CN100587071C - A kind of plant floral organ-specific promoter and application thereof - Google Patents

Abstract

The invention discloses a plant flower organ specific promoter and application thereof. The nucleotide sequence of the promoter is: (1), the nucleotide sequence described in Sequence 1 in the sequence listing; or (2), the nucleotide sequence complementary to the nucleotide sequence described in (1); Or (3), a nucleotide sequence having 60% or more homology with the nucleotide sequence described in (1) or (2); or (4), with (1), (2) or (3) The nucleotide sequence is a nucleotide sequence capable of hybridizing under stringent hybridization conditions. The promoter of the present invention is a flower organ-specific promoter with stability, strong driving force and high specificity. Utilizing the promoter of the present invention can cultivate new varieties of flower crops different from the recipient wild type in flower shape, flower color, flower fragrance and fertility, cultivate male sterile crop varieties and cultivate landscaping that eliminates or greatly reduces "flying catkins" plants etc.

Description

A kind of plant floral organ-specific promoter and application thereof

technical field

The invention relates to a plant promoter, in particular to a plant flower organ-specific promoter and application thereof.

Background technique

Promoter is the most important factor among gene expression regulators, which basically determines whether, when and where a gene is expressed. According to the mode of action and function, promoters can be roughly divided into three categories: constitutive promoters, specific promoters and inducible promoters [Wang Guanlin, Fang Hongyun, 2002, Principles and Technology of Plant Genetic Engineering (Second Edition), Beijing, Science and Technology Press]. This classification basically reflects the respective functional characteristics of different types of promoters, but in some cases, one type of promoter often has the characteristics of other types of promoters.

An inducible promoter (inducible promoter) means that the gene controlled by it can greatly increase the transcription level under the stimulation of certain specific physical, chemical and biological signals (collectively referred to as "inducer" or "inducible factor"). It is characterized in that the gene controlled by this type of promoter does not express or has very low expression (also called "background expression") in the absence of an inducing factor, but once induced by an inducing factor, the expression level of the gene rapidly and substantially increased. Inducible promoters are often classified and named according to their induction signals, for example: hormone-inducible promoters [Xu D et al., 1993, Plant Mol Biol, 22(4): 573-588; TaylorJE et al., 1995, Plant J, 7( 1): 129-134], chemically inducible promoters (Williams S et al., 1992, Biol/Technol, 10: 540-543; for review see: Padidam M, 2003, Curr Opinion in Plant Biol, 6169-177), light-inducible promoters Sub [Sheen JY et al., 1987, Plant Mol Biol, 8 (3): 227-238; Matsuoka M et al., 1994, Plant J, 6 (3): 311-319], heat inducible promoter [SchofflF et al., 1989, Mol Gen Genet, 217(2-3):246-53], wound-inducible promoter [Farmer EE et al., 1992, Plant Cell, 4:129-134; Carrera E et al., 1998, Plant J, 15(6): 765-771], fungal-inducible promoters (Fukuda Y et al., 1994, Plant Mol Biol, 24(3): 485-493) and symbiotic bacteria-inducible promoters (Miao GH et al., 1993, Plant Cell, 5: 781-786 )wait. Inducible promoters are often more or less organ and/or tissue specific. For example, the tobacco salicylic acid inducible promoter PR-1a mainly drives gene expression in leaves (Uknes S et al., 1993, Plant Cell, 5:159-169) .

A constitutive promoter means that under the control of this type of promoter, the expression of a structural gene is generally constant at a certain level, and there is no significant difference in the expression level in different organs and/or tissues. Its characteristics are: the expression of structural genes controlled by it is continuous, but not specific in time and space; the expression of RNA and protein is relatively constant, and is not induced by external factors, such as: corn Ubiquultin promoter, rice Actinl promoter (Wang et al., Molecular and Cellular Biology, 12(8):3399-3406(1992)), U.S. Patent No. 5641876) and cauliflower leaf virus (CaMV35SRNA) (Odell et al., Nature, 313:810-812(1985)) wait.

