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Limonium bicolor gene LbCPC and application thereof

A kind of two-color tonic blood grass, gene technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve desertification, agricultural yield decline, soil degradation and other problems

Active Publication Date: 2021-10-26
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Very few crops can survive in highly salinized areas, leading to a significant drop in agricultural yields and contributing to soil degradation and desertification

Method used

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  • Limonium bicolor gene LbCPC and application thereof
  • Limonium bicolor gene LbCPC and application thereof
  • Limonium bicolor gene LbCPC and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Cloning and bioinformatics analysis of LbCPC gene

[0058] 1 material

[0059] The seeds of Limonium bicolor (Bunge) O. Kuntze were collected from the saline-alkali land of the Yellow River Delta in Dongying City, Shandong Province, China. After fully drying, store in the refrigerator at 4°C. The seeds with uniform size and full grain were selected for irradiation mutagenesis treatment.

[0060] 2 Experimental methods

[0061] 2.1 LbCPC cloning and bioinformatics analysis

[0062] Total RNA was extracted from the leaves of Limonium bicolor at the salt gland development stage (stage A), and cDNA was obtained by reverse transcription kit. According to the previous second-generation transcriptome data, combined with 3'RACE and 5'RACE technology, primers were designed to clone the LbCPC gene of Limonium bicolor, and bioinformatics analysis was performed.

[0063] 2.1.1 Disinfection and cultivation of Limonium bicolor seeds

[0064] Put Limonium bicolor seeds...

Embodiment 2

[0131] Cloning and functional analysis of embodiment 2 gene LbCPC promoter

[0132] 1 Cloning of LbCPC Gene Promoter of Limonium bicolor

[0133] According to the DNA sequence of the gene, primers were designed to clone the DNA flanking sequence at the 5' end of the gene (Table 3).

[0134] Table 3 Primers used for cloning the 5' end DNA flanking sequence of LbCPC gene

[0135]

[0136] Use the TaKaRa Genome Walking Kit (Code No.6108) for the reaction, using the leaf genomic DNA as a template, and the reaction steps are as follows:

[0137] (1) 1st PCR reaction:

[0138] The first PCR reaction was performed with AP1 (AP2, AP3, and AP4 performed simultaneously) as the upstream primer and 5SP1 as the downstream primer.

[0139] ① Prepare 1st PCR reaction solution according to the following components.

[0140]

[0141] ②1st PCR reaction conditions are as follows:

[0142]

[0143] (2) 2nd PCR reaction:

[0144] After diluting the 1st PCR reaction solution by 1 to ...

Embodiment 3

[0173] Example 3 Construction of LbCPC Overexpression Vector and Transgenic Plants

[0174] 1LbCPC over-expression carrier construction

[0175] (1) Use Takra high-fidelity enzyme to clone the CDS sequence of the gene, add KpnⅠ and BamHI restriction sites and corresponding protective bases at both ends of the primers (Table 5), connect to the pEASY T3 cloning vector after gel cutting, and transform into Escherichia coli Afterwards, the plasmid was extracted and digested with pCAMBIA 1300-35S-sGFP expression vector. The reaction system was as follows: KpnⅠ(BamHI) 1 μl, vector or plasmid 1 μg, CutSmart Buffer 5 μl, H 2 O make up to 50 μl. React at 37°C for 30 minutes.

[0176] (2) After the digestion is completed, perform agarose gel electrophoresis on the digested product, and then cut out the target fragment in the agarose gel, then use the gel recovery kit to recover the DNA fragment, and use T4 ligase to connect the digested carrier and fragments, the ligation reaction sy...

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Abstract

The invention provides a limonium bicolor gene LbCPC and an application thereof. The gene LbCPC is cloned from limonium bicolor, the size of an open reading frame of the gene LbCPC is 321bp, and 106 amino acids are encoded. Through analysis, the LbCPC encodes an R3MYB transcription factor, and plays a role in inhibiting the initiation of epidermal hair and promoting the formation of root hair. Cloning and bioinformatics analysis on the LbCPC gene and a promoter show that the LbCPC possibly plays an important role in the growth and development process of the limonium bicolor. And a limonium bicolor LbCPC overexpression strain is further obtained, which shows that the salt gland structure of a leaf part is obviously changed, the number of constituent cells is obviously increased in groups, and a large number of 5, 6, 7 and 8 luminous point salt glands appear, so that the LbCPC is speculated to play a role in positively regulating the development of the salt glands. The invention lays a foundation for further exploring and revealing the development mechanism of salt glands.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to the gene LbCPC of Limonium bicolor and its application. Background technique [0002] Soil salinization is an environmental problem facing the world, affecting about 6.5% of the Earth's surface. Of the approximately 230 million hectares of irrigated farmland, about 20% of the land area is affected by salinization, and this proportion is increasing year by year due to improper irrigation practices. To expand the area of ​​cultivated land, it is necessary to transform and make full use of large areas of saline-alkali land. Few crops can survive in highly salinized areas, leading to a significant drop in agricultural yields and contributing to soil degradation and desertification. Therefore, in order to meet the challenge of salinization to agriculture, it is necessary to improve the salt tolerance of non-halophytes by transforming them with salt tolerance genes. Halophyt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/113C12N15/84A01H5/12A01H5/00A01H6/00
CPCC07K14/415C12N15/8273C12N15/8205
Inventor 袁芳赵博庆冷冰莹王宝山
Owner SHANDONG NORMAL UNIV
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