CN107338248B - Seed specific expression system and application thereof - Google Patents

Abstract

The present invention provides an isolated DNA having the sequence of SEQ ID NO: 1 or a similar sequence. The DNA is capable of directing the specific transcription and/or expression of a nucleic acid operably linked downstream thereof in a plant seed. The invention also provides an expression cassette and a transgenic plant containing the DNA.

Description

Seed specific expression system and application thereof

Technical Field

The present invention is in the field of plant transgenic breeding and, in particular, the present invention relates to isolated DNA capable of directing the specific transcription and/or expression in plant seeds of a nucleic acid operably linked downstream thereof. In addition, the invention also relates to an expression cassette, a plant and the like containing the DNA, and relates to application of the DNA.

Background

Although the genomic sequence of many plants (e.g., rice, particularly Oryza sativa. japonica, etc.) has been revealed (e.g., see GenBank accession No. AP003843.3, GenBank accession No. AP004670.4, etc.), the function of a number of genomic sequences remains unknown. In addition, U.S. patent application No. US2006075523a1 discloses polypeptides and polynucleotides related to abiotic stress responses from rice and other plants, wherein the polynucleotide sequences SEQ ID NO: 15442 is up to 2000 nucleotides in length and is buried in many of the sequences disclosed in this document without being assigned a specific function; while US patent US7365185B2 discloses genomic promoter sequences from rice and other plants, but wherein the polynucleotide sequence SEQ ID NO: 68961 is up to 2760 nucleotides in length and is buried in many of the sequences disclosed in this document and not assigned a specific function, let alone it is not known whether it is specific for the site of expression in a plant.

The foreign DNA sequence initiates expression in the plant host by linking to a specific promoter, the choice of which determines the timing and location of expression of the gene. At present, the constitutive strong promoters widely used in the field of agricultural biotechnology are mainly some constitutive strong promoters, such as CaMV35S promoter and maize Ubiquitin-1 promoter, however, when the promoters are used to induce target genes to transform crops such as rice and the like in order to improve the quality of the crops, the improvement effect is not obvious because the time (development stage specificity) or space (tissue and organ specificity) of the expression of the target genes cannot be well controlled, or the constitutive promoters induce too high expression of the genes to affect the growth and development of the plants, which are obstacles encountered when the constitutive strong promoters are used to combine with functional genes to improve the quality of the crops at present.

In addition, when some metabolic processes or regulation pathways are researched, more than two genes in the same pathway are often required to be transformed into the same strain, and a long time is required for transforming one of the genes to obtain a transgenic plant and then transforming the other gene, or for hybridizing two genes after the two genes are transformed respectively, so that the transformation time of multiple genes is shortened in order to improve efficiency, recently, it has been reported that the transformation of multiple genes can be carried out simultaneously by using a new vector, but if the same promoter is repeatedly used in the transformation of multiple genes, gene silencing can be caused due to high homology of the promoter sequence.

The seed specific expression system can be used for researching the influence of genes or regulatory sequences on important agronomic characters such as growth and development or yield of seeds, and the like, obtains a new seed specific expression promoter in practical production application, and has important significance for production application.

Therefore, the inventors have studied for a long time and surprisingly found a novel seed-specific promoter, which can provide more selectivity for the future development of agricultural genetic engineering.

Summary of The Invention

The present invention provides novel isolated DNA that functions as a seed-specific expression promoter. In addition, the invention also relates to an expression cassette and a transgenic plant containing the DNA, and relates to application of the DNA.

In particular, in a first aspect, the present invention provides an isolated DNA capable of directing the specific transcription and/or expression in a plant seed of a nucleic acid operably linked downstream thereof, the sequence of said isolated DNA being selected from the group consisting of:

(a) has the sequence shown in SEQ ID NO: 1;

(b) a DNA sequence capable of hybridizing to the DNA of sequence (a) under stringent conditions;

(c) a DNA sequence having at least 90% (preferably at least 95%) sequence identity to the sequence of (a);

(d) comprises the amino acid sequence of SEQ ID NO: 1 of at least 150 (preferably at least 200) contiguous nucleotides; and

(e) a DNA sequence complementary to any one of the sequences of (a) to (d).

