Application of rape tissue specific promoter for regulating target genes expressed in plant anther

A plant flower and plant pollen technology, applied in the field of plant biology, can solve problems such as few researches, large structures, and complexities

Inactive Publication Date: 2017-08-18
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there are relatively few rapeseed anther or pollen-specific promoters with high driving activity and good specificity. However, due to the large genome and complex structure of rapeseed, there are not many studies on the molecular mechanism of anther development. More is the reference model plant Arabidopsis related research

Method used

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  • Application of rape tissue specific promoter for regulating target genes expressed in plant anther
  • Application of rape tissue specific promoter for regulating target genes expressed in plant anther
  • Application of rape tissue specific promoter for regulating target genes expressed in plant anther

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Cloning of Rapeseed Tissue-Specific Promoters SEQ ID NO.1~SEQ ID NO.5 and Construction of Plant Expression Vectors

[0027] Genomic DNA of double 11 leaves in rapeseed was extracted by CTAB method, and the DNA was used as a template for PCR amplification with a reaction volume of 50 μl.

[0028] The primers for amplifying the promoter SEQ ID NO.1 are:

[0029] 1-F: 5'-(Sal I)CGCgtcgacCCATCTTACTCCAATAGTATTTTGG-3', 1-R: 5'-(SmaI)CcccggggCCGTTCTTCTTTCAATATTTAAAA-3;

[0030] The primers for amplifying the promoter SEQ ID NO.2 are:

[0031] 2-F: 5'-(Sal I)CGCgtcgacATGGAGAACTTAAATAGACAAATAC-3', 2-R: 5'-(SmaI)Ccccg ggCCGTTTCTTTCTTTCAATATTTAAAA-3;

[0032] The primers for amplifying the promoter SEQ ID NO.3 are:

[0033] 3-F: 5'-(Sal I)CGCgtcgacATGGAGAACTTAAATAGACAAATAC-3', 3-R: 5'-(SmaI)Ccccggg AATCAGATTATTTAAGCTGTCTAAA-3;

[0034] The primers for amplifying the promoter SEQ ID NO.4 are:

[0035] 4-F: 5'-(Sal I)CGCgtcgacATGAAACGAAAGCCACCTC-3', 4-R: 5'-(SmaI)CcccggggCCGTTT...

Embodiment 2

[0042] The application of promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 to regulate the specific expression of target genes in plant anthers:

[0043] 1) The expression vector of the promoter fusion reporter gene is transformed into Arabidopsis thaliana

[0044] Example 1 The successfully constructed monoclonal Escherichia coli bacterial liquid containing the promoter plant expression vector was activated at 37°C and the plasmid was extracted, and pG2NHL-H2BYFP-gusplus-Nost-1, pG2NHL-H2BYFP-gusplus-Nost-2, pG2NHL-H2BYFP-gusplus-Nost-3, pG2NHL-H2BYFP-gusplus-Nost-4 and pG2NHL-H2BYFP-gusplus-Nost-5 were respectively transformed into Agrobacterium GV3101 competent cells, in the presence of kanamycin (50μg / mL) and After culturing on a rifampin (50 μg / mL) plate, single clones were picked and detected with promoter cloning primers to determine positive clones. Activate the positive Agrobacterium liquid at 28°C, using the inflorescence dip method (Zhan...

Embodiment 3

[0075] Conservation of promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 in Brassica cabbage, cabbage and rapeseed

[0076] Based on the PCR reaction, use the primers in the embodiment case 1 to clone respectively promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 in Chinese cabbage, cabbage and rapeseed The homologous fragments in the sequence were sequenced, and their sequence homology was found to be as high as 100%, and the lowest was 99.78%, with only 1 bp difference, and the difference was not in the core promoter region. This shows that the sequences of promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 are highly conserved in Brassica cabbage, cabbage and rapeseed. It is predicted that the functions of the promoters SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 are also conserved. Just as in Example 2, the promoter of SEQ ID NO.5 drives the expression of GUS gene in Ara...

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Abstract

The invention belongs to the field of plant biotechnology, and relates to the separation and application of plant tissue specific promoters, in particular to application of a rape tissue specific promoter for regulating target genes expressed in plant anther. The promoter and the truncated fragments of the promoter are integrated with the reported genes and transformed into arabidopsis thaliana, and can drive the reported genes to express specifically in anther by GUS staining, and the sequences are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4; after further truncation, a promoter fragment with a sequence of pollen specific expression shown as SEQ ID NO.5 is obtained. The invention further discloses that the tissue specific promoter or the truncated promoter fragments provide new effective regulatory elements for controlling plant fertility, and have a good application prospect for genetic engineering breeding.

Description

technical field [0001] The invention belongs to the field of plant biotechnology. Specifically, the invention relates to the application of a rapeseed tissue-specific promoter in regulating the specific expression of a target gene in plant anthers. The rapeseed tissue-specific promoter provided by the invention can regulate the target gene Specifically transcribed or expressed in plant anthers or pollen. Background technique [0002] Gene regulation in higher plants is mainly carried out at the transcriptional level, and is controlled by the mutual coordination of various cis-acting elements and trans-acting factors. The exogenous DNA sequence is linked to a specific promoter to promote expression in the plant host, so the choice of promoter type determines the time and location of gene expression. In recent years, the more popular research objects are tissue-specific promoters. These tissue-specific promoters mainly include leaf, phloem, vascular bundle, root and other org...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/02
CPCC12N15/8231C12N15/8241
Inventor 涂金星王轩鹏夏胜前易斌沈金雄马朝芝文静傅廷栋
Owner HUAZHONG AGRI UNIV
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