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Two-color sea lavander herb gene LbHLH and application thereof

A two-color blood-enriching grass, gene technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of secondary soil salinization and high cost

Inactive Publication Date: 2021-09-07
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional methods of improving saline-alkali land, such as freshwater pressurization and underground pipe drainage, are costly and cause secondary salinization of soil[8]

Method used

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  • Two-color sea lavander herb gene LbHLH and application thereof
  • Two-color sea lavander herb gene LbHLH and application thereof
  • Two-color sea lavander herb gene LbHLH and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Cloning, bioinformatics analysis, subcellular localization and in situ hybridization of LbHLH gene

[0072] 1. Cloning and bioinformatics analysis of LbHLH gene

[0073]Collect a large number of first true leaves at different stages of leaf development, including undifferentiated stage (stage A; 4-5 days after sowing, 5000 leaves), salt gland differentiation stage (stage B; 6-7 days after sowing, 4000 leaves) , stomatal differentiation stage (C stage; 8-10 days after sowing, 3000 leaves), epidermal cell differentiation stage (D stage; 11-16 days after sowing, 1000 leaves), maturity stage (E stage; more than 17 days after sowing, 1000 leaves).

[0074] The total RNA of Limonium bicolor was extracted with a plant total RNA extraction kit (RC401-01; Novizan Biotechnology Co., Ltd.). According to the LbHLH sequence obtained from the reported transcriptome assembly sequence of Limonium bicolor as a reference, Primer 5.0 was used to design primers LbHLH-S and LbHL...

Embodiment 2

[0084] Cloning and Histochemical Analysis of Example 2 Gene LbHLH Promoter

[0085] The total DNA of Limonium bicolor was obtained using the Plant DNA Extraction and Separation Kit (Novizan Biotechnology Co., Ltd.), using the full-length promoter sequence of LbHLH in the genome of Limonium bicolor as a reference, and the extracted Limonium bicolor Using DNA as a template, the full-length promoter sequence of LbHLH was obtained by cloning with primers LbHLH-P-S and LbHLH-P-A (Table 1). The elements in the promoter were predicted by PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) and CSDS2.0 (http: / / gsds.gao-lab.org / ) A schematic diagram of the components is drawn.

[0086] In order to replace the 35S promoter in pCAMBIA3301 with the LbHLH promoter, the CaMV 35S promoter was excised from pCAMBIA3301 using restriction enzymes HindIII and NcoI to obtain a linear vector, and primers 3301-LbHLH-P-S and 3301-LbHLH- P-A (Table 1) was cloned to obtain the LbHL...

Embodiment 3

[0089] Example 3 Construction of Col-35S::LbHLH transgenic plants

[0090] Use primers LbHLH-OEAt-S and LbHLH-OEAt-A (Table 1) carrying NcoI restriction sites to clone to obtain the full-length CDS of LbHLH, and use restriction enzyme NcoI to single-digest pCAMBIA3301 to obtain a linear vector, and use homologous recombination reagents Cassette (Novazyme Biotechnology Co., Ltd.) ligated the linear vector pCAMBIA3301 and the full-length CDS of LbHLH to generate p35S::LbHLH. The p35S::LbHLH was introduced into Agrobacterium GV3101 to transform Arabidopsis Col-0. After 3 generations of screening with herbicide (Bsata herbicide, the main component is glufosinate-ammonium, 0.1%, v / v), positive transgenic lines were obtained and physiological assays were performed on Col-35S::LbHLH overexpression lines.

[0091] The positive transgenic plants were identified at the molecular level with primers LbHLH-OEAt-S and LbHLH-OEAt-A. Then, mRNA was extracted from different Col-35S::LbHLH tr...

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Abstract

The invention provides a two-color sea lavander herb gene LbHLH and application thereof. According to the invention, the full length of the gene LbHLH is obtained by performing cloning on a salt-secreting halophyte two-color sea lavander herb. The gene LbHLH comprises an open reading frame of 2067 bp, and is used for coding a protein consisting of 688 amino acids. The gene LbHLH has a typical helix-loop-helix (HLH) structural domain between amino acids 480-526. Experiments show that an arabidopsis thaliana plant for heterologous expression of the gene has the effects of promoting epidermal hair development, reducing root hair development and enhancing salt tolerance. A yeast two-hybrid experiment also shows that the gene LbHLH can interact with AtGL1, so that the gene LbHLH can play an important role in differentiation and salt resistance of hairy bodies and root hairs.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to the gene LbHLH of Limonium bicolor and its application. Background technique [0002] According to data from the Food and Agriculture Organization of the United Nations (FAO), 950 million hectares of land worldwide are affected by soil salinity, accounting for more than 6.5% of the world's total land area, and nearly half of the world's irrigated soil is affected by salinization. The intensification of salinization and the reduction of arable land have seriously affected food production and food security. Therefore, it is particularly urgent to develop and utilize saline-alkali land. Traditional methods of improving saline-alkali land, such as freshwater pressurization and underground pipe drainage, are costly and cause secondary salinization of soil [8]. Therefore, it is very important to develop biological methods to improve saline-alkali land. [0003] Halophytes ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00A01H5/06A01H6/20
CPCC07K14/415C12N15/8273C12N15/8271
Inventor 王茜袁芳王宝山
Owner SHANDONG NORMAL UNIV
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