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Plant glandular hair-specific expression gene hd-9, its expression vector and application

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: 2022-07-08
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, its expression vector and application
  • Plant glandular hair-specific expression gene hd-9, its expression vector and application
  • Plant glandular hair-specific expression gene hd-9, its expression vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0025] HD-Zip Ⅳ The family genes are widely involved in the regulation of plant epidermal hair formation, epidermal cell differentiation and anthocyanin accumulation. For a long time, the inventors have Nicotiana tobacum ) The gene members of HD-Zip Ⅳ have been deeply analyzed for tissue expression characteristics, and it has been found that HD-9 The gene is specifically expressed in glandular hairs.

[0026] 1. Tobacco HD-9 separation

[0027] (1) Total RNA of common tobacco was extracted 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 Gene cloning: According to the inventor's previous experimental results, the gene is specifically expressed for glandular hair HD-9 For cloning, PCR reaction conditions were 95 °C for 5 min; 95 °C for 40 s, 58 °C...

Embodiment 2

[0049] Embodiment 2: HD-9 Obtainment of gene overexpression lines

[0050] 1. Construction of the 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) Select Spe I and nru I two enzyme cleavage sites are ligase sites, which are utilized 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 restriction sites are Spe I and Nru I, respectively.

[0056] PCR reaction system: 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; 4 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, 72°C for 3 min; extension at 72°...

Embodiment 3

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

[0074] 1. Construction of Knockout Vectors

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

[0076] GGCCCAATGTGCGGCCA TTG , PAM area marked by underline, completed by Hangzhou Baige Biotechnology Co., Ltd.). Ligation of 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 E. coli, and the plasmid was extracted for sequencing and screening HD-9 -knock out gene editing vectors.

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

[0078] The method and process are the same as in Example 2.

[0079] 3. Obtaining knockout plants

[0080] Tobacco cultivar K326 was infected with Agrobacterium by leaf disk transformation. Screening of transgenic plants was carried out on the MS resistan...

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Abstract

The invention discloses a specific expression gene of plant glandular hair HD‑9 , its expression vector and its application, aiming at solving the technical problem of lack of regulation means for the development of plant gland hair head secretory cells. The present invention identifies a gene that can regulate the development of glandular hair cells HD‑9 , the overexpression vector and CRISPR / Cas9 vector of the gene were constructed, and the corresponding overexpression lines and gene knockout lines were obtained by transformation respectively. The identification and detection of phenotype and physiological characteristics showed that, HD‑9 Genes can regulate the development of glandular head cells of plant long stalk glandular hairs, which have important application value in regulating and increasing the secretion content of glandular hairs and directional improvement of glandular hair types.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a plant glandular hair-specific expression gene HD-9 , its expression vector and application. Background technique [0002] Plant epidermal hairs are specialized structures of epidermal cells. As a natural barrier between plants and the environment, they play an important role in plants responding to biotic and abiotic stresses in the environment. In addition, as secretory organs, glandular hairs can promote the excretion 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. According to the presence or absence of secretory glands, they can be divided into protective hairs (non-glandular hairs) and glandular hairs. [0003] Glandular hairs can specifically synthesize and secrete a variety of secondary metabolites, and terpenes are the most important c...

Claims

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Application Information

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Patent Type & Authority Patents(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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