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Tobacco KC1 gene, and preparation method and application thereof

A tobacco and gene technology, applied in the field of genetic engineering, can solve the problems of unknown function of tobacco KC1, large potassium consumption, etc., and achieve the effect of promoting potassium ion absorption and transport, and promoting absorption

Pending Publication Date: 2017-08-04
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 KC1 is unknown.

Method used

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  • Tobacco KC1 gene, and preparation method and application thereof
  • Tobacco KC1 gene, and preparation method and application thereof
  • Tobacco KC1 gene, and preparation method and application thereof

Examples

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preparation example Construction

[0019] The present invention provides a method for preparing the tobacco KC1 gene described in the above technical scheme, comprising the following steps: designing PCR amplification primers; extracting tobacco cell total RNA; synthesizing tobacco cell cDNA; using the tobacco cell cDNA as a template to perform PCR of the KC1 gene Amplify to obtain the target fragment, and obtain the KC1 gene sequence after sequencing.

[0020] In the present invention, the design of the PCR amplification primers is based on GenBank: AB196791.1 as a reference sequence and designed using the software primer 5. The sequence of the AB196791.1 is shown in Seq ID No.2.

[0021] In the present invention, the PCR amplification primer preferably includes a forward primer and a reverse primer, the nucleotide sequence of the forward primer is: 5'-ATGTCATACTCGGACACG-3', the length is 18bp; the The nucleotide sequence of the reverse primer is 5'-TCAAAAAATATACAAGTGATC-3'; the length is 21bp.

[0022] In th...

Embodiment 1

[0030] 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'-ATGTCATACTCGGACACG-3', the nucleotide sequence of the reverse primer is 5'-TCAAAAAATATACAAGTGATC-3', to synthesize the cDNA As a template, perform PCR amplification. The PCR amplification system is 20 μL, including 10 μL of Premix ExTaq, 0.5 μL of 10 μM forward primer, 0.5 μL of 10 μM reverse primer, 1 μL of tobacco cell cDNA, ddH 2 O 8 μL; the reaction program of the PCR amplification is: pre-denaturation at 95° C. for 5 minutes; denaturation at 95° C. for 30 seconds; annealing at 55° C. for 30 seconds; extension at 72° C. for 2 minutes; 35 cycles.

[0031] After the PCR amplification is complet...

Embodiment 2

[0033] The T-vector connected with the KC1 gene described in Example 1 and the expression vector P416 were subjected to double enzyme digestion (restriction sites: XbaI and SmaI), 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 The specific steps to R5421 are as follows: Streak the preserved R5421 yeast on the solid medium YPDA with an inoculation loop, and incubate at 28°C for 12 hours; pick a single colony of R5421 yeast into the Ep tube, add 1mL of YPDA culture medium and vortex; Transfer all the above bacterial solutions into the Erlenmeyer flask containing YPDA culture solution, shake the bacteria to ...

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Abstract

The invention provides a KC1 gene. A sequence is shown as Seq ID No. 1. A preparation method of the KC1 gene comprises the following steps: designing a PCR amplification primer; extracting tobacco cell total RNA; synthesizing a tobacco cell cDNA; and performing PCR amplification for the KC1 gene by adopting the tobacco cell cDNA as a template to obtain a target fragment, and sequencing to obtain a KC1 gene sequence. A full length of the KC1 gene is 1923bp, by virtue of functional verification, a recombinant yeast after the KC1 gene is transferred into a potassium absorption-defect yeast mutant strain R5421 has a potassium ion absorption and transferring function, the over-expressed KC1 gene in a tobacco plant can promote the absorption of the tobacco to the potassium ions. The KC1 gene of the invention has a function for facilitating the absorption and transferring of the potassium ions.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a tobacco KC1 gene and a preparation method thereof. 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] AtKC1 is the only Shaker family α subunit found so far that cannot generate current in a heterologous expression system. It is recognized as a protein that regulates other Shaker family members. Yeast two-hybrid experiments proved that the intracellular region of AtKC1 can interact with AKT1 and AKT2 (Pilot et al., 2...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8243
Inventor 任学良鲁黎明李立芹郭玉双张洁
Owner GUIZHOU TOBACCO SCI RES INST
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