A kind of overexpression can increase the gene of tobacco potassium content and its coded product and application

A technology of overexpression and potassium content, applied in the field of plant cells, can solve the problems of low potassium content in tobacco leaves, restricting tobacco leaf quality, etc., and achieve the effects of improving aroma quality and aroma quantity, huge economic benefit potential, and improving tobacco quality.

Active Publication Date: 2022-03-18
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the potassium content of tobacco leaves in my country's Huanghuai tobacco-growing areas is low, which restricts the improvement of tobacco leaf quality to a large extent.

Method used

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  • A kind of overexpression can increase the gene of tobacco potassium content and its coded product and application
  • A kind of overexpression can increase the gene of tobacco potassium content and its coded product and application
  • A kind of overexpression can increase the gene of tobacco potassium content and its coded product and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Discovery and cloning of PE1 gene

[0035] (1) Discovery of PE1 gene: Transcriptome sequencing analysis was performed on mature leaves of high-potassium tobacco line Z111 and low-potassium tobacco line Z121, and a gene transcript was found in high-potassium line Z111 through differential expression analysis of genes The transcript abundance of the low-potassium strain Z121 is 5 times that of the low-potassium strain Z121. Compared with the public tobacco database, no similar gene sequence and functional annotation information were found. The gene was preliminarily determined to be a new gene and named Potassium Enrichment 1, PE1;

[0036] (2) Tobacco first-strand cDNA synthesis process:

[0037] The isolation and purification of total RNA from tobacco high-potassium line Z111 mature leaves were carried out with reference to the instructions of the TRIZOL kit (ThermoFisher Scientific, USA), and 2 μg of total RNA was drawn into a 1.5ml centrifuge tube, followed by RevertA...

Embodiment 2

[0046] PE1 gene function confirmed,

[0047] Fluorescence quantitative PCR (QPCR) method was used to analyze the expression of PE1 gene in tobacco varieties (lines) with different potassium efficiency:

[0048] (1) The experimental samples were selected as mature leaves of 7 tobacco lines including 89112, Yunyan 87, Yuyan 6, Qinyan 96, China Tobacco 100, Z111 and Z121;

[0049] (2) In each sample, according to the method described in Example 1, 2 μg of total RNA was reverse-transcribed into first-strand cDNA as a template;

[0050] (3) According to the full-length cDNA sequence of the PE1 gene, the sequence of specific primers for fluorescent quantitative PCR is:

[0051] Upstream primer: 5'-TCCCGTATCAGAAGAAGTCC-3', as shown in SEQ ID NO.7,

[0052] Downstream primer: 5'-AAACTGTAAGGGTGCCAAGT-3'; as shown in SEQ ID NO.8;

[0053]1) QPCR reaction system (20 μl): 2×SYBR mix 10 μl, upstream primer 0.5 μl, downstream primer 0.5 μl, cDNA template 1 μl, ddH 2 O 8 μl;

[0054] 2)...

Embodiment 3

[0062] Construction of High Potassium Tobacco Strains Using PE1 Gene

[0063] Include the following steps:

[0064] S1, construction of tobacco transgene expression vector,

[0065] Using the full-length cDNA fragment of the PE1 gene constructed in Example 2 as a template, PCR amplification was performed with specific primers containing BamHI and XbaI linker sequences. Between the BamHI and XbaI sites behind the cauliflower mosaic virus (CaMV) 35S promoter of the meta-expression vector pWM101, the recombinant vector pWM101-35S-PE1 was obtained;

[0066] The above-mentioned specific primer sequences are:

[0067] Upstream primer: 5'-ACGGATCCATGGTGCTCGCGCCTGTTG-3', as shown in SEQ ID NO.5,

[0068] Downstream primer: 5'-CATCTAGATCAGGGCAAGCTAGCTGGC-3', as shown in SEQ ID NO.6;

[0069] S2, Agrobacterium-mediated leaf disc transformation into tobacco low potassium lines,

[0070] The recombinant vector constructed by S1 was transformed into a tobacco low-potassium strain by t...

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Abstract

The invention relates to the technical field of plant cells, and specifically discloses a gene that can be overexpressed to increase the potassium content of tobacco, its encoded product and its application. The gene is the PE1 gene, and the step of using the PE1 gene to construct a tobacco strain with high potassium content Including tobacco first-strand cDNA synthesis, recombinant vector pWM101-35S-PE1 construction and positive plant screening, recombinant vector pWM101-35S-PE1 construction through Agrobacterium activation, transfer, infection, co-cultivation, selective differentiation culture, rooting culture and transplanting steps. The potassium content in the transgenic high-potassium tobacco strain obtained by the method of the invention is greatly increased, and has broad application prospects in cultivating high-potassium tobacco varieties.

Description

technical field [0001] The invention relates to the technical field of plant cells, in particular to a gene for overexpression that can increase the potassium content of tobacco, its encoded product and its application. Background technique [0002] Potassium is an important nutrient element necessary for tobacco growth and an important evaluation index for tobacco leaf quality. The higher potassium content not only ensures the normal maturity and yellowing of the tobacco plants, but also improves the aroma and aroma of the tobacco leaves. At present, the potassium content of tobacco leaves in Huanghuai tobacco-growing areas in my country is relatively low, which restricts the improvement of tobacco leaf quality to a large extent. Therefore, it is an important problem to be solved urgently in the production of tobacco leaves to increase the potassium content of the main tobacco varieties. [0003] There are great differences in potassium absorption, distribution, transloca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/82
CPCC07K14/415C12N15/8243
Inventor 夏宗良张小全韦凤杰杨铁钊
Owner HENAN AGRICULTURAL UNIVERSITY
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