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Specific expression promoter for plant embryo and application thereof

A specific and promoter technology, applied in the fields of application, plant products, botanical equipment and methods, etc., can solve the problems of lack of embryo-specific molecular or cell markers, technical difficulties in cloning seed embryo-specific genes, etc.

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

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Problems solved by technology

At present, some progress has been made in the research on the morphology and physiology of seeds, but due to the technical difficulties in cloning seed embryo-specific genes, for example, the high-level expression of seed storage protein genes can easily cover up the information of some low-abundance genes (Goldberg et al., cell 1989, 56:149-160); due to the lack of some special molecular or cell markers in the embryonic development process, there is still little understanding of the development of seed embryos at the level of molecular biology

Method used

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  • Specific expression promoter for plant embryo and application thereof
  • Specific expression promoter for plant embryo and application thereof
  • Specific expression promoter for plant embryo and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1, the acquisition of plant embryo-specific expression promoter (16 kDa oleosin promoter)

[0038] According to the cDNA sequence of the rice 16 kDa oleosin gene (GenBank number L76464), the upstream sequence of the 16 kDa oleosin gene was searched from GenBank, and primers were designed to amplify the 16 kDa oleosin promoter. To facilitate vector construction, two restriction sites (underlined) were sequentially added to each pair of primers. The forward primer for the 16kDa oleosin promoter is F1: 5'-ACC AAGCTT CGCTGATGTGGTATCATAG-3' (Hind III) and reverse primer R1: 5'AA GTC GAC GGGGATCGAGCACACCTGCAG-3' (Sal I).

[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, Chinese Academy of Sciences) Using DNA as a template and using F1 and R...

Embodiment 2

[0040] Embodiment 2, plant embryo-specific expression promoter (16 kDa oleosin promoter) expression vector construction and genetic transformation

[0041] 1. Construction of plant expression vector with 16KDa oleosin promoter fused to GUS gene

[0042] pGPTV-35S-HPT was constructed with reference to the method described in the literature (Qu and Takaiwa, Plant Biotech J 2004, 2: 113-125), and was preserved by the Institute of Botany, Chinese Academy of Sciences. The pT7-oleP plasmid and the binary expression vector pGPTV-35S-HPT were digested with Hind III and Sal I. Recover an enzyme-digested fragment containing 1438bp of 16 kDa oleosin promoter, and connect this fragment between the Hind III and Sal 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 Hind III and Sal I, and a 1.4 kb fragment of 16 kDa oleosin promoter was obtained. Recombinant plasmid indicated by ...

Embodiment 3

[0048] Embodiment 3, Southern hybridization detection of transgenic rice plants

[0049] Using CTAB method to extract positive T cells identified by PCR 0 For the genomic DNA of rice transgenic plant leaves, about 40 μg of DNA was added to 50 units of EcoR I to fully digest, and after separation by 0.8% agarose gel electrophoresis, the DNA was transferred to a positively charged nylon membrane with 0.4N NaOH ( purchased from Amersham pharmacia). The transferred membrane was pre-hybridized in 0.5M sodium phosphate buffer containing 7% (W / V) SDS at 65°C for 5 hours, and the probe was labeled with [α-32P]dCTP random primer method (purchased from China Furui Co., Ltd. ). The hybridization probe used is a 441bp DNA fragment located inside GUS (the sequence is the 1344-1784th nucleotide sequence from GENBANK No. S69414.1, with the pGPTV-35S-HPT plasmid as a template, 5'-CTGCGACGCTCACACCGATACC- 3' and 5'-TCACCGAAGTTCATGCCAGTCCAG-3' are primers for PCR amplification products) as pr...

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Abstract

The invention discloses a specific expression promoter for a plant embryo and application thereof. The promoter is derived from (1) a DNA (Deoxyribonucleic Acid) sequence shown as a sequence 1 in a sequence table, (2) a DNA sequence having over 70 percent of homology with the DNA sequence defined by the sequence 1 in the sequence table and having the function of a specific expression promoter forthe plant embryo, and (3) a nucleotide sequence which can be hybridized with the DNA sequence defined by the sequence 1 in the sequence table under a highly serious condition. The promoter can be used for promoting specific expression of an exogenous gene in the plant embryo, and is suitable for any seed plant.

Description

technical field [0001] The invention relates to a plant milk-specific expression promoter and its application. Background technique [0002] The latest development of plant biotechnology not only realizes the improvement of traditional agronomic traits (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 produce ex...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 曲乐庆代玲玲
Owner INST OF BOTANY CHINESE ACAD OF SCI
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