CN114645017A - Function and application of specific expression promoter KT631P - Google Patents

Abstract

The invention belongs to the technical field of plant bioengineering and plant improvement genetic engineering, and particularly relates to functional identification and application of a plant flower specific expression promoter KT 631P. The invention discloses a method for regulating and controlling the expression of a heterologous nucleotide sequence in a plant body and a DNA construct. The DNA construct comprises a novel nucleotide sequence for a plant flower-specific expression promoter, and methods for preferentially expressing a heterologous nucleotide sequence in a plant floral organ using the flower-specific promoter sequences disclosed herein are provided.

Description

Function and application of specific expression promoter KT631P

Technical Field

The invention belongs to the technical field of plant bioengineering and plant improvement genetic engineering, and particularly relates to functional identification and application of a plant flower specific expression promoter KT 631P.

Background

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. The major constitutive strong promoters widely used in the agricultural biotechnology field at present are some strong constitutive promoters, such as the CaMV 35S promoter and the maize Ubiquitin-1 promoter (Battraw and Hall, 1990; Christensen et al 1992), however, when these promoters are used to induce target genes to transform rice and other crops in order to improve the quality of the crops, the improvement effect is not obvious because the time (development stage specificity) or space (tissue organ specificity) of the target gene expression cannot be well controlled, or the constitutive promoters induce too high gene expression amount to affect the growth and development of the plants, which are the 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, in the study of some metabolic processes or regulation pathways, more than two genes in the same pathway are often required to be transformed into the same strain, and a longer time is required for transforming one of the genes to obtain a transgenic plant and then transforming the other gene, or for two genes to be transformed respectively and then hybridized, so that the transformation time of multiple genes is shortened in order to improve the efficiency, recently, it has been reported that the transformation of multiple genes can be carried out simultaneously by using a new vector (Lin et al 2003; Chen et al 2006), but if the same promoter is repeatedly used in the process of multi-gene transformation, gene silencing can be caused due to high homology of the promoter sequence. Therefore, it is necessary to clone more tissue-organ-specific or specific condition-inducible promoters to overcome the above difficulties in transformation technology, so that different promoters can be selected as required to induce the desired gene to be expressed at a suitable time or space.

The tissue-specific promoter is also called organ-specific promoter, and under the driving of the promoter, the expression of the gene is often limited to certain specific organs or tissue parts, and the like, and the promoter shows the characteristics of developmental regulation and the like. The tissue-specific promoter not only can accumulate the expression product of the target gene in certain organs or tissue parts to increase the regional expression amount, but also can avoid unnecessary waste of plant nutrition. With the development of plant genetic engineering technology, the characteristic ensures that the tissue specific promoter is used as an important cis-acting element to be more widely applied in the fields of bioreactors, breeding of good varieties of transgenic plants with stress resistance characteristics such as insect resistance, disease resistance, drought resistance, hyperosmotic stress resistance and the like.

The formation and development of plant floral organs has been one of the hot problems of research. The development process of plant flowers is divided into 4 stages: flower formation induction, flower meristem formation, flower organ primordia production and flower organ maturation. The isolation of the flower-specific expression gene promoters in these processes plays an important role in controlling plant breeding and flower cultivation. Several floral organ-specific promoters have been isolated and identified in the world, for example, the PAL-valued zb8 promoter driving the expression of reporter GUS in anthers, flower buds, flower receptacles and filaments of transgenic rice [ Zhu Q, et al, 1995, Plant Mol. biol., 29:535-550], while others are specific for a particular tissue of the floral organ, such as some rice anther-specific promoters [ Tsuchiya T et al, 1994, Plant Mol. biol., 20:1189-1193], tomato pollen-specific promoter LAT52[ Twell D et al, 1989, Mol Gent, 217:240-245] and tobacco anther tapetum-specific promoter TA29[ Koltunow AM et al, 1990, Plant cell, 2: 1224] and the like.

Disclosure of Invention

The invention provides a promoter nucleotide sequence capable of driving specific transcription in plant floral organs, and a method for cloning and applying the promoter. The invention also provides methods for expressing nucleotide sequences in plants. In some embodiments, the method comprises (a) operably linking a nucleotide sequence to a polynucleotide comprising SEQ ID No:1 to produce an expression cassette; and (b) generating a transgenic plant comprising the expression cassette, whereby the nucleotide is expressed in the plant, more particularly in the floral organ of the plant. In some embodiments, the "generating" comprises transforming a plant cell with the expression cassette and regenerating a plant from the transformed plant cell.

