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Plant glandular hair specific expression gene HD-9 as well as expression vector and application thereof

A technique for expressing genes, HD-9, which is applied in the field of molecular biology and can solve problems such as the lack of developmental regulation means

Active Publication Date: 2021-06-22
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a plant glandular trichome-specific expression gene capable of regulating the development of glandular hair head secretory cells HD-9 , and use its expression vector to transform plants, in order to solve the current technical problem of lack of means for regulating the development of secretory cells in the head of plant glandular hairs, and then achieve the purpose of directional improvement of plant glandular hair types and / or increase the content of glandular hair secretions

Method used

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  • Plant glandular hair specific expression gene HD-9 as well as expression vector and application thereof
  • Plant glandular hair specific expression gene HD-9 as well as expression vector and application thereof
  • Plant glandular hair specific expression gene HD-9 as well as expression vector and application thereof

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Effect test

Embodiment 1

[0024] Example 1: Glandular hair-specific expression genes HD-9 Gene cloning and tissue expression analysis

[0025] HD-Zip IV The family genes are widely involved in the regulation of plant epidermal trichome formation, epidermal cell differentiation and anthocyanin accumulation. For a long time, inventors have been interested in common tobacco ( Nicotiana tobacum ) The gene members of HD-Zip Ⅳ conducted an in-depth analysis of tissue expression characteristics and found that HD-9 The gene is specifically expressed in glandular trichomes.

[0026] 1. Tobacco HD-9 separation

[0027] (1) Extract the total RNA of common tobacco with TRIZOL.

[0028] (2) Reverse transcription of cDNA: RNA was reverse transcribed into cDNA using PrimerScript RT reagent Kit (TaKaRa, Japan).

[0029] (3) HD-9 Cloning of the gene: according to the inventor's preliminary experimental results, the gene specifically expressed in the glandular trichomes HD-9 For cloning, the PCR reaction cond...

Embodiment 2

[0049] Embodiment two: HD-9 Acquisition of gene overexpression lines

[0050] 1. Construction of overexpression vector

[0051] tobacco HD-9 Gene (the nucleotide sequence is shown in SEQ ID NO.1, and the expression vector adopts pCAMBIA-NPT vector).

[0052] (1) selection Speech I and Nru I two enzyme cutting sites are ligase sites, utilize as follows HD-9 Cloning primers for PCR amplification (see SEQ ID NO.5-6):

[0053] F: 5'-AGGACTAGTCACCATTTAGATAGAAAAGAGTGACC-3',

[0054] R: 5'-GAGATCGCGAAACCTTGGAATGCAACTAACC-3';

[0055] The underlined enzyme cleavage sites are Spe I and Nru I, respectively.

[0056] The PCR reaction system is: 2 μl genomic DNA (100 ng / μl); 1 μl Primer Star DNA polymerase; 2 μl primer 1 (10 μM); 2 μl primer 2 (10 μM); 10 μl 5× PCR reaction buffer; μl dNTPs (2.5mM); 29 μl water; total volume 50 μl;

[0057] The reaction program was: pre-denaturation at 94°C for 3 min; 35 cycles of 94°C for 20 s, 65°C for 20 s, and 72°C for 3 min; extension a...

Embodiment 3

[0073] Embodiment three: HD-9 Obtaining knockout plants

[0074] 1. Construction of the knockout vector

[0075] obtained by sequencing HD-9 Genome sequence, first design and synthesize gRNA target sequence (see SEQ ID NO.7):

[0076] GGCCCAATGTGCGGCCA TTG , the underlined PAM region was completed by Hangzhou Bioge Biotechnology Co., Ltd.). Ligate Oligo dimer with CRISPR / Cas9 vector: CRISPR / Cas9 vector 2.0 μL, Oligo dimer 1.0 μL, Enzyme Mix 1.0 μL, sterile water H 2 O supplemented to 10.0 μL. The ligation product was transformed into Escherichia coli, and the plasmid was extracted for sequencing screening HD-9 -knock out gene editing vectors.

[0077] 2. HD-9 -knock out vector into GV3101 Agrobacterium

[0078] Method process is the same as embodiment 2.

[0079] 3. Obtaining of Gene Knockout Plants

[0080] Tobacco cultivar K326 was infected with Agrobacterium by leaf disk transformation method. The transgenic plants were screened on the MS resistant medium conta...

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Abstract

The invention discloses a plant glandular hair specific expression gene HD-9 as well as an expression vector and application thereof, and aims to solve the technical problem of lack of plant glandular hair head secretory cell development regulation means at present. The gene HD-9 capable of regulating and controlling development of glandular hair cells is identified, an overexpression vector and a CRISPR / Cas9 vector of the gene are constructed, and a corresponding overexpression strain and a gene knockout strain are obtained through conversion respectively. Phenotype and physiological feature identification and detection show that: the HD-9 gene can regulate and control the development of glandular head cells of plant long-stem glandular hair, and has important application value in the aspects of regulating and increasing the content of glandular hair secretions, directionally improving glandular hair types and the like.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a plant glandular trichome-specific expression gene HD-9 , its expression vector and application. Background technique [0002] Plant epidermis is a specialized structure of epidermal cells, which acts as a natural barrier between plants and the environment, and plays an important role in plants' response to biotic and abiotic stresses in the environment. In addition, as a secretory organ, glandular trichomes can promote the secretion of heavy metals and reduce the accumulation of heavy metals in plants. The structure and shape of plant epidermal hairs are diverse, consisting of single to multiple cells, and can be divided into protective hairs (non-glandular hairs) and glandular hairs according to the presence or absence of secretory glands. [0003] Glandular trichomes can specifically synthesize and secrete a variety of secondary metabolites, and terpenoids are the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00A01H6/82
CPCC07K14/415C12N15/8218C12N15/8205C12N15/8241Y02A50/30
Inventor 崔红张洪映王召军闫筱筱
Owner HENAN AGRICULTURAL UNIVERSITY
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