;

[0124] (6) Inverted culture at 28°C for 3 days

[0125] (7) Identification of positive clones.

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Abstract

The invention discloses a gene of transcription factor LcbHLH16421 of Liriodendron tulipifera and its application, belonging to the technical field of plant genetic engineering. The present invention analyzes the sequence of the Liriodendron bHLH family through bioinformatics tools, and then clones and obtains the LcbHLH16421 transcription factor gene through sequencing and homologous comparison. An overexpression vector was constructed for the LcbHLH16421 gene, transformed into Arabidopsis thaliana, and T1 generation seeds were obtained; the LcbHLH16421 transgenic Arabidopsis T2 generation plants and wild-type plants were placed in a low temperature treatment at 4°C for 3 days at the same time, and the phenotype observation found that the growth of the transgenic plants was affected by the low temperature. The impact of stress was small, while the growth of wild-type plants was inhibited, and the leaves were wilting. The results show that the LcbHLH16421 gene of Liriodendron thaliana can enhance the tolerance of Arabidopsis plants to low temperature stress, and has important application value in molecular breeding for improving low temperature stress of plants.

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 LcbHLH16421 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 di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82C12N5/10C07K14/415A01H5/00A01H6/20
CPCC07K14/415C12N15/8273
Inventor 杨立明朱礼明高敏施季森郝兆东陈金慧
Owner NANJING FORESTRY UNIV
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