Organ or tissue-specific promoter (organ-and/or tissue-specific promoter) means that the expression of its regulatory gene often occurs only in one or some specific organs and/or tissues of the plant, or often occurs only At one or some specific stages of plant growth and development. Its characteristic is that the gene expression controlled or regulated by it has obvious temporal and spatial characteristics, and often exhibits the characteristics of developmental regulation. For example, root-specific promoters (Yamamoto YT et al., 1991, Plant Cell, 3:371-382), leaf-specific promoters [Taylor, WC, 2001, Plant Mol Biol, 46(3):325-333), Fruit-specific promoter (Pear JR et al., 1989, Plant Mol Biol, 13:639-651), flower-specific promoter (Van tunen et al., 1988, EMBO J., 7:1257), endosperm-specific promoter [ Colot V et al., 1987, EMBO J, 6(12):3559-3564], cotton fiber-specific promoter (Ma DP et al., 1997, Biochem Biophys Acta, 1344:111-114) and phloem-specific promoter [Bostwick DE et al., 1994, Plant Mol Biol, 26:887-897] and the like.

Reproductive growth is an important stage in the life history of higher plants, and the formation and development of floral organs, which are important organs for reproductive processes, have been widely concerned by biologists and agronomists. The floral development process of plants can be divided into four stages (Koornneefm et al; Ann. Rev. Plant Physiol Plant MolBiol; 1998, 49: 345-370): floral induction, floral meristem formation, floral organ primordium production and The floral organs are mature. Therefore, the development of floral organs is a highly complex and orderly process of physiology, biochemistry and morphogenesis. During this process, a large number of specific genes are expressed.

At present, some floral organ-specific promoters have been identified, isolated and functionally studied in the world. Some floral organ-specific promoters drive the expression of exogenous genes in multiple relatively independent units of floral organs. For example, the zb8 promoter of the PAL family drives the expression of the reporter gene GUS in anthers, flower buds, receptacles and filaments of transgenic rice ( Zhu Q et al, 1995, Plant Mol.Biol., 29:535-550), while other flower organ-specific promoters have the specificity of a specific unit of flower organ, for example, the rice anther-specific promoter ( Tsuchiya T et al., 1994, Plant Mol Biol, 20:1189-1193; Wu Xiaohuai et al., 2003, Science Bulletin, 48:2154-2161), tomato pollen-specific promoter LAT52 (Twell D et al., 1989, Mol GenGenet.217: 240-245), TomA108 (Xu XS and Chen RD, 2006, Physiol and Biochem, 25: 231-240) and the tobacco anther tapetum-specific promoter TA29 (Koltunow AM et al., 1990, PlantCell, 2: 1201-1224 )etc. Studies have also found that many genes in the anthocyanin metabolism pathway and the metabolism of volatile chemical substances in floral fragrance are specific to floral organs. For example, the petunia chalcone (CHS) gene promoter has strong petal-specific expression properties (Van Tunen et al., 1988, EMBO J., 7:1257).

The study of floral organ-specific genes and their promoters not only has important theoretical significance for understanding the control genes and networks of floral organ differentiation, formation, growth and development, but also has important application value for improving plants by using genetic engineering technology. Especially in genetic engineering of crop artificial male sterility (Mariani et al., 1990, Nature, 347:737-741; Xiao et al., 2004, Chinese invention patent, ZL00109108.5), flower shape, flower color and flowering period regulation of flowers and other genetic engineering ( Li et al., 2003, Chinese Bioengineering Journal, 23:42-46) and the shortening of the childhood period of fruit trees and the genetic engineering aspects such as adjusting the time to market of fruit by advancing the flowering period have broad application prospects.

Cabbage (Brassica oleracea L.) is an important vegetable crop of the genus Brassica, which is cultivated as the main vegetable in many countries in the world. In addition, its limited hybrid compatibility with other Brassicaceae plants such as rapeseed has made great contributions to the improvement of varieties such as rapeseed. For example, Brassica napus is already one of the main types of rapeseed in many countries and regions including my country.

