Identification and application of a plant endosperm-specific expression promoter posens100

A promoter and endosperm technology, applied in the field of DNA application and isolated DNA, can solve the problems of waiting for a long time, high homology gene silencing, and the effect of improving the expression time and space of the target gene is not obvious. The effect of high expression

Active Publication Date: 2022-02-11
深圳广三系农业科技有限公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

At present, some constitutive strong promoters are widely used in the field of agricultural biotechnology, such as the CaMV35S promoter of cauliflower mosaic virus and the Ubiquitin-1 promoter of maize. When it is expected to improve the quality of crops, the improvement effect is not obvious because the time (developmental stage-specific) or space (tissue-organ-specific) of the expression of the target gene is not well controlled, or because these constitutive promoters induce gene The expression level is too high to affect the growth and development of plants. These are the obstacles encountered when using constitutive strong promoters combined with functional genes to improve crop quality
[0004] In addition, when studying certain metabolic processes or regulatory pathways, it is often necessary to transform two or more genes on the same pathway into the same strain. If one of the genes is transformed to obtain a transgenic plant and then another gene is transformed, or After the two genes are transformed separately and then hybridized, it takes a long time to wait.
In order to improve efficiency and shorten the time for multiple gene transformation, it has recently been reported that new vectors can be used to transform multiple genes at the same time, but if the same promoter is used repeatedly in multigene transformation, it will also be difficult due to the high homology of the promoter sequence. Sex may lead to gene silencing

Method used

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  • Identification and application of a plant endosperm-specific expression promoter posens100
  • Identification and application of a plant endosperm-specific expression promoter posens100
  • Identification and application of a plant endosperm-specific expression promoter posens100

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1, Tissue expression pattern analysis of OsEnS100 gene

[0043] In the research, the inventor accidentally found that the expression level of the rice OsEnS100 gene is significantly different in different tissues and organs. The roots, stems, leaves, palea, palea, pistil, anthers (Stage 6-12) and endosperm (DAP 7 and DAP25) of rice plants were taken respectively, RNA was extracted, reverse transcribed into cDNA as a template, and rice ACTIN gene As an internal reference, primers for OsEnS100 and ACTIN genes were designed respectively, and the expression of OsEnS100 gene in various tissues of rice was analyzed by Quantitative Real time-PCR (qRT-PCR) method.

[0044] The qRT-PCR primers for OsEnS100 gene are:

[0045] Primer 1: 5'-TGCGAGGCGATCAGCCACAT-3' (SEQ ID NO: 3);

[0046] Primer 2: 5'-GCCAGCCAGTAGCAGACACC-3' (SEQ ID NO: 4);

[0047] The qRT-PCR primers for the ACTIN gene are:

[0048] Primer 3: 5'-GCTATGTACGTCGCCATCCA-3' (SEQ ID NO: 5);

[0049] Primer 4...

Embodiment 2

[0055] Embodiment 2, the cloning of fluorescent reporter gene EGFP and promoter pOsEnS100

[0056] The primers required for cloning the fluorescent reporter gene EGFP are:

[0057] Primer 5: 5'-ATCCTCTAGAGTCGACATGGTGAGCAAGGGCGAGGAGCTGT-3' (SEQ ID NO: 7);

[0058] Primer 6: 5'-GGCCAGTGCCAAGCTTTTACTTGTACAGCTCGTCCATGCCG-3' (SEQ ID NO: 8);

[0059] The primers required for cloning the promoter pOsEnS100 are:

[0060] Primer 7: 5'-CCATGATTACGAATTCCGTGGCATGGTGCCCGGGGGTGGGG-3' (SEQ ID NO: 9);

[0061] Primer 8: 5'-TGCTCACCATGTCGACATTTTTGTTGCAGAAAATCTTAACT-3' (SEQ ID NO: 10);

[0062] Among them, the underlined sequence in primers 5 and 8 is the restriction site of SalI; the underlined sequence AAGCTT in primer 6 is the restriction site of HindIII; the underlined sequence in primer 7 is the restriction site of EcoRI. The 10 bases on the left side of the restriction sites of primers 5, 6, 7 and 8 are backbone vector recombination fragments, which are used for homologous recombinati...

Embodiment 3

[0065] Embodiment 3, the construction of expression vector pSZYJY-06

[0066] Such as figure 2 As shown, the reporter gene EGFP sequence recovered from the PCR gel in Example 2 was connected under the action of homologous recombinase In-fusion into the vector pCAMBIA1300 double-digested with SalI and HindIII, and the colonies were picked for PCR detection. The positive colonies were sequenced, and the sequence of the EGFP reporter gene was shown as SEQ ID NO: 2. After the sequence verification was correct, the corresponding positive clone plasmid was extracted and named pSZYJY-05.

[0067] Then, under the action of homologous recombinase In-fusion, the pOsEnS100 promoter sequence recovered from the gel in Example 2 was connected into the vector pSZYJY-05 which was double-digested with EcoRI and SalI again, and colonies were picked for PCR detection. Colonies with positive PCR results were sequenced. The sequence of the pOsEnS100 promoter was shown as SEQ ID NO: 1. After the ...

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Abstract

The invention discloses a plant endosperm-specific expression promoter pOsEnS100 The identification and application of the invention belong to the field of plant biotechnology, and specifically relate to the isolation, functional identification and application of a rice endosperm-specific expression promoter. The promoter disclosed in the present invention is specifically expressed in the endosperm of plants, and is applicable to plants with endosperm in any seed, especially endosperm-type monocotyledonous plants, especially capable of driving the specific expression of exogenous genes in rice endosperm, and in plant transgenic The field has a good application prospect.

Description

technical field [0001] The present invention belongs to the field of plant biotechnology, in particular, the present invention relates to isolated DNA, which can guide the specific transcription and / or expression of nucleic acid operably linked downstream of it in plant endosperm. In addition, the present invention also relates to expression cassettes and plants containing the DNA, and to applications of the DNA. Background technique [0002] Plant gene regulation is mainly carried out at the transcriptional level, which is coordinated by a variety of cis-acting elements and trans-acting factors. The promoter is an important cis-acting element. It is a DNA sequence located in the upstream region of the 5' end of the structural gene to regulate gene transcription. It can activate RNA polymerase to accurately combine with the template DNA to ensure accurate and efficient transcription. play a key role in transcriptional regulation. According to the different characteristics ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/11
CPCC12N15/8234C07K14/415
Inventor 唐晓艳王梦龙严维陈竹锋彭小群
Owner 深圳广三系农业科技有限公司
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