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Potassium transport protein KUP3 from tobacco and coded gene and application thereof

A gene and tobacco technology, applied in the field of potassium transporter KUP3 and its coding gene and application, can solve the problems of lack of absorption capacity, decrease and increase of potassium ion concentration, and achieve the effect of promoting potassium ion absorption and transport

Active Publication Date: 2019-02-19
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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  • Potassium transport protein KUP3 from tobacco and coded gene and application thereof
  • Potassium transport protein KUP3 from tobacco and coded gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Obtaining of KUP3 gene

[0045] 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'-ATGGATATCGACTACGGGAA-3'; the nucleotide sequence of the reverse primer is 5'-TTAAACAATGTAGACCATCC-3', using the synthetic cDNA as a template , for PCR amplification.

[0046] 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 O 8 μL.

[0047] 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.

[0048] After t...

Embodiment 2

[0049] Example 2 The role of KUP3 gene in promoting the absorption and transport of potassium ions

[0050] The T-vector and the expression vector P416 connected with the KUP3 gene described in Example 1 were subjected to double enzyme digestion (restriction sites: SmaI and XhoI), and the target gene and expression vector P416 were recovered, and then ligated with ligase , transfer the ligated recombinant yeast expression vector into Escherichia coli DH5α competent cells, carry out PCR amplification and enzyme digestion on the transformed Escherichia coli single colony to verify whether the construction is successful, and transfer the successfully constructed recombinant yeast expression vector into into R5421.

[0051] The specific steps are as follows: Streak the preserved R5421 yeast on the solid medium YPDA with an inoculation loop, culture at 28°C for 12 hours; pick a single colony of R5421 yeast in the Ep tube, add 1mL of YPDA culture solution and vortex; Transfer all t...

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Abstract

The invention relates to the field of genetic engineering, and particularly discloses a potassium transport protein KUP3 from tobacco and a coded gene and application thereof. A KUP3 gene separated from the tobacco is provided for the first time, the full length of the KUP3 gene is 2364 bp, functional verification proves that after the provided KUP3 gene is transferred to a potassium uptake defected type yeast mutant strain R5421, the function of potassium ion absorbing and transferring is regained by a recombinant yeast expressing the KUP3 gene, and therefore the provided KUP3 gene has the function of promoting potassium ion absorbing and transferring.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a potassium transporter KUP3 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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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/82A01H6/20
CPCC07K14/415C12N15/8243
Inventor 王仁刚任学良李立芹鲁黎明郭玉双张洁林世锋
Owner GUIZHOU TOBACCO SCI RES INST
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