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Tobacco auxin transport protein NtPIN4 and application thereof

A technology for transporting protein and auxin, which is applied in the field of plant genetic engineering and can solve problems such as branching development of tobacco auxin transporting protein that have not yet been seen

Inactive Publication Date: 2017-09-19
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As an important model crop for scientific research, tobacco is also an important economic crop and agricultural crop. The branch development of tobacco has a very close relationship with the yield and internal quality of tobacco leaves. A detailed report on the development of proteins and their branches

Method used

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  • Tobacco auxin transport protein NtPIN4 and application thereof
  • Tobacco auxin transport protein NtPIN4 and application thereof
  • Tobacco auxin transport protein NtPIN4 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] This embodiment mainly NtPIN4 The gene cloning process is briefly described as follows.

[0048] (1) Prepare cDNA as a cloning template

[0049] Take 100 mg of stems of Tobacco (Safflower Dajinyuan) as a sample, fully grind in liquid nitrogen, extract total RNA according to the instructions of the RNA extraction kit, and then reverse-transcribe it into cDNA for future use;

[0050] (2) Design primers for PCR amplification

[0051] Designed to amplify the tobacco auxin transporter gene NtPIN4 The primer sequences are as follows:

[0052] NtPIN4-F: 5’-ATGATCACTTGGCACGATCTA-3’,

[0053] NtPIN4-R: 5'-TTATAATCCAAGAATGATGTAGTACAC-3';

[0054] The cDNA prepared in step (1) was used as a template, and the above primers were used for PCR amplification. The PCR amplification conditions were: 94°C pre-denaturation for 4 min; 94°C denaturation for 30s, 56°C annealing for 30s, and 72°C extension for 40s, a total of 25 Cycle; then extend at 72°C for 10 min; PCR amplification ...

Embodiment 2

[0066] In this example, the inventors treated and collected different tissues and organs with different exogenous plant hormones, and used fluorescence quantitative PCR to NtPIN4 The gene expression patterns were analyzed, and the related experiments were briefly described below.

[0067] The tobacco seeds were placed in a petri dish, and Hoagland solution was added for cultivation (light / dark = 18 / 6 h, 23 °C ~ 28 °C), and after germination for 2 weeks, the seedlings were moved to the solutions of different hormones and placed in the solution of different hormones. The leaves were sprayed with corresponding concentrations of hormones, and the treatment time was 5 h. The collected samples were quickly frozen in liquid nitrogen and then placed in a -80 °C refrigerator for storage for future use; in addition, after normal cultivation, the roots, stems, leaves, axillary buds and flowers of the budding tobacco were collected. As a sample, it was quick-frozen in liquid nitrogen and...

Embodiment 3

[0086] for further understanding NtPIN4 The regulatory role of genes in plant growth, the inventors constructed a knockout NtPIN4 The CRISPR / Cas9 expression vector of the gene, the following is a brief introduction to the construction process as follows.

[0087] First known from Example 1 NtPIN4 Genomic and coding region sequences, according to target site design criteria, are NtPIN4 A target site with a length of 20 bp is designed on the first exon sequence of the gene (schematic as image 3 shown), the knockout primer sequences PIN4-K-F and PIN4-K-R were designed as follows:

[0088] PIN4-K-F: 5’-GATTGCATATGGTTCTGTCCGATGG-3’,

[0089] PIN4-K-R: 5’-AAACCCATCGGACAGAACCATATGC-3’;

[0090] Design the reaction system to obtain DNA duplexes (annealing) at the target site. The 20 μL reaction system is designed as follows:

[0091] Annealing Buffer for DNA OLigos (5×), 4 μL;

[0092] Upstream and downstream primers (PIN4-K-F, PIN4-K-R), 4 μL each (50 μmoL / μL);

[0093] ...

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Abstract

The invention belongs to the technical field of plant genetic engineering and particularly relates to a tobacco auxin transport protein NtPIN4 and an application thereof. A gene NtPIN4 as a coding gene of the tobacco auxin transport protein comprises 1953bp, and a base sequence is shown as SEQ ID NO.1. The amino acid sequence of the tobacco auxin transport protein NtPIN4 is shown as SEQ ID NO.2. After the gene NtPIN4 is silenced, lateral branches of tobacco are obviously increased in gene silenced plants, and the growing number of axillary buds is obviously increased, so that tobacco plant type can be regulated. In normal tobacco plants, expression quantity of the gene NtPIN4 in stems and the axillary buds is higher. For mutant strains with the gene NtPIN4 knockout, branches are increased obviously, and axillary buds are increased actively. Deep research for the gene NtPIN4 shows that the gene NtPIN4 has important application value in tobacco growth regulation and plant type regulation.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a patent application for a tobacco auxin transporter NtPIN4 and its application. Background technique [0002] Auxin (IAA) is one of the most important hormones in plants, and it is also one of the earliest hormones that can regulate plant branching. It plays an important role in many biological processes such as embryo formation and root morphogenesis. [0003] In the process of plant growth, branch development has the phenomenon of apical dominance, that is, terminal buds can inhibit the occurrence of lateral buds, and removal of terminal buds (topping) will promote the production of lateral buds. It has long been thought that the reason for this inhibition of lateral bud development is auxin. The main reasons are: the study found that after the removal of the terminal bud, the IAA content in the stem of the plant decreased significantly, and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N9/22C12N15/90A01H5/00
CPCC12N9/22C12N15/102C12N15/8251C12N15/902C07K14/415
Inventor 谢小东杨军魏攀蔡联合李泽锋罗朝鹏张剑锋武明珠李锋王中
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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