Significant progress has been made in the cloning of flower organ-related genes and their promoters in cabbage. Self-incompatibility related genes Thl1, Thl2, ARC1 and male sterility have been cloned. These specific genes, especially their promoters, have been applied to genetic engineering breeding (Bhalla et al., 1998, Mol Breeding, 4(12):531-541). At the same time, some progress has been made in the improvement of cabbage using other plant flower-specific promoters. For example, Zhu Yuying et al. (Acta Agriculture Ahanghai, 2001, 17(1): 79-82) introduced the fusion of TA29 and Barnase genes into cabbage, and observed Male sterility and sterile plants with degenerated stamens appeared in the transgenic plants. With the improvement of awareness of environmental protection and the improvement of food quality requirements, people's requirements for using plant-derived genes to improve plants or crops are becoming stronger and stronger.

Contents of the invention

The object of the present invention is to provide a plant floral organ specific promoter and its application.

The plant floral organ-specific promoter provided by the present invention has a nucleotide sequence of:

(1), the nucleotide sequence described in sequence 1 in the sequence listing; or

(2), a nucleotide sequence complementary to the nucleotide sequence described in (1); or

(3), a nucleotide sequence having 60% or more homology with the nucleotide sequence described in (1) or (2); or

(4) A nucleotide sequence capable of hybridizing with the nucleotide sequence described in (1), (2) or (3) under stringent hybridization conditions.

The above-mentioned stringent hybridization conditions can be in 2×SSC, 0.1% SDS solution, hybridize at 68°C and wash the membrane twice, 5min each time; or in 0.5X SSC, 0.1% SDS solution, at 68°C Hybridize and wash the membrane twice, 15min each time.

Sequence 1 in the sequence listing consists of 1244 deoxynucleotides; the 1156-1167 deoxynucleotides from the 5' end are TATA boxes. The 1197th deoxynucleotide from the 5' end is the transcription initiation site.

The expression cassette containing the above-mentioned flower-specific promoter also belongs to the protection scope of the present invention.

In the expression cassette, the downstream of the floral organ-specific promoter is connected with a structural gene, a regulatory gene, an antisense gene of a structural gene, an antisense gene of a regulatory gene, or a small RNA capable of interfering with endogenous gene expression, for Drive the expression of structural genes, regulatory genes, antisense genes of structural genes, antisense genes of regulatory genes, natural small RNAs or artificially synthesized small RNAs.

The recombinant expression vector containing the above-mentioned floral organ-specific promoter also belongs to the protection scope of the present invention, and the recombinant expression vector is a recombinant vector constructed by containing the above-mentioned expression cassette and a plasmid, virus or carrier expression vector; the recombinant expression vector It is a recombinant plant expression vector; the recombinant plant expression vector comprises the above expression cassette and can transfer the expression cassette into plant host cells, tissues or organs and their progeny and can or at least facilitate the integration of the expression cassette into the host In the genome, it includes but not limited to binary vectors, coinciding vectors.

The above-mentioned recombinant expression vectors can transform plant cells or tissues or organs by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated or gene guns, to obtain transgenic plants Cells or tissues or organs and the complete plants differentiated and regenerated therefrom and their clones or their progeny.

The present invention also provides a method for obtaining the above-mentioned floral organ-specific promoter, which uses the genomic DNA of cabbage as a template, and uses the nucleotide sequence of sequence 2 in the sequence listing and the nucleotide sequence of sequence 3 in the sequence listing A pair of primers were used to amplify the floral organ promoter by PCR.

Experiments have proved that the flower-specific promoter Bofs of the present invention can initiate the specific expression of the reporter gene GUS in the petals, anthers, stigmas and pollen of the floral organs of transgenic tobacco, but no expression was detected in the vegetative organs, and its offspring showed the same It shows that the promoter Bofs provided by the present invention not only has a strong drive, but also has good floral organ specificity, and this strong drive and specificity can be stably transmitted to offspring. Therefore, the promoter Bofs isolated from Brassica oleracea L provided by the present invention is a flower organ-specific promoter with stability, strong driving force and high specificity.