The plant flower specific expression promoter provided by the invention contains SEQ ID No:1, and further comprises a nucleotide sequence identical to SEQ ID No:1, and can drive the specific expression of the operably linked nucleotide sequence in the floral organ of the plant.

The promoter nucleotide sequence of the embodiments can be used to isolate the corresponding sequence from other organisms, such as other plants (monocots or dicots, etc.). Based on the sequence homology between these corresponding sequences and those listed herein, techniques such as PCR, hybridization, and the like are used to identify and isolate these corresponding sequences. Accordingly, corresponding fragments isolated on the basis of their sequence similarity to the complete KT631P promoter sequence (or fragments thereof) as set forth herein are also included in the embodiments. The promoter region of the embodiments can be isolated from any plant, including, but not limited to, rice, brassica, maize, wheat, sorghum, crambe, white mustard, castor bean, sesame, cottonseed, linseed, soybean, arabidopsis, phaseolus, peanut, alfalfa, oat, rapeseed, barley, oat, Rye (Rye), millet, milo, triticale, einkorn, Spelt, emmer, flax, grasses (Gramma grass), tripsacum, sorghum, orchis, fescue, perennial wheat, sugarcane, ruscus carota, papaya, banana, safflower, oil palm, cantaloupe, apple, cucumber, dendrobium, gladiolus, chrysanthemum, liliacea, cotton, eucalyptus, sunflower, canola, sugar beet, coffee, yam, ornamental plants, pine species, and the like.

The term "promoter" as used herein refers to a DNA regulatory sequence, typically containing a TATA box, which directs RNA polymerase II to initiate transcription of a particular coding sequence at a suitable transcription initiation site. Promoters may additionally contain other recognition sequences, which are usually located upstream or 5' to the TATA box, referred to as upstream promoter elements, which can influence the rate of transcription. It will be appreciated that once the nucleotide sequence of the promoter regions disclosed herein has been identified, it is within the skill of the art to isolate and identify additional regulatory elements upstream of the particular promoter region identified herein. Thus, the promoter regions disclosed herein may additionally comprise upstream regulatory elements, such as elements responsible for tissue-and time-specific expression, elements and enhancers that regulate constitutive expression, and the like.

The term "specifically express" as used herein means that a desired gene is expressed at a specific time or in a specific tissue or organ. A "tissue-specific promoter," also known as an "organ-specific promoter," is one under which a gene is often expressed only in a particular tissue or organ. The plant floral organ-specific expression promoter as used herein refers to a promoter that specifically expresses in a plant floral organ.

In general, a promoter that drives expression of a particular gene is considered tissue or organ specific if the amount of mRNA that is the transcription product of that gene is at least 5-fold, preferably at least 10-fold, more preferably at least 100-fold, and most preferably at least 1000-fold higher in that tissue or organ than in other tissues or organs. The activity and strength of the promoter can be determined based on the content of mRNA or protein of the gene that it drives.

Also included in embodiments of the invention are DNA constructs comprising a promoter operably linked to a heterologous nucleotide sequence, the promoter comprising a sequence disclosed herein and being capable of driving expression of the heterologous nucleotide sequence in a plant cell. Embodiments of the invention also provide expression vectors, and plants or plant cells stably comprising the DNA constructs described above in their genomes. "operably linked" refers to a linkage that places a heterologous nucleotide sequence under the action of a promoter, and also to the joining of two nucleotide sequences such that the coding sequences for each DNA segment are maintained in proper reading frame. "heterologous nucleotide sequence" refers to a sequence which is not in its native state operably linked to the promoter sequence KT631P described herein, which may be homologous to a plant host or heterologous.

The KT631P floral organ specific promoter disclosed herein and variants and fragments thereof are useful in plant genetic engineering, for example in the preparation of transformed or transgenic plants to produce a phenotype of interest. "transformed plant" or "transgenic plant" refers to a plant that contains within its genome a heterologous nucleotide sequence. Usually the transformed plant or transgenic plant genome stably contains these heterologous nucleotide sequences, which can be stably inherited to the next generation. These heterologous nucleotide sequences may be present in the genome alone or together with the recombinant DNA construct. As used herein, "transgenic event" includes any cell, cell line, callus, tissue, plant part or whole plant, so long as its genotype is altered by the presence of exogenous nucleic acid, including starting hosts altered by transgenic manipulation, and progeny produced by sexual or asexual propagation of such starting hosts. As used herein, "transgenic event" does not include plants having an altered genome (chromosomal or extra-chromosomal) by traditional plant growing methods or natural events such as random crossing, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

A "transgenic event" is obtained by transforming a plant cell with an exogenous DNA construct (containing a nucleic acid expression cassette, which in turn contains a gene of interest), re-culturing the plant cell with the exogenous DNA construct inserted into its genome to obtain a plurality of plant bodies, and selecting a desired positive transgenic line based on the inserted exogenous gene. A typical phenotypic characteristic of a transgenic event is the expression of a gene of interest. At the genetic level, a "gene of interest" is a part of the genome composition of a plant. "transgenic event" also refers to progeny of a transgenic plant that contain foreign DNA resulting from crossing another plant.