Preferably the DNA of the first aspect of the invention has a sequence length of less than 2000 nucleotides, preferably less than 1500 nucleotides, more preferably less than 1200 nucleotides.

Preferably, the DNA of the first aspect of the invention is capable of directing the specific transcription and/or expression of a nucleic acid operably linked downstream thereof in a plant seed.

In a particular embodiment of the invention, the DNA sequence of the first aspect of the invention is as set forth in SEQ ID NO: 1 is shown.

In a second aspect, the present invention provides an expression cassette comprising:

(a) the DNA of the first aspect of the present invention; and

(b) a nucleic acid operably linked downstream of the DNA of the first aspect of the invention. Preferably the expression cassette of the second aspect of the invention, wherein the nucleic acid is capable of conferring to a plant a trait selected from the group consisting of:

(a) an increase in the yield of a protein encoded by said nucleic acid;

(b) an increase in the amount of inhibitory RNA encoded by the nucleic acid;

(c) an increase in the resistance to plant diseases and insect pests, particularly against diseases and insect pests occurring in plant seeds;

(d) improvement in nutritional quality, including enhancement of the content of nutrients naturally present in the plant and the impartation of nutrients not naturally present in the plant; and

(e) increased plant yield, particularly in economic parts of the plant such as seeds.

In a third aspect, the present invention provides a transgenic plant, plant seed, plant tissue or plant cell transformed with an expression cassette of the second aspect of the invention, preferably wherein the DNA of the first aspect of the invention is capable of directing the specific transcription and/or expression in a plant seed of a nucleic acid operably linked downstream thereof.

Preferably, the plant, plant seed, plant tissue or plant cell of the third aspect of the invention, wherein the plant is a monocotyledonous or dicotyledonous plant, such as maize, rice, wheat, barley, sorghum, soybean, oilseed rape, cotton, tomato, potato, sugarcane, sugar beet, tobacco, oilseed rape or Arabidopsis thaliana.

In a fourth aspect, the present invention provides a method of producing a plant of the third aspect of the invention, comprising:

(1) constructing an expression cassette of the second aspect of the invention;

(2) introducing the expression cassette obtained in step (1) into a plant cell;

(3) regenerating a transgenic plant; and

(4) selecting a transgenic plant wherein the DNA of the first aspect of the invention is capable of directing the specific transcription and/or expression in a plant seed of a nucleic acid operably linked downstream thereof; and is

(5) Optionally, propagating the plant obtained in step (4) to obtain progeny.

In a fifth aspect, the present invention also provides a method for conferring traits to plants, comprising

(1) Selecting for a nucleic acid capable of conferring a plant trait:

(2) constructing an expression cassette of the second aspect of the present invention using the nucleic acid obtained in step (1);

(3) introducing the expression cassette obtained in step (2) into a plant cell;

(4) regenerating a transgenic plant; and

(5) selecting a transgenic plant conferring a plant trait; and is

(6) Optionally, propagating the plant obtained in step (5) to obtain progeny,

wherein the trait is selected from the group consisting of:

(a) an increase in the yield of a protein encoded by said nucleic acid;

(b) an increase in the amount of inhibitory RNA encoded by the nucleic acid;

(c) the improvement of the capability of resisting plant diseases and insect pests, particularly the capability of resisting the plant diseases and insect pests generated in plant seeds;

(d) improvement in nutritional quality, including enhancement of the content of nutrients naturally present in the plant and the impartation of nutrients not naturally present in the plant; and

(e) increased plant yield, particularly in economic parts of plants.

Drawings

FIG. 1 is a plant expression vector of the Arabidopsis AT4G28520 gene promoter-driven GFP gene constructed in the present invention, wherein LB and RB are the left and right borders of T-DNA, respectively; hpt represents the hygromycin resistance gene; cam35SP represents the promoter of CamV35S gene; NOS represents a terminator of the NOS gene; GFP represents a GFP protein (green fluorescent protein) gene; 3A represents a terminator of the 35S gene; 520P is the AT4G28520 promoter obtained in example 1.

