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Tobacco KUP8 protein and encoding gene and application thereof

A technology that encodes genes and tobacco, applied in the field of genetic engineering, can solve problems such as lack of absorption capacity, reduced survival rate, enhancement, etc., and achieve the effect of promoting potassium ion absorption and transport

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

[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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  • Tobacco KUP8 protein and encoding gene and application thereof
  • Tobacco KUP8 protein and encoding gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] The acquisition of embodiment 1 KUP8 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'-ATGGATCGGCAAACAGGAAG-3', and the nucleotide sequence of the reverse primer is 5'-TCAAGCCTCATAAAGCATAC-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 O 8 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 ...

Embodiment 2

[0044] The role of embodiment 2 KUP8 gene in promoting potassium ion uptake and transport

[0045] The T-vector connected with the KUP8 gene described in Example 1 and the expression vector P416 (P416 yeast free type shuttle expression vector, TEF constitutive promoter, CYC1 terminator, CEN6ARSH4 replication origin, the selection marker in yeast is URA3, The screening marker in Escherichia coli is Amp. It is recorded in Functional Expression of aω-3Fatty Acid Desaturase Gene from Glycine max in Saccharomyces cerevisiae) to carry out double enzyme digestion (restriction site: SmaI and XhoI), and recover the target gene and expression vector P416, Then connect with ligase, transfer the ligated recombinant yeast expression vector into the competent cells of Escherichia coli DH5α, carry out PCR amplification and enzyme digestion on the transformed Escherichia coli single colony to verify whether the construction is successful, and construct the successful recombinant The yeast exp...

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Abstract

The invention relates to the field of genetic engineering and particularly discloses a tobacco KUP8 protein and an encoding gene and application thereof. A KUP8 gene separated from tobaccos is provided for the first time, a nucleotide sequence of the KUP8 gene is shown as SEQ ID NO.2, and the encoded nucleotide sequence of the KUP8 gene is shown as SEQ ID NO.1. It is shown through functional identification that the provided KUP8 gene is transferred into a potassium absorption deficient yeast mutant strain R5421, then a recombinant yeast expressing the KUP8 gene has functions of potassium ion absorption and transport. It is explained that the KUP8 gene provided by the 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 KUP8 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 Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/82A01H6/20
CPCC07K14/415C12N15/8243
Inventor 王仁刚任学良鲁黎明李立芹王自力张洁耿召良
Owner GUIZHOU TOBACCO SCI RES INST
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