The plant floral organ-specific promoter of the present invention can be expressed and stably inherited in different plants or crops. The plants or crops refer to flowering plants. According to different plant classification methods, the plants or crops can include but Not limited to angiosperms and gymnosperms, monocots and dicots, herbaceous plants, vines and woody plants, annuals and perennials, aquatic and terrestrial plants and sexually and vegetatively A classification of plants, said plants or crops are preferably terrestrial plants.

The antisense gene of the endogenous gene, the small RNA (including natural and artificially synthesized small RNA) or foreign gene that can interfere with the expression of the endogenous gene are connected downstream of the promoter of the present invention, and the expression cassette is constructed and used with different expression vectors Linking, transforming into plants, using its floral heterosexual expression activity, specifically expressing the antisense gene, foreign gene or small RNA controlled or directed by it in floral organs. In transgenic plants, the expression of antisense genes of endogenous genes, small RNAs (including natural and synthetic small RNAs) or exogenous genes that can interfere with the expression of endogenous genes are limited to flowers, excluding these genes in the flowers. Expression in other parts of the plant.

The floral organ-specific promoter of the present invention not only provides important molecular elements for theoretical research on the molecular mechanism of differentiation, formation, growth and development of floral organs, but also provides important molecular elements for plant genetic engineering breeding, especially flower shape, flower color, Genetically engineered breeding for aroma and flowering regulation provides key molecular elements. Using the promoter to drive the antisense gene of the endogenous gene, the small RNA that can interfere with the expression of the endogenous gene, or the exogenous gene to transform the cells or tissues of the plant and its progeny cells, can cultivate different flower shape, flower color, flower fragrance and fertility, etc. New varieties of flower crops based on the wild type of recipients, cultivation of male sterile crop varieties, and cultivation of landscaping plants that eliminate or greatly reduce "flying catkins", etc.

Description of drawings

Fig. 1 is the electrophoresis of the Bofs clone with a floral organ-specific promoter.

Figure 2 is the DNA sequence of the floral organ-specific promoter Bofs. In the figure, the underlined ones are the primer sequences, the italicized ones are the sites recognized by the additional restriction endonucleases, the 5' end is the Hind III site, and the 3' end is the Bam HI site.

Fig. 3 is a flowchart of the construction of the plant expression vector pRDBofsG driven by the flower organ-specific promoter Bofs gene (ie Bofs::GUS fusion gene).

Fig. 4 is an enzyme digestion identification map of the plant expression vector pRDBofsG containing the floral organ-specific promoter Bofs driving the GUS gene (ie Bofs::GUS fusion gene).

Fig. 5 is a PCR identification diagram of pRDBofsG transgenic tobacco T0 generation plants.

Fig. 6 is the GUS staining photos of the petals of pRDBofsG transgenic tobacco T0 generation plants and non-transgenic control plants.

Figure 7 is the GUS staining photos of the stigmas of the pRDBofsG transgenic tobacco T0 generation plants and non-transgenic control plants.

Figure 8 is the GUS staining photos of the anthers of the pRDBofsG transgenic tobacco T0 generation plants and non-transgenic control plants.

Figure 9 is the GUS staining photos of the pollen of pRDBofsG transgenic tobacco T0 generation plants and non-transgenic control plants.