FIG. 2 is the observation of the GFP gene driven by the AT4G28520 promoter of the present invention in transgenic Arabidopsis seeds. The left image is the image of transgenic arabidopsis seeds of generation T1 in white transmitted light, and the right image is the image of transgenic arabidopsis seeds of generation T1 in ultraviolet excited light.

Detailed Description

In a first aspect, the present invention provides an isolated DNA which is capable of directing the specific transcription and/or expression in a plant seed of a nucleic acid operably linked downstream thereof, in particular which is capable of directing the specific transcription and/or expression in a plant seed of a nucleic acid operably linked downstream thereof.

In this context, such isolated DNA may also be referred to as a seed-specific promoter, belonging to cis-acting elements. Under the drive of the seed-specific promoter, the expression of the gene downstream of and operably linked to the seed-specific promoter is often limited to the seed organ or tissue site thereof, and exhibits developmental regulation and other properties. The seed-specific promoter not only enables the expression product of the target gene to be accumulated mainly in the plant seed or its tissue site, for example, the amount of downstream nucleic acid mRNA produced in the seed or its tissue is 5 times, 10 times, 100 times, 200 times, or even 1000 times that of other plant body sites, increasing the regional expression amount, but also can avoid unnecessary waste of plant nutrition. In a specific embodiment of the present invention, it is directly determined by visual staining that the seed-specific promoter of the present invention directs transcription and/or expression in plant seeds and that this regulation persists throughout the plant development stage, whereas in other parts of the plant there is no transcription and/or expression that is clearly distinguishable to the naked eye.

In this context, the term "operably linked" refers to a linkage such that transcription and/or expression of a nucleic acid linked downstream is initiated and regulated by the seed-specific promoter of the invention. Typically, the operably linked nucleic acids are contiguous. The downstream nucleic acid can be linked downstream of the seed-specific promoter with a small spacing of nucleotides, under the control of which it is regulated, while maintaining the reading frame.

As used herein, the term "isolated" DNA means that the DNA has been isolated from its natural environment (e.g., the genome of a plant cell), or has been chemically synthesized or recombinantly expressed, and that the DNA is free or substantially free of other nucleic acids, cells, and culture media from which it is derived. The isolated DNA of the present invention has a shorter nucleic acid sequence, preferably a sequence length of less than 2000 nucleotides, preferably less than 1500 nucleotides, more preferably less than 1200 nucleotides. In a specific embodiment of the invention, the isolated DNA of the invention is SEQ ID NO: 1, or a fragment thereof.

The isolated DNA of the present invention also includes a DNA having the sequence of SEQ ID NO: 1, i.e. a DNA having the sequence shown in SEQ ID NO: 1, which is still capable of directing the specific transcription and/or expression in plant seeds of a nucleic acid operably linked downstream thereof, of the variant sequence of SEQ ID NO: 1, or a fragment thereof. Variant sequences include sequences that are capable of hybridizing to a polypeptide having SEQ ID NO: 1, or a DNA sequence which hybridizes to the DNA of the sequence shown in 1. "stringent conditions" as used herein are well known and include, for example, hybridization at 60 ℃ for 12 to 16 hours in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH6.4) and 1mM EDTA, followed by washing with a washing solution containing 0.1SDS, and 0.1% SSC at 65 ℃ for 15 to 60 minutes.

Variant sequences also include sequences identical to SEQ ID NO: 1, or a DNA sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. The percentage of sequence identity can be obtained by well-known Bioinformatics algorithms, including the Myers and Miller algorithms (Bioinformatics, 4 (1): 11-17, 1988), Needleman-Wunsch global alignment (J.mol.biol., 48 (3): 443-53, 1970), Smith-Waterman local alignment (J.mol.biol., 147: 195-197, 1981), Pearson and Lipman similarity search (PNAS, 85 (8): 2444: (2448, 1988), Karlin and Altschul algorithms (Altschul et al, J.mol.biol., 215 (3): 403: 410, 1990; PNAS, 90: 5873-5877, 1993). This is familiar to the person skilled in the art.

Variant sequences also include SEQ ID NOs: 1, such as SEQ ID NO: 1 of at least 150, 200, 300, 500, 800, up to 999 contiguous nucleotides.

