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Plant endosperm specificity expression promoter and its application

A specific, promoter technology, applied in the fields of application, plant gene improvement, botanical equipment and methods, etc., can solve the problem of consuming bioenergy, adversely affecting metabolites in biological growth and development, and the expression intensity cannot meet the needs of transgenic industrialization, etc. problems, to achieve great application prospects, increase the added value of science and technology, and improve the quality of seeds

Inactive Publication Date: 2007-10-31
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 specificity expression promoter and its application
  • Plant endosperm specificity expression promoter and its application
  • Plant endosperm specificity expression promoter and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the acquisition of plant endosperm-specific expression promoter (GluB-5 promoter)

[0036] According to the glutelin gene GluB-5 cDNA sequence of rice (GenBank number is AB016501; Mitsukawa et al., 1998, Plant Biotechnol 15: 205-211), search the upstream sequence of GluB-5 gene from GenBank, and design primers to amplify GluB-5 Promoter. To facilitate vector construction, two restriction sites (underlined) were sequentially added to each pair of primers.

[0037] Forward primer for GluB-5 promoter is F1: 5'-AAAG GTC GAC GAGAAAAGAAGATTTGCTGAC-3'( Sal I ), the reverse primer is R1: 5'-AAA CCCGGG CTATTTTATTGAAAG GATAATGG-3'( Sma I )

[0038] A small amount of genomic DNA was extracted from rice (wild-type rice Kitaake) leaves by CTAB method, and the GluB-5 promoter sequence was amplified by PCR using it as a template and F1 and R1 as primers. The reaction system is: 1 μl of 10 μM forward and reverse primers, 2 μl of 10×Ex Buffer, 1 μl (10 ng) of geno...

Embodiment 2

[0039] Embodiment 2, plant endosperm-specific expression promoter (GluB-5 promoter) expression vector construction and genetic transformation

[0040] 1. Construction of plant expression vector with GluB-5 promoter fused to GUS gene

[0041] Use Sal I and Sma I to double digest the pT7-GluB-5 plasmid, reclaim the 2269bp enzyme-cut fragment containing the GluB-5 promoter, and insert the fragment into pGPTV-35S-HPT (according to literature Qu and Takaiwa, PlantBiotech J 2 : between the Sal I and Sma I enzyme recognition sites constructed by the method described in 113-125). On the basis of PCR identification, the resulting recombinant plasmid was identified by double enzyme digestion using Sal I and EcoR I to obtain a fragment containing the 4.5 kb GluB-3 promoter, and the recombinant plasmid that was identified by enzyme digestion and identified as containing the GluB-5 promoter was named as GluB-5-pGPTV-35S-HPT (the schematic diagram of its structure is shown in Figure 2). A...

Embodiment 3

[0048] Example 3, Functional verification of plant endosperm-specific expression promoter (GluB-5 promoter)

[0049] transgenic GluB-5-pGPTV-35S-HPT rice T 0 Plants were subjected to histochemical staining. Specific steps: Transplant GluB-5-pGPTV-35S-HPT rice T 0 Substitute plant or wild-type rice Kitaake (control) root, leaf, leaf sheath, stem, cut into small pieces, the seeds of the filling stage 7 days, 12 days, 17 days after flowering were cut longitudinally from the middle with a scalpel, soaked In GUS reaction solution (0.1M NaPO 4 buffer, pH 7.0, 10 mM EDTA, pH 7.0, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1.0 mM X-Gluc, 0.1% Triton X-100), react at 37°C. The results showed that GUS expression was not observed in the roots, leaves, leaf sheaths, and stems of rice transfected with GluB-5-pGPTV-35S-HPT; the aleurone layer, subaleurone layer, and outer endosperm of the seeds turned blue 7 days after flowering, and the center of the endosperm Parts and ...

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Abstract

The invention discloses a special expressing promoter of plant endosperm and appliance, which is characterized by the following: 1) possessing DNA sequence of sequence 1 in sequence table; 2) possessing 70% or above 70% homologous property with limited DNA sequence of sequence 1 in sequence table; possessing DNA sequence with same function; 3) possessing crossing nucleic acid sequence with limited NDA sequence of sequence 1 in sequence table under high strict condition. This promoter can start special expression of external source gene in plant endosperm, which is fit for any plant with endosperm such as monocotyledon or 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 seeds to p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82
Inventor 曲乐庆
Owner INST OF BOTANY CHINESE ACAD OF SCI
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