Plant endosperm specificity promoter and its application

A specific, promoter-based technology, applied in the fields of application, plant genetic improvement, botany equipment and methods, etc., can solve the problem of metabolites that adversely affect biological growth and development, consume bio-energy, and the expression intensity cannot meet the needs of transgenic industrialization, etc. problems, to achieve the effect of increasing added value of science and technology, great application prospects, and improving seed quality

Active 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 promoter and its application
  • Plant endosperm specificity promoter and its application
  • Plant endosperm specificity promoter and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0036] According to the glutelin gene GluC cDNA sequence of rice (GenBank No. AB016501; Mitsukawa et al., 1998, Plant Biotechnol 15: 205-211), the upstream sequence of the GluC gene was searched from GenBank, and primers were designed to amplify the GluC promoter. To facilitate vector construction, two restriction sites (underlined) were sequentially added to each pair of primers.

[0037] Forward primer for GluC promoter is F1: 5'-GGG AAGCTT GTTCAAGATTTTATTTTTGG-3'( Hind III ), the reverse primer is R1: 5'-ACGC GTC GAC AGTTATTCACTTAGTTTCCC-3'( Sal I )

[0038] A small amount of genomic DNA was extracted from rice (wild-type rice Kitaake) leaves by CTAB method, and the GluC 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 ge...

Embodiment 2

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

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

[0041] Digest the pT7-GluC plasmid with Hind III and Sal I, reclaim the 2331bp enzyme-cut fragment containing the GluC promoter, and insert the fragment into pGPTV-35S-HPT (according to the literature Qu and Takaiwa, PlantBiotech J 2: 113-125 between the Hind III and Sal I enzyme recognition sites. On the basis of PCR identification, the obtained recombinant plasmid was identified by double enzyme digestion with Hind III and Sal I, and a fragment containing 4.5kb GluC promoter was obtained; the recombinant plasmid identified by enzyme digestion and identified as containing GluC promoter was named GluC-pGPTV -35S-HPT (the schematic diagram of which is shown in Figure 2). Among them, the double enzyme digestion identification map of GluC-pGPTV-35S-HP...

Embodiment 3

[0049] Example 3, Functional verification of plant endosperm-specific expression promoter (GluC promoter)

[0050] transgenic GluC-pGPTV-35S-HPT rice T 0 Plants were subjected to histochemical staining. Specific steps: Transplant GluC-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 GluC-pGPTV-35S-HPT; the aleurone layer, sub-aleurone layer, and endosperm of the seeds turned blue 7 days after flowering, and the embryos were not stained. Blue; the blue color ...

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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 曲乐庆宋艳茹
Owner INST OF BOTANY CHINESE ACAD OF SCI
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