Tobacco chloride ion channel protein gene NtCLC-F and application thereof

A chloride ion channel, chloride ion content technology, applied in the chloride ion channel protein gene NtCLC-F and its application field, can solve the problem of low transportation speed of lotus

Inactive Publication Date: 2021-05-07
CHINA TOBACCO HENAN IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Low-chlorine-enriched lotus flowers transport faster i

Method used

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  • Tobacco chloride ion channel protein gene NtCLC-F and application thereof
  • Tobacco chloride ion channel protein gene NtCLC-F and application thereof
  • Tobacco chloride ion channel protein gene NtCLC-F and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] This example briefly introduces the process of cloning the tobacco NtCLC-F gene and constructing the silencing vector as follows.

[0036] (1) Tobacco NtCLC-F gene cloning

[0037] According to the previous research on the tobacco genome and related NtCLC-F genes, the specific coding sequence was selected as the target fragment, and the primer sequences for PCR amplification were designed as follows:

[0038] NtCLC-F-F: 5'-CTGGTAGGGAGCTTCTC-3',

[0039] NtCLC-F-R: 5'-CCTCCTAAAGAGAATGG-3'.

[0040] The cDNA of tobacco K326 (tobacco variety) leaves was used as a template, and NtCLC-F-F and NtCLC-F-R were used as primers to perform PCR amplification to obtain the NtCLC-F gene.

[0041] The PCR amplification program was: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, and after 34 cycles, complete extension at 72°C for 5 min.

[0042] The PCR amplification products were detected by agarose gel ele...

Embodiment 2

[0052] On the basis of Example 1, the constructed recombinant TRV2-NtCLC-F vector was further transformed into tobacco plants using Agrobacterium-mediated VIGS technology, and the phenotypic changes of related plants were verified and analyzed. The specific experimental process is as follows.

[0053] (1) Transformation of Agrobacterium

[0054] It should be noted that referring to the operation of Example 1 and the prior art, TRV2-GFP and TRV2-PDS recombinant vectors were prepared as transgenic positive and negative controls at the same time, and the specific transformation process was as follows:

[0055] The positive cloning plasmids of TRV2-GFP (vector control), TRV2-PDS (VIGS efficiency control) and TRV2-NtCLC-F were transformed into Agrobacterium GV3101 competent cells by electric shock transformation respectively, using 50mg / L Kan and The YEB plates with 50 mg / L Rif were cultured and screened, and after inverted culture at 28°C for 2 days, the Agrobacterium with the tar...

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Abstract

The invention belongs to the technical field of tobacco gene engineering, and particularly relates to a tobacco chloride ion channel protein gene NtCLC-F and application thereof. The base sequence of the gene is as shown in SEQ ID NO.1. The tobacco chloride ion channel protein NtCLC-F is composed of 726 amino acid residues, wherein the 181st-500th amino acids are conserved chloride ion channel protein structural domains. The protein is related to the chloride ion content of plant leaves, and after the expression of the protein is reduced, the chloride ion content of the leaves is obviously increased. Preliminary research on a specific tobacco chloride ion channel NtCLC-F shows that the specific tobacco chloride ion channel NtCLC-F is highly related to the content of the tobacco chloride ions, and after the gene is silenced, the content of the tobacco chloride ions is obviously increased. Based on the characteristic, a certain application basis and reference can be provided for cultivation of a new tobacco variety with the chlorine ion content regulated and controlled.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to the technical field of tobacco genetic engineering, and specifically relates to a chloride channel protein gene NtCLC-F and an application thereof. Background technique [0002] Tobacco raw materials are the foundation of the cigarette industry. In recent years, the overall production level of tobacco leaf raw materials in my country has been significantly improved, but there is still a certain gap between the quality of tobacco leaf raw materials and the needs of cigarette process formula, especially in some tobacco areas such as flue-cured tobacco from Huanghuai Tobacco Area and Hainan cigars. Among them, the chloride ion of tobacco leaves in the Huanghuai tobacco area has been far higher than the national level for many years, which has seriously affected the application of tobacco leaf raw materials in this area in high-end products. Outside the range of ions, it will a...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N15/84A01H5/12A01H6/82
CPCC07K14/415C12N15/8218C12N15/8243
Inventor 刘超王根发马波波刘向真杨永锋刘茂林赵森森彭玉富金立锋王燃
Owner CHINA TOBACCO HENAN IND
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