Plant endosperm specific expression promoter and application thereof

A specific, promoter technology, applied in applications, plant products, botanical equipment and methods, etc., can solve problems such as biological growth and development adversely affecting metabolites, expression intensity unable to meet the needs of transgenic industrialization, and bioenergy consumption. , to increase the added value of science and technology, improve the quality of seeds, and achieve the effect of great application prospects.

Inactive Publication Date: 2011-11-23
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the widely used promoters (including CaMV35S, ubiquitin1, and Actin1) have high expression efficiency, they can express foreign genes in almost all tissues because their expression is not limited by time and space. During the expression process, it will drive the expression of genes in other tissues outside the seed, which will not only consume bioenergy, but also lead to the synthesis of some metabolites that may adversely affect the growth and development of organisms
Moreover, the expression intensity of the above-mentioned promoters cannot meet the needs of transgenic industrialization.

Method used

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  • Plant endosperm specific expression promoter and application thereof
  • Plant endosperm specific expression promoter and application thereof
  • Plant endosperm specific expression promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the acquisition of plant endosperm-specific expression promoter (GluB-7 promoter)

[0038] According to the amino acid sequence alignment method of rice glutenin GluB-1, a gene cDNA sequence GluB-7 (GenBank No. AY987390) that may encode glutenin was searched from the database. Search the upstream sequence of GluB-7 gene from GenBank, and design primers to amplify the GluB-7 promoter. To facilitate vector construction, two restriction sites (underlined) were sequentially added to the primers. GluB-7XbaF: 5'-TAA TCTAGA TCACACTACTTCATTATTGGG-3'( Xba I ), is the forward primer GluB-7SmaR of the promoter: 5'-TTAA CCCGGG AGCTATTTGAGGATGTTATTGG-3'( Sma I ) is the reverse primer.

[0039] The CTAB method was used to extract a small amount of genome from the leaves of rice (wild type rice Taichung No. 65, which was bred in Taiwan Province of my country in 1936; Qu Leqing et al., Acta Botany, 2001, 43: 1167-1171; preserved by the Institute of Botany, Chines...

Embodiment 2

[0040] Embodiment 2, plant endosperm-specific expression promoter (GluB-7 promoter) expression vector construction and genetic transformation

[0041] 1. Construction of plant expression vector with GluB-7 promoter fused to GUS gene

[0042] The binary expression vector pGPTV-35S-HPT was constructed according to the method described in Qu and Takaiwa, Plant Biotech J 2: 113-125, and was preserved by the Institute of Botany, Chinese Academy of Sciences. The pMD-GluB-7 plasmid and pGPTV-35S-HPT were digested with Sma I and Xba I. A 2310bp enzyme-digested fragment containing the GluB-7 promoter was recovered, and this fragment was ligated between the Sma I and Xba I enzyme recognition sites of pGPTV-35S-HPT. On the basis of PCR identification, the obtained recombinant plasmid was identified by double enzyme digestion with Sma I and Xba I, and a fragment containing 2.3kb GluB-7 promoter was obtained. The double-enzyme digestion identification shows that the recombinant plasmid c...

Embodiment 3

[0047] Example 3, Functional verification of plant endosperm-specific expression promoter (GluB-7 promoter)

[0048] Transgenic GluB-7-pGPTV-35S-HPT rice T 0 Plants were subjected to histochemical staining. Specific steps: Transplant GluB-7-pGPTV-35S-HPT rice T 0 Part of the leaves, roots, stem tissues of the generation plants, and the seeds at the filling stage 11 days, 15 days, and 17 days after flowering that were cut longitudinally from the middle with a scalpel were soaked in GUS reaction solution (0.1M NaPO 4 Buffer, pH7.0, 10mM EDTA, pH7.0, 5mM potassium ferricyanide, 5mM potassium ferrocyanide, 1.0mMX-Gluc, 0.1% Triton X-100), react at 37°C. The stained tissues were preserved in 70% ethanol, observed, and photographed under a dissecting microscope. The results showed that GUS expression was not observed in the roots, stems and leaves of transgenic GluB-7-pGPTV-35S-HPT rice. Image 6 Shown; the outer endosperm of the 11-day-after-anthesis seeds turned blue, but the ...

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Abstract

The invention discloses a plant endosperm specific expression promoter and an application thereof. The promoter is: 1) a DNA sequence of sequence 1 in the sequence table; 2) a DNA sequence which has homology of above 70% to the DNA sequence defined in sequence 1 in the sequence table, and has the function of a plant endosperm specific expression promoter; 3) a nucleotide sequence which can be hybridized with the DNA sequence defined in sequence 1 in the sequence table under high stringency conditions. The promoter of the invention can start specific expression of exogenous genes in plant endosperm, and is applicable to any plant whose seeds have endosperm, that is, monocotyledon or endosperm-type dicotyledon.

Description

technical field [0001] The invention relates to a plant endosperm specific expression promoter and application thereof. Background technique [0002] The latest development of plant biotechnology not only realizes the improvement of traditional agronomic shapes (such as increasing crop yield, enhancing disease resistance, insect resistance, herbicide resistance, improving quality, etc.), but also makes plants become bioreactors for biomedicine and industrial products (Daniell et al., Trends Plant Sci. 2001, 6: 219-226; Giddings et al., Nature Biotech. 2000, 18: 1151-1156; Hood and Jilka, Curr. Opin. Biotechnol. 1999, 10: 382-386 ; Hood and Woodard, Plants as Factories for Protein Production 2002, pp.119-135. Netherlands: Kluwer Academic). For most gramineous crops, due to its high yield, low production cost, storage resistance and easy control of production scale, it is an ideal carrier for the second generation of transgenic products. In recent years, the use of rice seed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 曲乐庆徐秀萍董祥柏柴志坚
Owner INST OF BOTANY CHINESE ACAD OF SCI
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