"plant" as used herein includes whole plants, plant tissue organs (e.g., leaves, roots, stems, etc.), seeds, plant cells, and progeny of same. In embodiments, the term "part plant" of a transgenic plant is understood to include plant cells, protoplasts, tissues, calli, embryos of the transgenic plant or its progeny, and flowers, stems, fruits, ovules, leaves or roots of the transgenic plant or its progeny, and the like.

As used herein, "plant cell" includes, but is not limited to, seed suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametes, pollen, sporophytes, and microspores. The class of plants useful in the methods disclosed herein includes all higher plants that can be transformed, including monocots and dicots.

The promoter sequences disclosed herein can regulate expression of any heterologous nucleotide sequence in a host plant. Thus, the heterologous nucleotide sequence can be a structural gene (encoding a protein of interest) operably linked to a promoter as disclosed herein. The target gene in the embodiment includes genes involved in the regulation of signal transduction such as transcription factors, kinases, etc., and housekeeping genes such as heat shock protein genes. More specifically, the class of transgenes includes, for example, resistance genes that encode proteins that confer tolerance to abiotic stresses including drought, temperature, salt and toxins (pesticides and herbicides), among others, in plants. Or resistance genes which encode proteins which confer tolerance to biotic stresses on plants, such as fungal, viral, bacterial, insect and nematode insults, and diseases caused by such stresses. The phenotype encoding includes altering the expression of a gene in the plant, thereby altering the defense mechanism of the plant against pathogens, insects, etc., or increasing the resistance of the plant to herbicides, altering the growth and development process of the plant depending on the environment, etc. These changes can be obtained by expressing exogenous genes in plants or by increasing the expression of specific endogenous genes. Or by reducing the expression products of one or more endogenous genes, such as enzymes, transporters, cofactors, etc., in the plant, by influencing the metabolic mechanisms of the plant to obtain the corresponding phenotype.

As can be seen from the above, any desired gene can be operably linked to the promoter sequence of the embodiments and expressed in plants, preferably in plant floral organs.

The heterologous nucleotide sequence operably linked to the herein disclosed promoter for floral organ specific expression of KT631P may be an antisense sequence to some target gene according to the RNA interference technique (RNAi). "antisense nucleotide sequence" refers to a double-stranded RNA molecule that is complementary to the nucleotide sequence of a target gene. Transcription of the antisense DNA sequence prevents normal expression of the target DNA sequence when introduced into a plant cell. The RNA transcript encoded by the antisense nucleotide sequence may be complementary to and may hybridize with an endogenous mRNA transcribed from the target gene, whereby synthesis of the native protein encoded by the target gene is restricted, thereby achieving the corresponding phenotype.

The invention will now be described in further detail by means of the following detailed description, taken in conjunction with the accompanying drawings, without in any way limiting the scope of the invention.

Drawings

FIG. 1 is a T-DNA region map of expression vector pHPG. LB and RB are the left and right borders of the T-DNA, respectively; hpt represents the hygromycin resistance gene; pnos represents a promoter of the nos gene; tnos represents a terminator of the nos gene; GUS represents GUS protein gene; t35s represents the terminator of the 35s gene; HindIII and BamHI represent the restriction sites of restriction enzymes HindIII and BamHI, respectively; the specific promoter of the floral organ is the specific expression promoter of the floral organ separated and identified by the invention.

FIG. 2 shows GUS staining of tissue organs of KT631P transgenic rice. A is a blade; b is a stem; c is a root; d is the floral organ.

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 T4 DNA ligase was purchased from Promega, all methods being performed according to the methods provided by the kits. The pHPG vector used in the experiment is obtained by modifying the experiment, and the basic skeleton is derived from pCAMBIA1303 of CAMBIA company.

1. Isolation and characterization of promoter KT631P

Designing primers required for cloning promoter KT 631P:

primer 1: 5' -aagcttTTAATAGAATATTACCCCATCGTC -3'

Primer 2: 5' -ggatccGCCCACCCCCTCCTCCTCG -3'

The sequence aagctt in primer 1 is the restriction site of HindIII, and the sequence ggatcc in primer 2 is the restriction site of BamHI.