In a second aspect, the present invention provides an expression cassette comprising:

(a) the DNA of the first aspect of the present invention; and

(b) a nucleic acid operably linked downstream of the DNA of the first aspect of the invention.

The expression cassette of the present invention comprises the DNA of the first aspect of the present invention, a nucleic acid operably linked downstream of the DNA of the first aspect of the present invention, and optionally a transcriptional and translational termination region (e.g., a transcription termination element or polyadenylation signal) in the 5 '-3' direction of transcription. The expression cassettes of the invention may also include an origin of replication required for replication in bacteria (e.g., the ORI region from pBR322 or the P15A ORI), elements required for Agrobacterium T-DNA transfer (e.g., the left and/or right borders of T-DNA). Other components that may be included in an expression cassette of the invention include enhancers, introns, multiple cloning sites, operators, repressor binding sites, transcription factor binding sites, and the like. Exemplary enhancers include enhancer elements from the CaMV35S promoter, octopine synthase gene, rice actin I gene, maize alcohol dehydrogenase gene, maize dwarf I gene, TMV omega element, and promoters of yeast. Viral leaders may also be employed as a long-range with enhancer utility, such as leaders from Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV), among others. Exemplary plant introns include introns from Adh 1, bronze 1, actin 2, and sucrose synthase introns.

Herein, the nucleic acid operably linked downstream of the DNA of the first aspect of the invention encodes a polynucleotide of a heterologous protein, a polynucleotide encoding a fusion protein, an antisense sequence, a polynucleotide encoding a dsRNA sequence, and the like. Preferably a nucleic acid capable of conferring a beneficial trait in a plant (particularly a plant seed), said trait comprising:

(a) an increase in the yield of a protein encoded by said nucleic acid;

(b) an increase in the amount of inhibitory RNA encoded by the nucleic acid;

(c) the improvement of the capability of resisting plant diseases and insect pests, particularly the capability of resisting the plant diseases and insect pests generated in plant seeds;

(d) improvement in nutritional quality, including enhancement of the content of nutrients naturally present in the plant and the impartation of nutrients not naturally present in the plant; and

(e) increased plant yield, particularly in economic parts of plants.

Accordingly, the nucleic acid operably linked downstream of the DNA of the first aspect of the invention may encode a gene for a protein, such as an insect resistance gene, a bacterial disease resistance gene, a fungal disease resistance gene, a viral disease resistance gene, a nematode disease resistance gene, a herbicide resistance gene, a gene affecting seed composition or quality, a nutrient utilization gene, a mycotoxin reduction gene, a male sterility gene, a selectable marker gene, a screenable marker gene, a fluorescent marker gene, a negative selectable marker gene, a positive selectable marker gene, a gene affecting plant agronomic characteristics (e.g., yield), an environmental stress resistance gene (e.g., a gene conferring resistance or tolerance to drought, heat, cold, freezing, excessive moisture, salt stress, or oxidative stress), a gene improving starch characteristics or quantity, oil quantity or quality, amino acid or protein composition, and so on. In addition, the nucleic acid operably linked downstream of the DNA of the first aspect of the invention may also encode a dsRNA, an antisense RNA, a siRNA, a miRNA which is complementary to a target DNA (e.g., a plant promoter whose expression is to be down-regulated, a plant gene necessary for survival, growth or reproduction of a parasitic plant organism) and which results in down-regulation of expression of these polynucleotides.

The expression cassettes of the invention may be inserted into plasmids, cosmids, yeast artificial chromosomes, bacterial artificial chromosomes or any other vector suitable for transformation into a host cell. Preferred host cells are bacterial cells, in particular for cloning or storing polynucleotides, or for transforming plant cells, such as E.coli, Agrobacterium tumefaciens and Agrobacterium rhizogenes. When the host cell is a plant cell, the expression cassette or vector may be inserted into the genome of the transformed plant cell. The insertions may be localized or random insertions. Preferably, the insertion is effected by e.g. homologous recombination. In addition, the expression cassette or vector may be maintained extrachromosomally. The expression cassette or vector of the invention may be present in the nucleus, chloroplast, mitochondria and/or plastid of a plant cell. Preferably, the expression cassette or vector of the invention is inserted into the chromosomal DNA of the plant cell nucleus.

