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Tobacco HKT 1 gene and preparation method and application thereof

A tobacco and gene technology, applied in the field of genetic engineering, can solve the problems of large potassium consumption and unknown function of tobacco HKT1, and achieve the effect of promoting absorption, potassium ion absorption and transport

Active Publication Date: 2017-08-25
GUIZHOU TOBACCO SCI RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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 HKT1 is unknown.

Method used

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  • Tobacco HKT 1 gene and preparation method and application thereof
  • Tobacco HKT 1 gene and preparation method and application thereof
  • Tobacco HKT 1 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 HKT1 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 HKT1 gene Amplify to obtain the target fragment, and obtain the HKT1 gene sequence after sequencing.

[0020] In the present invention, the design of the PCR amplification primers is based on NCBI Reference Sequence: XM_009601690.1 as a reference sequence, using the software primer 5 to design, and the sequence of the XM_009601690.1 is shown in SeqID No.2.

[0021] In the present invention, the PCR amplification primer preferably includes a forward primer and a reverse primer, and the nucleotide sequence of the forward primer is: 5'-ATGATCCTTTCATTGTTAGG-3', with a length of 20bp; The nucleotide sequence of the primer is 5'-TTATAATACTTTTCCAGCCC-3', and the len...

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 the reverse primer, the nucleotide sequence of the forward primer is: 5'-ATGATCCTTTCATTGTTAGG-3', the nucleotide sequence of the reverse primer is 5'-TTATAATACTTTTCCAGCCC-3', with the synthetic cDNA as a template , PCR amplification, PCR amplification system is 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; 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 completed, use a DN...

Embodiment 2

[0033] The T-vector connected with the HKT1 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 tobacco HKT1 gene. The sequence of the tobacco HKT 1 gene is shown in Seq ID No.1. A preparation method of the HKT1 gene comprises the followings steps of designing PCR amplification primers; extracting total RNA of tobacco cells; synthesizing tobacco cell cDNA; using the tobacco cell cDNA as a template to perform PCR amplification of the HKT1 gene and obtaining a target fragment, and after sequencing, obtaining the HKT1 gene sequence. The total length of the HKT1 gene is 1488 bp. After functional verification, the HKT1 gene is transferred into a potassium absorption defect type yeast mutant R5421, the recombinant yeast has absorption and transport functions. HKT1 gene overexpression in tobacco plants can promote adsorption for potassium ions of tobaccos, and the HKT1 gene has the functions of promoting adsorption and transport of the potassium ions.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a tobacco HKT1 gene and its preparation method 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] high affinity K + Transporter HKT (High-affinity K + Transporter) belongs to the membrane transport protein family, and its main function is to mediate K + and Na + co-transport or only mediate Na + of transshipment. This gene has been cloned from many plants (wheat, Arabidopsis, rice, etc.) (Schachtma...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/10C12N15/82A01H5/12
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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