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Tobacco trichome (TTR1) promoter as well as expression vector and application thereof

A technology of expressing vectors and promoters, applied in the field of molecular biology, can solve problems such as the negative impact on normal plant growth and development, the slow progress in research on the molecular mechanism of glandular hair generation, the lack of theoretical support and material basis for variety selection and cultivation measures optimization , to achieve the effect of reducing the impact and increasing the effect

Active Publication Date: 2018-05-25
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] For a long time, most of the studies on glandular hairs have focused on the effects of agronomic cultivation measures and environmental factors on the density of glandular hairs and the chemical composition of leaves. The research on the molecular mechanism of glandular hairs has been slow. Variety selection and cultivation measures optimization for chemical composition improv

Method used

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  • Tobacco trichome (TTR1) promoter as well as expression vector and application thereof
  • Tobacco trichome (TTR1) promoter as well as expression vector and application thereof
  • Tobacco trichome (TTR1) promoter as well as expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment one: tobacco cultivation

[0031] Tobacco seeds were sterilized with 75% alcohol, and evenly spread on MS solid medium for cultivation. The cultivation conditions were 25°C, 14 hours of light per day, cultivation for 4 to 7 weeks, and set aside.

Embodiment 2

[0032] Example 2: PTTR1-PH7GW2 vector construction

[0033] Tobacco glandular hairs TTR1 The nucleotide sequence of the promoter is shown in SEQ ID NO.1, and the expression vector is pH7WG2 vector.

[0034] (1) Restriction site analysis: yes TTR1 The restriction site analysis of the promoter sequence found that there is no Sac I and Speech 1 two enzyme cutting sites, therefore, select these two sites for use as the ligase site;

[0035] (2) Enzyme digestion: Excise the P35s promoter in the pH7WG2 vector with endonucleases SacI and SpeI. The enzyme digestion reaction system is: 25 μl pH7WG2 plasmid (200 ng / μl); 1 μl restriction endonuclease 1 (15 U / μl); 1ul restriction enzyme 2 (15U / μl); 5 μl restriction enzyme reaction 10× Buffer; 18 μl water; the total volume is 50 μl; 37℃ water bath for 3 hours;

[0036] (3) Gel-cutting to recover the vector skeleton: run gel electrophoresis, perform gel-cutting to recover the plasmid skeleton fragments, and then use a DNA recovery ki...

Embodiment 3

[0050] Example 3: Construction of PTTR1-GUS-PH7GW2 vector

[0051] Using the Gateway method will GUS Gene insertion into PTTR1-PH7GW2 vector TTR1 downstream of the promoter sequence. The specific primer sequences used are shown in Table 1.

[0052] Gateway method import GUS The specific steps of the gene are as follows:

[0053] (1) Perform with GUS-F / GUS-R primers GUS The first round of PCR amplification of the gene, the specific reaction system is as amplified in Example 2 TTR1 promoter sequence, get GUS The first-round PCR product of the gene;

[0054] (2) to GUS The first-round PCR product of the gene was used as a template, and the GUS Gene second-round PCR primers for second-round PCR amplification;

[0055] (3) After the PCR reaction, cut the gel and recover GUS Gene DNA fragment, spare;

[0056] (4) Carry out BP reaction, the specific reaction system is: 1.5 μl GUS DNA fragment recovery product; 0.5 μl pDONR201; 1.0 μl BP clonase Ⅱ enzyme Mix; 2.0 μl...

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Abstract

The invention discloses a tobacco trichome (TTR1) promoter as well as an expression vector and application thereof. The nucleotide sequence of the TTR1 promoter is SEQ ID NO.1. The eukaryotic expression vector designed for plant genetic transformation has a TTR1 promoter sequence; the TTR1 promoter is applied to promotion of target gene expression, a development regulation mechanism for tobacco trichome tissues, formation of aroma substances in the trichome and improvement of tobacco quality; a method for promoting GUS gene expression by the TTR1 promoter is designed; a promoter is inserted into an expression vector containing a GUS gene so as to change a conventional promoter in the expression vector to obtain recombinant plasmid; the recombinant plasmid is transported into a receiver plant and is cultivated. The TTR1 gene promoter disclosed by the invention can drive the GUS gene to be specifically expressed in the tobacco trichome, can be used as an expression exogenous gene of plant genetic transformation and an expression element for a tobacco bioreactor, and has important value for development regulation of the tobacco trichome tissues, formation of the aroma substances in the trichome and improvement of the tobacco quality.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a tobacco glandular hair TTR1 Promoter, its expression vector and its application. Background technique [0002] Plant epidermis is divided into two forms: unicellular and multicellular. It covers most of the ground epidermal tissues of plants and plays an important role in repelling aphids, defending against virus invasion, and responding to adversity stresses such as drought and ultraviolet radiation. The epidermis of Arabidopsis thaliana is a unicellular structure, while the epidermal trichomes of tobacco, snapdragon, and tomato are multicellular glandular trichomes. [0003] Tobacco leaf glandular hair secretions constitute an important part of leaf chemistry, among which, the main secretion of tobacco glandular hairs - cebradane compounds are important aroma precursors of tobacco leaves. Therefore, tobacco glandular hairs not only affect the stress resistance of ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/12A01H6/82
CPCC07K14/415C12N15/8225
Inventor 崔红吴敏亮王召军张洪映葛莉崔丽鹏陈晓芬张松涛杨永霞贾宏昉陈亮
Owner HENAN AGRICULTURAL UNIVERSITY
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