Detailed ways

The promoter nucleotide sequence described in the present invention may be a nucleotide sequence in which one or more nucleotides are substituted, deleted, inserted or inverted, that is, an artificial mutant of the isolated nucleotide sequence or "Natural" mutant, which retains its promoter function; it can also be a fusion sequence of said nucleotide sequence with other promoter sequences or conservative regulatory sequences ("motif" or "box") in the promoter region. The "promoter" described in the present invention records the upstream of the start site (+1) and has the function of instructing RNA polymerase to initiate transcription at the correct position, but is not limited to the part near this region; in addition, it also contains the same Another essential region associated with proteins other than RNA polymerase for regulation of expression. The "promoter region" mentioned in the present invention is defined as a region containing the promoter defined above. The "promoter activity" mentioned in the present invention means that when a gene is linked downstream of the promoter in an expressible manner and introduced into a host, the host shows the ability to produce the gene inside or outside the host When the ability and function of the product are determined, the promoter has the activity of initiating. Usually, a gene (reporter gene) encoding a protein that is easily detected qualitatively or quantitatively is connected downstream of the promoter, the gene is introduced into the host, and the expressed protein is detected to determine the activity or presence of a specific promoter The promoter or the efficacy of the promoter. The structural gene mentioned in the present invention refers to a nucleotide sequence capable of encoding a certain protein or other active substance function, including RNA or DNA sequence. The regulatory gene refers to a nucleotide sequence that encodes a certain RNA or protein or other active substances that can regulate or regulate the expression of other structural genes, including RNA or DNA sequences. The sense gene includes the above-mentioned structural genes and regulatory genes. The antisense gene refers to the RNA or DNA sequence complementary to the RNA encoded by the above-mentioned structural gene or regulatory gene. The endogenous gene refers to the gene from the host itself, including RNA or DNA sequence. The exogenous gene refers to any nucleic acid sequence, which has the function of encoding a certain protein or other active substances, including natural and artificial RNA or DNA sequences, which are not combined with anther-specific promoters under normal conditions . The small RNA refers to an RNA sequence fragment isolated from an organism or artificially synthesized, and its length is usually 20-26 deoxynucleotides or can be cut into 20-26 deoxynucleotides after being introduced into dormitory cells RNA fragments, which themselves are non-toxic or have very low toxicity to organisms.

The expression vector described in the present invention refers to any plant vector known in the prior art and capable of expressing in plants, such as pBin19, pBI121 (product of ClonTech, USA) and pCAMBIA series (product of CAMBIA Center, Australia) wait.

The transformation described in the present invention refers to any plant transformation method known in the prior art that can introduce exogenous genes into plant cells or plant tissues, such as Agrobacterium-mediated method and gene gun.

The host cells or host tissues or host organs and their progeny cells mentioned in the present invention refer to all plant cells or plant tissues or plant organs or whole plants that are matured through tissue differentiation or asexual embryo regeneration by these cells, tissues or organs (including seeds).

The term "nucleic acid sequence" or "nucleotide sequence" refers to a sequence comprising naturally occurring nucleotide or nucleoside monomers. The sequences also include modified or substituted sequences of non-naturally occurring monomers or portions thereof that serve a similar function.

The term "floral organ-specific promoter" refers to a promoter that expresses a gene under the control of the promoter in the floral organ of a plant with no or basic expression but not in other organs of the plant.

The term "sequence having 60% or more homology" refers to those nucleic acid sequences that have 60% or more nucleotide sequences identical or similar to the sequence in (1) or (2) or Nucleotide sequences that function in essentially the same way and are capable of driving anther-specific expression of their downstream genes, their differences from the sequences in (1) or (2) may be due to local structural modifications or mutations, Including artificial mutations and non-artificial mutations.

The term "hybridizing sequence" refers to a nucleic acid sequence that can hybridize to the sequence of (1), (2), (3) or (4) under stringent hybridization conditions. "Stringent hybridization conditions" refers to those known to those skilled in the art, or can be found in molecular biology or genetic engineering experiment guidelines, such as "Molecular Cloning" (3 ^rd Ed) (Sambrook et al., Cold Spring Harbor Laboratory, New York, 2001 ) (hereinafter referred to as "Molecular Cloning" third edition"), specifically in the solution of 2×SSC, 0.1% SDS, hybridization at 68°C and washing the membrane twice. Hybridize and wash the membrane twice at 68°C in a solution of 0.5X SSC and 0.1% SDS for 5min each time. 15 minutes each time.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The percentages in the following examples are all mass percentages unless otherwise specified.

The T0 generation represents the transgenic plants and their clones obtained from the callus, and the T1 represents the seeds produced by selfing of the T0 generation and the plants and clones grown from it.