In a third aspect, the invention provides a transgenic plant, plant seed, plant tissue or plant cell transformed with an expression cassette of the second aspect of the invention.

The specific embodiments of the present invention have demonstrated the plant utility of the seed-specific promoters of the present invention through efficient application in two representative monocots and dicots, and thus the seed-specific promoters of the present invention can be used to transform any plant species, including monocots and dicots, such as plants from the genera: medicago, Lycopersicon, Brassica, Cucumis, Solanum, Juglans, Gossypium, Malus, Vitis, Gomphrena, Populus, Fragaria, Arabidopsis, Picea, Capsicum, Chenopodium, Chrysanthemum, Pharbitidis, Pinus, Pisum, Oryza, Zea, Triticum, triticale, Secale, Lolium, Hordeum, Glycine, Taxus, kalanchum, beta, Helianthus, Nicotiana, Cucurbita, Rosa, Fragaria, Lotus, Ardisia, Plantago, Trigonella, vigna, Citrus, Linum, Geranium, Manihot, Trigonella, Raphanus, Sinapis, Atropa, Gymnolia, Hyoscyamus, Nicandra, petunia, Digitalis, Helicoverum, Asparagus, Crataegus, Narcica, Nasturtia, Pelargonium, Narcissus, Narcina, Pelargonium, Narcina, Narcissus, Pelargonium, Na, Panicum, pennisetum, ranunculus, senecio, tubaeus, coeruleus, phaseolus, avena and allium, preferred plants include maize, rice, wheat, barley, sorghum, soybean, oilseed rape, cotton, tomato, potato, sugar cane, sugar beet, tobacco or arabidopsis, more preferably rice.

In a fourth aspect, the present invention provides a method of producing a plant of the third aspect of the invention, comprising:

(1) constructing an expression cassette of the second aspect of the invention;

(2) introducing the expression cassette obtained in step (1) into a plant cell;

(3) regenerating a transgenic plant; and

(4) selecting a transgenic plant wherein the DNA of the first aspect of the invention is capable of directing the specific transcription and/or expression in a plant seed of a nucleic acid operably linked downstream thereof; and is

(5) Optionally, propagating the plant obtained in step (4) to obtain progeny.

The transgenic plants of the invention are prepared using transformation methods known to those skilled in the art of plant biotechnology. Any method can be used to transform the recombinant expression vector into a plant cell to produce a transgenic plant of the invention. The transformation methods may include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, and microinjection, among others. In a specific embodiment of the invention, Agrobacterium-based transformation techniques are used (see Horsch RB et al (1985) Science 225: 1229; WhiteFF, Vectors for Gene Transfer in high Plants, Transgenic Plants, Vol.1, Engineering and Ultilization, Academic Press, 1993, pp.15-38; Jenes B et al technical ques for Gene Transfer, Transgenic Plants, Vol.1, Engineering and Ultilization, Academic Press, 1993, pp.128-143, et al). Agrobacterium strains (e.g., Agrobacterium tumefaciens or Agrobacterium rhizogenes) contain plasmids (Ti or Ri plasmids) and T-DNA elements, which are transferred to plants after transfection with Agrobacterium, and the T-DNA is integrated into the genome of the plant cells. The T-DNA may be located on a Ri-plasmid or a Ti-plasmid or may be contained independently in a so-called binary vector. Agrobacterium-mediated transformation methods are described, for example, in. Agrobacterium-mediated transformation is most suitable for dicotyledonous plants, but is also suitable for monocotyledonous plants. Transformation of plants by agrobacterium is described, for example. Transformation can result in transient or stable transformation and expression. Although the nucleotide sequence of the present invention may be inserted into any plant and plant cell falling within these broad categories, it is particularly suitable for use in crop plant cells.

