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Potassium translocator TPK1-1 from tobacco and coding gene and application thereof

A gene and tobacco technology, applied in the field of potassium transporter TPK1-1 and its coding gene and application, can solve the problems of unknown function of tobacco TPK, large potassium consumption, etc., and achieve the effect of promoting the absorption and transport of potassium ions

Active Publication Date: 2019-02-22
GUIZHOU TOBACCO SCI RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[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 ion channels in tobacco, and the function of tobacco TPK is unknown.

Method used

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  • Potassium translocator TPK1-1 from tobacco and coding gene and application thereof
  • Potassium translocator TPK1-1 from tobacco and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Obtaining of TPK1-1 gene

[0046] 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'-ATGGAGAAAGAGCCTCTTCT-3'; the nucleotide sequence of the reverse primer is 5'-TCAATGGTGGTGGCTTTCCA-3', using the synthetic cDNA as a template , for PCR amplification.

[0047] 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.

[0048] 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.

[0049] After ...

Embodiment 2

[0050] Example 2 The role of TPK1-1 gene in promoting potassium ion absorption and transport

[0051] The T-vector connected with the TPK1-1 gene described in Example 1 and the expression vector P416 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 Enzyme ligation, transfer the ligated recombinant yeast expression vector into Escherichia coli DH5α competent cells, perform PCR amplification and enzyme digestion on the transformed Escherichia coli colony to verify whether the construction is successful, and construct the successful recombinant yeast expression vector Transfer to R5421.

[0052] 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 the liquid into t...

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Abstract

The invention relates to the field of genetic engineering, and specifically discloses a potassium translocator TPK1-1 from tobacco and a coding gene and application thereof. The TPK1-1 gene separatedfrom the tobacco is provided for the first time. The TPK1-1 gene is 1287bp in length. According to functional verification, after the TPK1-1 gene is transferred into the potassium absorption deficientyeast mutant R5421, recombinant yeast expressing the TPK1-1 gene has the functions of potassium ion absorption and transportation again. Therefore, the TPK1-1 gene has the functions of promoting potassium ion absorption and transportation.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a potassium transporter TPK1-1 from tobacco and its coding gene and application. Background technique [0002] Potassium ion channels are ion channels that allow potassium ions to specifically permeate the plasma membrane, while blocking the permeation of other ions - especially sodium ions. These channels are generally composed of two parts: one part is the channel region, which selects and allows potassium ions to pass through, while blocking sodium ions; the other part is a gating switch, which switches the channel on and off according to the signal in the environment. [0003] In the prior art, studies on potassium ion channel genes are extensive in the model plant Arabidopsis thaliana. For example, studies have shown that the TPK / KCO channel in the model plant Arabidopsis includes two-pore potassium channel TPK (also known as tandem pore channel) and a Kir -type channel. ...

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王自力林世锋
Owner GUIZHOU TOBACCO SCI RES INST
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