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NtCCC (nicotiana tabacum cation/chloride cotransporter) and application thereof

A chloride ion and cation technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of plant growth inhibition, inability to grow, and poor soil physical and chemical properties.

Active Publication Date: 2017-10-24
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In salinized soil, because the soil contains a large amount of saline-alkali components, the physical and chemical properties of the soil are often poor, which leads to the inhibition of plant growth, or even the inability to grow

Method used

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  • NtCCC (nicotiana tabacum cation/chloride cotransporter) and application thereof
  • NtCCC (nicotiana tabacum cation/chloride cotransporter) and application thereof
  • NtCCC (nicotiana tabacum cation/chloride cotransporter) and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] This embodiment is mainly about tobacco cation / chloride co-transport gene NtC The acquisition process is briefly introduced as follows.

[0043] Using the cultivar tobacco leaf as a sample, the total RNA of the tobacco leaf was extracted with an RNA extraction kit, and reverse-transcribed into cDNA for future use;

[0044] Through the method of homologous comparison, refer to Arabidopsis At CCC The sequence of the gene and the known partial gene sequence of tobacco, the designed amplification primer sequence is as follows:

[0045] F: 5'-CGCGAGCTCGGTACCATGACCTGCAATAAACAT-3',

[0046]R: 5'-GCTCACCATGGATCCCTATGTAAACAAAGTGAC-3';

[0047] Using the cDNA prepared above as a template, use the above primers to perform PCR amplification, purify the amplified product and then sequence it to obtain the tobacco cation / chloride ion co-transporter gene NtC The gene sequence, whose base sequence is shown in SEQ ID No.1, includes a total of 1113 bp bases, of which the 5th to 477t...

Embodiment 2

[0051] Utilize the tobacco cation / chloride co-transport gene obtained in Example 1 NtC , the inventor further constructed a vector for transient gene silencing, and the related construction process is briefly introduced as follows.

[0052] Select a relatively specific nucleic acid fragment in this gene (the 5th-477th nucleotide sequence of SEQ ID NO.1 in the sequence table) as the guide sequence of VIGS, design the primer sequence as follows, and amplify this sequence;

[0053] The primer sequence design for VIGS transient silencing vector construction is as follows:

[0054] NtCCC-F: 5'-CGACGACAAGACCCTCCTGCAATAAACATCCAAT-3',

[0055] NtCCC-R: 5'-GAGGAGAAGAGCCTTACTGCCTTGTTTGCAAC-3';

[0056] Amplified length: 473 bp.

[0057] Use the In-Fusion method to connect the above-mentioned amplified fragments to the TRV vector (50°C ligation for 15 minutes) to construct and obtain TRV- NtC Carrier (recorded as TRV2- NtC carrier).

[0058] After the sequencing was correct, the re...

Embodiment 3

[0060] Using the VIGS transient silencing vector constructed in Example 2, taking Nicotiana benthamiana as an example, the inventors further carried out cultivation experiments to silence the tobacco cation / chloride ion co-transport gene in plants NtC , and the related experimental procedures are briefly introduced as follows.

[0061] Tobacco seeds were sown in seedling pots to grow seedlings, and the seedlings were divided two weeks after germination, planted in plastic pots (10cm×10cm), and daily fertilizer and water management was carried out at 22°C, 16h light / 8h dark conditions, and the growth was 4~ 5w, select 12 pots of tobacco seedlings with the same growth for future use.

[0062] Agrobacterium single colonies containing TRV1, TRV2, TRV2-PDS (positive control) and TRV2-NtCCC were inserted into YEB (5 mL) medium (kanamycin, 50 μg / mL), 28 °C, 250 r / min After shaking overnight for about 48 hours, transfer to 50 mL of YEB;

[0063] Shake culture overnight at 28°C, cent...

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Abstract

The invention relates to nicotiana tabacum chloride transport related genes, in particular to an application of an NtCCC (nicotiana tabacum cation / chloride cotransporter) in improvement of saline-alkali soil. The contransporter is related to chloride transport and has a base sequence shown in SEQ ID NO.1, wherein nucleotides at 5-477 sites are specific nucleic acid fragments. The NtCCC is a key regulator in nicotiana tabacum chloride metabolism, is found to be expressed in all tissue of nicotiana tabacum through real-time PCR and has higher expression in stems and leafbuds of nicotiana tabacum. Gene silenced plants with increased chloride content can be obtained through VIGS (virus-induced gene silence) or NtCCC knockout, making breeding of the nicotiana tabacum with higher chloride content possible, and a new technological approach is provided for adaptation of the nicotiana tabacum to different planting environments and improvement of saline-alkali soil.

Description

technical field [0001] The present invention relates to genes related to tobacco chloride ion transport, in particular to a tobacco cation / chloride ion co-transport gene NtC Application in saline-alkali soil improvement. Background technique [0002] At present, soil salinization has become a global problem. Saline-alkali land is a general term for various saline soils and alkaline soils, as well as salinized and alkalized soils to varying degrees. Because the soil contains a large amount of saline-alkali components and has bad physical and chemical properties, the growth of knowledge plants is inhibited, or even unable to grow. According to the incomplete statistics of UNESCO and FAO, the area of ​​saline-alkali land in the world is 954 million hectares, and our country is about 99 million hectares, ranking third in the world, but only one-sixth of the nicotine soil has been reclaimed and planted. In addition, the salinized area of ​​cultivated land in my country account...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01B79/02
CPCA01B79/02C07K14/415C12N15/8251
Inventor 张慧金立锋徐国云周会娜李锋翟妞刘萍萍陈霞陈千思郑庆霞申晓晔
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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