In a fifth aspect, the present invention also provides a method for conferring traits to plants, comprising

(1) Selecting for a nucleic acid capable of conferring a plant trait:

(2) constructing an expression cassette of the second aspect of the present invention using the nucleic acid obtained in step (1);

(3) introducing the expression cassette obtained in step (2) into a plant cell;

(4) regenerating a transgenic plant; and

(5) selecting a transgenic plant conferring a plant trait; and is

(6) Optionally, propagating the plant obtained in step (5) to obtain progeny,

wherein the trait is selected from the group consisting of:

(a) an increase in the yield of a protein encoded by said nucleic acid;

(b) an increase in the amount of inhibitory RNA encoded by the nucleic acid;

(c) improvement of stress resistance (including drought resistance, cold resistance, high temperature resistance, and salt tolerance);

(d) the improvement of the capability of resisting plant diseases and insect pests, particularly the capability of resisting the plant diseases and insect pests generated in plant seeds;

(e) improvement in nutritional quality, including enhancement of the content of nutrients naturally present in the plant and the impartation of nutrients not naturally present in the plant; and

(f) increased plant yield, particularly in economic parts of plants.

Herein, "selection" may be field or greenhouse selection, and may also be genetic selection using a genetic marker. In this context, "proliferation" may be sexual or asexual. In sexual reproduction, it is clear that additional crossing steps can be performed and progeny that inherit the transgenic trait selected.

The present invention incorporates publications which are intended to describe the invention more clearly and which are incorporated herein by reference in their entirety as if reproduced in their entirety.

For the sake of understanding, the present invention will be described in detail below by way of specific examples. It is to be expressly understood that the description is illustrative only and is not intended as a definition of the limits of the invention. Many variations and modifications of the present invention will be apparent to those skilled in the art in light of the teachings of this specification.

Detailed Description

The methods used in the following examples are conventional methods unless otherwise specified, the primers used were synthesized by Shanghai Yingjun Biotechnology, the sequencing was performed by Beijing Huada gene, the PCR kit and the endonuclease used in the vector construction process were purchased from Baoji bioengineering Co., Ltd, the pEASY-T1 ligation kit was purchased from Beijing Quanjin Biotechnology, and the T4DNA ligase was purchased from Promega, all methods being performed according to the method recommended by the kit supplier. The vector pCAMBIA1303 used in the experiments was purchased from CAMBIA.

Example 1 isolation and characterization of the promoter AT4G28520

Primers required for cloning the promoter AT4G28520 were designed as follows:

primer 1: 5'-AATCTCgAgTGGTCTGCCATGGGACGT-3' (SEQ ID NO:2)

Primer 2: 5'-CGCATCGATCATTTTCTTTTTGTTTG-3' (SEQ ID NO:3)

The sequence ctcgag in primer 1 is the restriction site of Xho I, and the sequence atcgat in primer 2 is the restriction site of ClaI.

The amplification is carried out by using forward and reverse primers of a promoter (wherein the sequence of an underlined part is a promoter sequence) and arabidopsis thaliana (Columbia) genomic DNA extracted by a plant genomic DNA extraction kit (Tiangen Biochemical technology, Beijing) Co., Ltd.) as a template, and the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds; annealing at 55 ℃ for 30 seconds; extension at 72 ℃ for 2 min; 30 cycles; extension at 72 ℃ for 10 min. After the reaction is finished, the PCR product is detected and recovered through 1% agarose gel electrophoresis, the product is connected with a pEASY-T2 vector, and positive clone is screened and sequenced for verification. The result shows that the amplified sequence is the expected AT4G28520 promoter sequence, and the sequence is shown as SEQ ID NO: 1 is shown.

Example 2 construction of expression vectors

The pEASY-T2 plasmid, into which the AT4G28520 promoter sequence has been inserted by sequencing verification, is cut by Xho I and ClaI, and is ligated into pHPG, which is a vector modified from pCAMBIA1300, and has a GFP gene linked to the back of its multiple cloning site. And selecting colonies with positive PCR results for PCR detection, selecting the colonies with positive PCR results for sequencing, and extracting corresponding positive cloning plasmids which are named as 520GFP after sequencing verification is correct. Wherein, the primers required by colony PCR detection are pHPG carrier primers which are positioned at two sides of the cloned promoter fragment, the amplified fragment is about the length of the promoter, the bacterial liquid is used as a template, the amplification detection is carried out, and the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds; annealing at 55 ℃ for 30 seconds; extension at 72 ℃ for 2 min; 34 cycles of treatment; extension at 72 ℃ for 10 min.