The amplification was carried out using forward and reverse primers for the promoter (wherein the sequence of the underlined part was the promoter sequence, as shown in Seq ID NO: 1) and the genomic DNA of rice (Zhonghua 11) extracted with a plant genomic DNA extraction kit (Tiangen Biochemical technology, Beijing, Ltd.) as a template, under the reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds; annealing at 55 ℃ for 30 seconds; extension at 72 ℃ for 1 min; 30 cycles; extension at 72 ℃ for 10 min. After the reaction is finished, detecting and recovering the PCR product through 1% agarose gel electrophoresis, connecting the product into pEASY-T1, screening positive clone and carrying out sequencing verification, wherein the result shows that: the amplified sequence was the expected KT631P promoter sequence.

2. Construction of expression vectors

The plasmid inserted with KT631P promoter sequence is cut with HindIII and BamHI enzyme, and connected to HindIII and BamHI enzyme cut vector pHPG, colony PCR result positive is selected for sequencing, and after correct sequencing, corresponding positive cloning plasmid is extracted and named pHPG-KT 631P.

Primers required for colony PCR detection:

primer 3: 5'-TCTCCGCTCATGACGATAAT-3'

And (4) primer: 5'-GACGTAACATGGTGAAGGGG-3'

Primer 3 and primer 4 are primers on pHPG carrier, located at two sides of cloned promoter segment, the length of amplified segment is about that of promoter, bacteria liquid is used as template, amplification detection is carried out, 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 1 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; pnos represents a promoter of the nos gene; tnos represents a terminator of the nos gene; GUS represents GUS protein gene; T35S represents the terminator of the 35S gene; HindIII and BamHI respectively represent the restriction sites for cutting HindIII and BamHI; the flower specific promoter is the flower organ specific expression promoter cloned by PCR.

3. Agrobacterium co-transformation

Plasmid pHPG-KT631P is transferred into Agrobacterium AGL0 strain by means of heat shock process, rice is co-transformed by means of Agrobacterium mediating process, and Arabidopsis thaliana plant is transformed by means of Agrobacterium inflorescence infecting process.

4. Functional identification

Separating each tissue organ from the transgenic plant, detecting GUS activity, placing each tissue organ in an EP tube containing GUS staining buffer, placing the tube in an incubator at 37 ℃ for incubation overnight, and then decoloring and storing the tube in absolute ethyl alcohol at room temperature.

4.1 tissue organ staining of transgenic Rice seedlings

GUS staining experiments are carried out on the hygromycin-resistant rice callus obtained by transforming the KT631P promoter, and the result is shown in FIG. 2B, and the resistant callus has basically no GUS gene expression. The tissue organs, such as leaves, stems, roots and flowers, of the rice KT631P transgenic rice seedling are respectively subjected to GUS staining, and the result is shown in figure 2, the GUS gene only has strong expression in the flower organ (figure 2D) of a plant, shows strong blue, and basically does not detect the expression of the GUS gene in other tissue organs.

Sequence listing

<110> Beijing unknown KaiTuo crop design center, Inc

<120> function of specific expression promoter KT631P and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1000

<212> DNA

<213> Rice (Oryza sativa L.ssp.japonica)

<400> 1

ttaatagaat attaccccat cgtccatcca gttaataaat aaataaaaag aatgtatttt 60

tagcgtttcc agatgcatag tcagaaataa actatttttt ggacagagat atataaggat 120

tgtaagataa gtcctcctat gtgttgtagg tccatgtttc atatatacag cttaaatata 180

tatggttttt tttctctttt gtacgcgtag caattttagc tttgtctgaa ctgaagcaac 240

tatcaaaagg caattagtag acgactaact aactagtttc tccctgcata tgtgaacacc 300

tagctagcta cccttcgtgc ccaacagccc ataccttacc atgcatctgc atgcagcagc 360

agacagcatc atatcgatga tccatccatc tctggatcga tccagttgca aatgcgaatt 420

aggcgaagca ataatcaaag ctgagatatg atcgagccga gatagatcga gatcgatcat 480

ctgttaggta atcactggtt aggtcagtgt gtcctgaaat taatgcattg ccactgaaag 540

aaagaaaatg ctcgacgaac tgtgcatcca tcgatccatg gctgtacagt acatgcgcgc 600

gtgcgtgtgc atcatcagcg atgcatgcga acgagcatga catgatcagc cgctgagctc 660

atcatcgctc gagatcacgg cctaattagc gagttatggc gtgccacatg gcatctggca 720

cgcgaaccaa gaaaagtctc tctctcctct cctctcttct tcatcaacac acactggttg 780

accaaccagc tagctgatcg agttcgttgt ttaccatcct attaataccc cttcctctct 840

ctcttctccc ctcctgaatt tctctcttgg tagaggtaga attgatctcc tttcttgttc 900

ttcttcgagt tcttcttaat tggagttctt ggagcagcat cggtagtcgt cgtcttcgtc 960

gatccagcat ctcaaggaat tcgaggagga gggggtgggc 1000

<210> 2

<211> 915

<212> DNA

<213> Rice (Oryza sativa L.ssp.japonica)