Embodiment 1, the cloning and separation of Bofs of flower organ-specific promoter of cabbage (Brassica oleracea L)

1. Extraction of Total DNA from Cabbage (Brassica oleracea L)

Total DNA was extracted from 1-2 g of young leaves of the cabbage variety "Heiye Xiaopingtou" (purchased from the Institute of Vegetable and Flower Research, Chinese Academy of Agricultural Sciences) potted in the greenhouse, and extracted by CTAB method (Molecular Cloning, third edition). Take 1-5ul DNA sample, measure its concentration and purity with a UV spectrophotometer, and use 0.8% agarose gel electrophoresis to detect DNA purity and integrity. Store the extracted DNA at -20°C.

2. PCR amplification and recovery of amplified fragments

The following two primers (primer 1 and primer 2) were designed and synthesized to amplify the flower organ-specific promoter of cabbage. In order to facilitate future cloning and construction, restriction enzymes HindIII and Bam were added to the 5' ends of the two primers, respectively. HI recognition sequence sites (underlined sequences in the following primer sequences)

Primer 1: 5'- AAGCTT AGCAGCACGAATGAAGTTC-3' ( Hind III )

Primer 2: 5'- GGATCC TTGTAGTGAGAAAACTCGGGGAA-3' ( Bam HI )

Using the cabbage genomic DNA extracted in step 1 as a template, carry out PCR reaction:

The reaction system is:

Template DNA: 800ng;

10X Exbuffer: 2ul;

dNTP mixture (2.5mM): 2ul;

ExTaq DNA Polymerase (5U/ul): 0.5ul;

Primer 1 (10uM): 2ul;

Primer 2 (10uM): 2ul;

Sterile double distilled water (sddH2O): Make up to 20ul.

PCR reaction program: first pre-denaturation at 94°C for 5min; then 94°C for 30sec, 50°C for 30sec, 72°C for 1min and 30sec, 35 cycles; finally 72°C for 10min.

After the PGR reaction is completed, take 15ul of the amplified product and go through 1.0% agarose gel electrophoresis, quickly cut out the specific fragment at about 1200bp with a disposable blade under ultraviolet light, recover and purify it with a DNA fragment recovery kit (Easy-NA Gel Extraction Kit, product of Omeg-Bio/TEK, Germany), dissolved in 50ulddH ₂ O, and stored at -20°C for future use.

3. Subcloning and sequencing of recovered fragments

The fragment recovered in step 2 was connected to the plasmid vector pUCm-T (product of Shanghai Sangon Bioengineering Technology Service Co., Ltd.), and the resulting recombinant plasmid was transformed into the large intestine by the "freeze-thaw method" (Molecular Cloning, third edition) Bacillus strain DH5α competent cells were cultured overnight at 37°C on LB solid medium containing 100 mg/L ampicillin, and the white colonies grown on the plate were picked and inserted into LB liquid medium containing 100 mg/L ampicillin. Incubate overnight at 37°C. When the concentration of the bacterial solution reached OD ₆₀₀ of 0.6, the bacterial cells were collected by centrifugation, and the plasmid was extracted according to a small amount of alkaline lysis method ("Molecular Cloning" third edition), and after double digestion with restriction enzymes HindIII and Bam HI, with 1.0% Agarose gel electrophoresis, the correct recombinant vector should be visible under the ultraviolet light as two bands with molecular sizes of about 2800bp (carrier band) and 1200bp (target band) respectively (Figure 1, in the figure, lane 1 is the DNA size molecule standard I---MarkerI; Lane 2 is the HindIII+BamHI restriction map of the cloning vector inserted into the above-mentioned PCR amplified fragment). The plasmid (pUBofs) containing the 1200bp insert fragment identified by the above enzyme digestion or the DH5α containing the plasmid was sent to a commercial sequencing company for sequencing (formerly Shanghai Boya Biotechnology Service Co., Ltd., now incorporated into Introgen Company), and the sequence will show that it is correct. The recombinant vector was named pUBofs. Sequencing results show that the above PCR-amplified fragment is the specific promoter of the flower organ of cabbage, and the sequence is shown in Figure 2. The specific promoter of the flower organ of cabbage has the nucleotide sequence of sequence 1 in the sequence list, and will have the sequence 1 in the sequence list The nucleotide sequence of the Brassica oleracea flower organ-specific promoter was named Bofs. In Fig. 2, the underlined ones are the primer sequences used (primer 1 and primer 2 above), and the italics are the additional restriction endonuclease recognition sites (HindIII and BamHI).

