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A kind of tobacco kup5 protein and its coding gene and application

A protein and tobacco technology, applied in the field of genetic engineering, can solve the problems of lack of absorption capacity, enhancement, and reduced survival rate, and achieve the effect of promoting the absorption and transport of potassium ions

Active Publication Date: 2021-10-15
GUIZHOU TOBACCO SCI RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing technology studies on potassium transporter genes are extensive in the model plant Arabidopsis thaliana. For example, studies have shown that Arabidopsis mutant kup6 has a lower survival rate under drought stress than wild-type plants, while 35S::KUP6 overexpression The drought tolerance of transgenic plants was significantly enhanced (Shabala et al., 2007); while the mutant seedlings of KUP7 gene exhibited a low-potassium-sensitive phenotype of yellowing leaves under low-potassium stress, and the potassium ions in the roots The content decreased significantly, and the loss of KUP7 gene function reduced the ability of Arabidopsis seedlings to absorb potassium under low potassium conditions, and the concentration of potassium ions in xylem juice decreased (Min et al., 2016)
[0004] Tobacco is a crop that consumes a lot of potassium. The potassium content of tobacco leaves is an important indicator to measure the quality of tobacco leaves. At present, there are few studies on potassium transporter genes in tobacco, and the function of tobacco KUP is unknown.

Method used

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  • A kind of tobacco kup5 protein and its coding gene and application
  • A kind of tobacco kup5 protein and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Obtaining of KUP5 gene

[0040] Get 0.5g of fresh tobacco leaves, use the Trizol method to extract the total RNA of tobacco cells, then use the cDNA synthesis kit of TaKaRa company to synthesize cDNA, and further use the Primer5.0 software to design and obtain primers through artificial optimization. The primers include forward primers and a reverse primer, the nucleotide sequence of the forward primer is: 5'-ATGGGGAAAGGAGAGATAGA-3', and the nucleotide sequence of the reverse primer is 5'-TCAAACCATGTATGTCATTC-3'. Using the synthesized cDNA as a template, PCR amplification was carried out.

[0041] The PCR amplification system is a 20 μL system, including: Premix ExTaq 10 μL, 10 μM forward primer 0.5 μL, 10 μM reverse primer 0.5 μL, tobacco cell cDNA 1 μL, ddH 2 08 μL.

[0042]The PCR amplification reaction program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, and 35 cycles.

...

Embodiment 2

[0044] Example 2 The role of KUP5 gene in promoting the absorption and transport of potassium ions

[0045] The T-vector connected with the KUP5 gene described in Example 1 and the expression vector P416 (p416 yeast free type shuttle expression vector, TEF constitutive promoter, CYC1 terminator, CEN6ARSH4 origin of replication, the selection marker in yeast is URA3, The screening marker in Escherichia coli is Amp. Excerpted from Functional Expression of aω-3Fatty Acid Desaturase Gene from Glycine max in Saccharomyces cerevisiae) were subjected to double enzyme digestion (restriction sites: SmaI and XhoI), and the target gene and expression vector P416 were recovered, and then Ligate with ligase, transfer the ligated recombinant yeast expression vector into the competent cells of E. coli DH5α, perform PCR amplification and enzyme digestion on the transformed E. coli single colony to verify whether the construction is successful, and construct the successful recombinant yeast Th...

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Abstract

The invention relates to the field of genetic engineering, and specifically discloses a tobacco KUP5 protein, its coding gene and its application. The present invention provides for the first time the KUP5 gene isolated from tobacco. The nucleotide sequence of the KUP5 gene is shown in SEQ ID NO.2, and the amino acid sequence of the protein KUP5 encoded by it is shown in SEQ ID NO.1. After functional verification, After the KUP5 gene provided by the invention is transferred into the potassium absorption-deficient yeast mutant strain R5421, the recombinant yeast expressing the KUP5 gene has the functions of potassium ion absorption and transport. It shows that the KUP5 gene provided by the present invention has the function of promoting the absorption and transport of potassium ions.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a potassium transporter KUP5 from tobacco and its coding gene and application. Background technique [0002] Potassium transporter is a carrier protein that can transport potassium ions into cells when the external potassium ion concentration is extremely low. Potassium transporters are usually some transmembrane proteins, which complete the transmembrane transport of potassium ions through conformational changes. It is an active transport process that requires ATP to provide energy, usually K + / H + or K + / Na + Collaborative transport. [0003] Existing technology studies on potassium transporter genes are extensive in the model plant Arabidopsis thaliana. For example, studies have shown that Arabidopsis mutant kup6 has a lower survival rate under drought stress than wild-type plants, while 35S::KUP6 overexpression The drought tolerance of transgenic plants was significa...

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 龙明锦任学良李立芹鲁黎明王仁刚张洁王自力
Owner GUIZHOU TOBACCO SCI RES INST
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