The map of the T-DNA region of the constructed expression vector is shown in FIG. 1, in which: LB and RB are the left and right borders of the T-DNA, respectively; hpt represents the hygromycin resistance gene; cam35SP represents the promoter of CamV35S gene; NOS represents a terminator of the NOS gene; GFP represents a GFP protein (green fluorescent protein) gene; 3A represents a terminator of the 35S gene; 520P is the AT4G28520 promoter obtained in example 1.

Example 3 plant transformation and functional characterization

The plasmid 520GFP is transferred into an agrobacterium AGL0 strain by a heat shock method, arabidopsis thaliana is infected by an agrobacterium dipping method, the arabidopsis thaliana is cultured for 2-3 days in the dark, and then the arabidopsis thaliana is cultured normally until seeds are harvested.

Selecting transgenic positive plants, and carrying out expression analysis of the promoter. As a result, as shown in the right panel of FIG. 2, under the ultraviolet excitation light, the seeds of the transgenic positive plants emit green fluorescence, and no green fluorescence is detected in any other tissues and organs. Thus, the AT4G28520 promoter provided by the invention is a seed-specific expression promoter.

The invention also verifies the expression characteristics of the AT4G28520 promoter in crops such as rape, rice and the like, and the result shows that the promoter also has the characteristic of seed specific expression in other crops like in Arabidopsis.

SEQUENCE LISTING

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<120> seed specific expression system and application thereof

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caaaacaaaa tgactgaaaa tgggaggagt agaagcagag taccatctga agatcacggc 180

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gcaccataga gaccaagccc accaataatc ccaaccttaa gcaacgtcga aatggcacca 660

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ccgagcagaa aactctcacc cacctccgaa attcacgtct tcactaaaat tttcgaaagg 1020

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ggtttaagct cagacttcta ttggagtaaa tgggacggtg tcacattttc cgttttggaa 1200

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Claims (5)

1. A promoter, the sequence of which is as shown in SEQ ID NO: 1 is shown.

2. An expression cassette, comprising:

(a) the promoter of claim 1; and

(b) a nucleic acid operably linked downstream of the promoter of claim 1.

3. The expression cassette of claim 2, wherein the nucleic acid is capable of conferring to a plant a trait selected from the group consisting of:

(a) the capability of resisting plant diseases and insect pests is improved;

(b) improvement in nutritional quality, including enhancement of the content of nutrients naturally present in the plant and the impartation of nutrients not naturally present in the plant; and

(c) an increase in plant yield, wherein said increase in plant yield is an increase in yield of an economic part of a plant.

4. A method of producing a plant, comprising:

(1) constructing an expression cassette according to any one of claims 2 to 3;

(2) introducing the expression cassette obtained in step (1) into a plant cell;

(3) regenerating a transgenic plant; and

(4) selecting a transgenic plant, wherein the promoter of claim 1 is capable of directing the specific transcription and/or expression of a nucleic acid operably linked downstream thereof in a plant seed; and is

(5) Optionally, propagating the plant obtained in step (4) to obtain progeny,

wherein the plant is Arabidopsis thaliana.

5. A method of imparting a trait to a plant comprising

(1) Selecting for a nucleic acid capable of conferring a plant trait:

(2) constructing an expression cassette according to any one of claims 2 to 3 using the nucleic acid obtained in step (1);

(3) introducing the expression cassette obtained in step (2) into a plant cell;

(4) regenerating a transgenic plant; and

(5) selecting a transgenic plant conferring a plant trait; and is

(6) Optionally, propagating the plant obtained in step (5) to obtain progeny,

wherein the trait is selected from the group consisting of:

(a) the capability of resisting plant diseases and insect pests is improved;

(b) improvement in nutritional quality, including enhancement of the content of nutrients naturally present in the plant and the impartation of nutrients not naturally present in the plant; and

(c) an increase in plant yield, wherein said increase in plant yield is an increase in yield of an economic part of a plant;

wherein the plant is Arabidopsis thaliana.

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