Promoter for early specific expression of plant anther and application of promoter

A technology of promoters and plant flowers, applied in the field of plant genetic engineering, can solve problems such as few and few, affecting male fertility, etc.

Active Publication Date: 2022-07-01
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, gene reports have been reported to be involved in the regulation of pollen tapetum, pollen wall and pollen development, thereby affecting male fertility. The gene types include E3 ubiquitin ligase, MYB transcription factor, cysteine-rich class receptor kinases, RNases, and long non-coding or small RNAs, etc., but there are few studies on the regulation of miRNAs in early pollen development

Method used

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  • Promoter for early specific expression of plant anther and application of promoter
  • Promoter for early specific expression of plant anther and application of promoter
  • Promoter for early specific expression of plant anther and application of promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Cloning of AT4G14811 gene promoter fragment EASEpro:

[0024] The length of 1.409 kb upstream of the ATG site of the initiation codon of the Arabidopsis AT4G14811 gene was selected as the candidate promoter region, and the Arabidopsis col-0 genomic DNA was used as the template, and primers F1 and R1 (below) were used for amplification. Subsequently, the amplified PCR products were constructed into the pENTR intermediate vector by TOPO reaction, and the LR reaction was carried out with the pKGWFS7 and pK7FWG0 final vectors respectively to construct the analysis vectors EASEpro-GUS and EASEpro-GFP with the EASEpro promoter fused with GUS and GFP. Among them, TOPO enzyme and LR enzyme were purchased from Invitrogen Company, and pKGWFS7 and pK7FWG0 vectors were purchased from Ghent University.

[0025] F1: 5'-CACCGTGAATAGGATGGCGAGAAGAG-3' (SEQ ID NO: 2)

[0026] R1: 5'-AAGAATTTTTCTTGAAGGCTCTTTG-3' (SEQ ID NO: 3)

Embodiment 2

[0027] Example 2, the acquisition of transgenic plants

[0028] (1) Cultivate Arabidopsis thaliana that can be infected by Agrobacterium:

[0029] The plump col-0 Arabidopsis thaliana seeds were selected, sterilized with 15% sodium hypochlorite solution or 75% absolute ethanol for 15 minutes, replaced with water 4 times, and evenly placed on sterilized MS solid medium. Disproportionate in a refrigerator at 4°C for 2-3 days, continue to cultivate for 10 days in a long-day incubator at 22°C with 16 hours of light / 8 hours of darkness to form seedlings, transfer the seedlings to the soil and cultivate until bolting.

[0030] (2) Agrobacterium transformation:

[0031] The pKGWFS7 and pK7FWG0 vectors with the EASEpro promoter fragment were transformed into Agrobacterium GV3101 competent cells by electroshock method, coated with solid LB medium with 50 μg / L rifampicin and spectinomycin, and cultured at 28°C for 2 days in the dark , screen for positive clones. The obtained positive...

Embodiment 3

[0036] Example 3. Activity analysis of EASpro promoter in anther tissue

[0037] The flowers and seedlings at different reproductive stages of positive transgenic Arabidopsis were taken and treated with pre-cooled 90% acetone for 20 minutes; the acetone was aspirated and discarded, and 50 mM phosphate buffer (pH 7.2) was added to rinse twice; the phosphate buffer was aspirated and discarded, Add an appropriate amount of GUS staining solution (50 mM phosphate buffer at pH 7.2, potassium ferricyanide 2 mM, potassium ferrocyanide 2 mM, X-gluc 1 mM) to completely immerse the plant material in the staining solution, and vacuum for 1 hr; Put it into a 37°C incubator for dyeing. After the plant material has developed color, aspirate the GUS staining solution, add 1 mL of 70% ethanol, and mix gently to decolorize. Observe and photograph under a stereo microscope.

[0038] The results showed that the GUS staining was darker in the flowers closer to the middle of the inflorescence, whi...

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Abstract

The invention discloses a promoter for early specific expression of plant anther and application of the promoter. The promoter is a promoter of an arabidopsis thaliana miRNA780 gene (AT4G14811), can drive a target gene to be specifically expressed in the early anther development stage of a plant, namely a tetrad stage, a microspore stage and a two-cell stage, and can be applied to research in the aspects of plant breeding and gene function analysis.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to a promoter sequence specifically and highly expressed in the early development stage of anther of Arabidopsis thaliana, a model plant, and the application of the promoter in plant transgenic. Background technique [0002] Arabidopsis thaliana (Arabidopsis thaliana) is a dicotyledonous model crop for the study of plant gene function. The study of its molecular mechanism will be crucial to the yield of plants, especially crops, including soybean, cotton, rice, and wheat. important role. As the world's population grows year by year, the area of ​​arable land is gradually decreasing. How to efficiently increase the unit yield of crops has become a topic of common concern for scientists all over the world. [0003] Because heterosis can produce higher growth rate and metabolic efficiency than both parents, increase the yield and body size of progeny, and show strong...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H5/10A01H6/20
CPCC12N15/8231Y02A40/146
Inventor 瞿礼嘉黄佳颖张立顾红雅
Owner PEKING UNIV
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