<400> 2

atgagcggga tgaattcgct gagcatggtg gaggcgaggc tgccgccggg gttcaggttc 60

cacccgcgag acgacgagct cgtgctggac tacctggaaa ggaagctcct cgacggcggc 120

gtgggcggcg ccgcggcggc ggcggcggcg gtcaccatct acggctgccc ggtgatggtc 180

gacgtcgatc tcaacaagtg cgagccatgg gaccttcctg agatcgcttg cgttggtggc 240

aaggagtggt acttctatag ccttagggat aggaagtatg caactggcca acgaacaaat 300

agagcaaccg aatcgggcta ctggaaggcc acaggaaaag atcgcccaat aagccggaaa 360

ggattgctcg tcggtatgcg aaaaaccctg gtgttctaca aaggtagagc ccctaagggg 420

aagaagaccg agtgggtcat gcatgaattc cgcaaagaag gacaagggga tccgatgaag 480

ttgcctctca aggaggactg ggtcttgtgt agagtcttct acaagagtag gacaaccatt 540

gccaagctgc caacggaggg tagctacaac aatattgaca gtgtggccac aacttcactg 600

cctcccctca ctgacaacta cattgcattt gatcagcctg gttcaatgca aaacctagag 660

ggttatgagc aagtgccctg cttctccaat aatccctctc aacagccatc gtcgtcgatg 720

aatgttccgt tgacatcggc catggttgat caagagcaaa acaatatggg tagggcgatc 780

aaggatgtgc tgagccaatt caccaagttt gaaggcaatg tgaagaggga ggcccttcaa 840

agtaattttt cccaggatgg atttgattac ttggctgaga gtggcttcac acaaatgtgg 900

aactcactca gttga 915

Claims (16)

1. A transformed plant cell comprising a promoter nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOs: 1, or the complementary strand thereof.

2. The transformed plant cell of claim 1, wherein the promoter nucleic acid molecule is operably linked to a heterologous nucleotide sequence.

3. The transformed plant cell of claim 2, wherein the heterologous nucleotide sequence is comprised in a sequence not naturally operably linked to said promoter sequence, which may be homologous or heterologous to the plant host.

4. The transformed plant cell of claim 2, wherein the heterologous nucleotide sequence encodes a gene product that confers to the plant resistance to herbicides, salts, cold, drought, pathogens, or insects, or that regulates plant growth and development, male sterility.

5. The transformed plant cell of claim 2, wherein the heterologous nucleotide sequence is a homologous sequence to a target gene that inhibits expression of a gene endogenous or exogenous to the plant by means of RNA interference techniques.

6. The transformed plant cell of claim 1, wherein said transformed plant cell is from a monocot.

7. The transformed plant cell of claim 6, wherein said monocot plant cell is from a gramineae plant, preferably rice.

8. The transformed plant cell of claim 1, wherein said transgenic plant cell is from a dicot.

9. The transformed plant cell of claim 8, wherein said dicotyledonous plant cell is from the family Brassicaceae, preferably Arabidopsis thaliana.

10. A method of expressing a nucleotide sequence in a plant, said method comprising introducing into a plant a DNA construct comprising a promoter and a heterologous nucleotide sequence of interest operably linked to said promoter, wherein said promoter comprises a nucleotide sequence selected from the group consisting of:

a) comprises the amino acid sequence shown in SEQ ID NO:1, a promoter nucleotide sequence set forth in 1;

b) comprises a sequence similar to that of SEQ ID NO:1, wherein said promoter nucleotide sequence is capable of initiating specific transcription and expression of a heterologous nucleotide sequence in a floral organ of a plant.

11. A nucleic acid molecule comprising a sequence selected from SEQ ID NOs: 1.

12. A vector comprising the nucleic acid molecule of claim 11.

13. A cell comprising the vector of claim 12.

14. The cell of claim 13, wherein the cell comprises a bacterial cell, a mammalian cell, an insect cell, a plant cell, or a fungal cell.

15. The cell of claim 14, wherein the bacterial cell is from agrobacterium tumefaciens or escherichia coli.

16. The cell of claim 13, wherein the plant cell is a rice cell.

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