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Specific expression promoter pEnd1 for endosperm of paddy rice and application of specific expression promoter

A promoter and specific technology, applied in the field of biotechnology and plant genetic engineering, can solve the problem that the promoter cannot meet the needs of genetic engineering to improve the quality of rice, and achieve the added value of science and technology, the level of expression and accumulation, and the large-scale application. Foreground effect

Active Publication Date: 2016-12-07
CHINA NAT RICE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some tissue-specific promoters have been invented so far, these promoters are still far from meeting the needs of genetic engineering to improve rice quality, so the promoter of endosperm-specific expression isolated by the present invention has a certain effect on the improvement of plant quality. important meaning

Method used

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  • Specific expression promoter pEnd1 for endosperm of paddy rice and application of specific expression promoter
  • Specific expression promoter pEnd1 for endosperm of paddy rice and application of specific expression promoter
  • Specific expression promoter pEnd1 for endosperm of paddy rice and application of specific expression promoter

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Embodiment 1

[0033] Such as figure 1 As shown, a probable endosperm was first discovered from the CREP (http: / / crep.ncpgr.cn) (Wang et al., 2010) of the rice whole growth period expression profiling database of the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University. Specifically expressed candidate gene, the sequence of this gene is obtained on the website NCBI (http: / / www.ncbi.nlm.nih.gov / ) of the National Institute of Biology, as shown in SEQ ID No: 2, a total of 1278bp, with The 1278bp sequence design primers to do qRT-PCR reaction to further determine its expression profile, qRT-PCR results show ( figure 2): The gene is expressed only in the endosperm. On this basis, a promoter candidate fragment named pEnd1 was amplified from the rice Nipponbare genome by PCR with specific primers, and the promoter candidate fragment pEnd1 was loaded into the binary vector pCAMBIA1305.1( image 3 ), assembled into pEnd1-GUS vector ( Figure 5 ), the pCAMBIA1305.1 ve...

Embodiment 2

[0034] The acquisition of embodiment 2 promoter

[0035] (1) Screening of rice seed endosperm-specific expression genes

[0036] Fluorescent quantitative PCR screening of genes specifically expressed in rice seed endosperm

[0037] Select Nipponbare rice roots, stems, leaves, leaf sheaths, young ears, and rice seed endosperms at different developmental stages (6, 9, 12, 15, 18, 21, and 24 days), respectively extract mRNA, reverse transcribe and synthesize cDNA, and use fluorescence Quantitative PCR identified the expression profile of rice endosperm-specific expression gene (gene number: Os02g0202400) in different tissues and developmental stages, and confirmed that the gene was specifically expressed in rice endosperm.

[0038] (2) Cloning of rice seed endosperm-specific expression promoter fragment

[0039] Genomic DNA from leaves of rice Nipponbare (Oryza sativa L. publicly available from the China Rice Research Institute) was extracted as a template, and primer 1:5'- C...

Embodiment 3

[0042] Functional verification of the promoter of embodiment 3

[0043] 1. Obtaining of transgenic pEnd1 rice

[0044] (1) Obtaining the recombinant vector

[0045] With the PCR product pEnd1 of 2141bp obtained by embodiment 1 and expression vector pCAMBIA1305.1 (the public can obtain from the Rice Research Institute of China, the structural representation of the vector is as follows image 3 The connection shown) is dependent on the In-fusion recombination method: first use EcoR I and NcoI to cut the vector pCAMBIA1305.1, cut off the 35S promoter, and then use the PCR product with the linker and EcoR I and NcoI enzyme The cut vector was subjected to In-fusion recombination at 50°C to obtain the recombinant vector, which was named pEnd1-GUS ( Figure 5 ). The expression vector pCAMBIA1305.1 was digested with EcoR I and Spe I, and the 35S and catalase intron fragments on the vector were excised together, and then the catalase intron fragment was amplified with EcoR I and Spe...

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Abstract

The invention relates to the technical fields of biological techniques and plant genetic engineering, in particular to a specific expression promoter for an endosperm of paddy rice and application of the specific expression promoter. The promoter can drive a target gene to be expressed in the endosperm in a transgenic regulation and control system of the paddy rice; a nucleotide sequence of the promoter is as shown by SEQ ID NO: 1. The specific expression promoter for an endosperm of a plant, which is provided by the invention, is a pEnd1 promoter which is separated and obtained from the paddy rice by designing a primer, using genomic DNA of the paddy rice as a template and by utilizing a PCR method; shown by an expression specificity experiment of the specific expression promoter in the paddy rice, the promoter provided by the invention makes a reporter gene of beta-glucuronidase (GUS) specifically expressed only in an endosperm of a seed of the paddy rice. The promoter provided by the invention is shown to be capable of promoting the expression of the specificity of an exogenous gene in the endosperm of the plant, is applicable to any plants of which the seeds have the endosperms, i.e., monogenus plants or endosperm type dicotyledonous plants.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and plant genetic engineering. Specifically, the invention relates to a rice endosperm-specific expression promoter and its application. The promoter can drive the expression of a target gene in the endosperm in a rice transgene regulation system. Background technique [0002] The endosperm of plant seeds is not only the main site for the storage of the main source of carbohydrates for humans and animals, but also serves as a bioreactor for the production of proteins of great value in medical and industrial fields. For example, vitamin A, GLP with the function of stimulating insulin secretion to prevent diabetes, glycinin with hypoglycemic effect, hepatitis B vaccine and pollen allergy vaccine have all been successfully expressed and accumulated at high levels in rice (Goto et al., Nature Biotech1999,17:282-286; Ye et al., Science 2000,287:303-305; Katsube et al., Plant Physiol 1999,120:106...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00
CPCC12N15/113C12N15/8234
Inventor 魏祥进胡培松李三峰唐绍清焦桂爱圣忠华谢黎虹邵高能
Owner CHINA NAT RICE RES INST
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