Example 2, Construction of flower organ-specific promoter-driven GUS gene (Bofs::GUS) plant expression vector pRDBofsG

The construction process of the plant expression vector pRDBofsG driven by a flower organ-specific promoter driving the GUS fusion gene (Bofs::GUS) is shown in Figure 3, and the specific method is as follows:

After pUBofs was digested with restriction endonucleases Hind III and Bam HI, the Bofs fragment (about 1200bp) was frozen and thawed ("Molecular Cloning" third edition) or DNA fragment recovery kit (Easy-NAGel Extraction Kit , Germany Omeg-Bio/TEK product) recovered and purified the flower organ-specific promoter Bofs fragment, and connected with the large fragment (about 13.6kb) of the plant expression vector pRD410 (Canada PBI product) that had been digested with Hind III and Bam HI , to obtain the recombinant plasmid. The resulting recombinant plasmid was transformed into Escherichia coli strain DH5a by "freeze-thaw method" (Molecular Cloning, third edition). Transformed DH5α was cultured overnight at 37°C on LB solid medium containing 50 mg/L kanamycin, picked a single colony grown on the plate, and inserted it into LB liquid medium containing 50 mg/L kanamycin. Incubate overnight at 37°C with shaking. When the concentration of the bacterial solution reaches OD600 value of 0.5-0.6, collect the bacterial cells by centrifugation, extract the plasmid according to the above-mentioned small amount of alkaline lysis method, and perform double digestion with restriction endonucleases Hindll and Bam HI, and electrophoresis on 1.0% agarose gel , two bands with molecular sizes of about 13.5kb (pRD410 carrier band) and 1200bp (Bofs) can be seen under ultraviolet light, and they were verified by sequencing. Digestion and sequencing showed that they contained the floral organ-specific promoter Bofs fragment and pRD410 enzyme The recombinant expression vector of the obtained large fragment was named pRDBofsG. The GUS gene in pRDBofsG requires a floral organ-specific promoter to drive its expression.

Embodiment 3, the identification of transforming Bofs fusion gene (Bofs::GUS) tobacco

1. Transformation of Agrobacterium and identification of transformants

Competent cells of Agrobacterium tumefaciens strain LBA4404 (product of LifeTechnology, USA) were prepared by the CaCl ₂ method (Molecular Cloning, third edition). The plant expression vector pRDBofsG containing the Bofs fusion gene (Bofs::GUS) was transferred into the prepared LBA4404 competent cells by freeze-thaw method (Molecular Cloning, third edition). Inoculate the transformed LBA4404 cells into YEB solid medium containing streptomycin (Str) 100mg/L and kanamycin (Kan) 50mg/L, culture in the dark at 28°C for 48-72h, pick the plate A single colony was inserted into YEB liquid medium containing 100 mg/L streptomycin and 50 mg/L kanamycin, and cultured overnight at 28°C with shaking. When the concentration of the culture reaches the _OD600 value of 0.4-0.6, get a small amount of bacterial solution (1.5-2ml), extract the plasmid according to the above-mentioned small amount of alkali lysis method, identify it with restriction endonuclease HindIII and BamHI double enzyme digestion, in 1.0% agarose gel electrophoresis, under the ultraviolet light, the visible molecular size is about 13.6kb (carrier band) and 1200bp (Bofs) two bands (Fig. 4, in Fig. 4, swimming lane 1 is Hind III+Bam HI of pRDBofsG Double-digestion map; Lane 2 is the double-cut λDNA size standard --- λDNA/HindIII+EcoRI), which was identified by sequencing. The results showed that the plant expression vector pRDBofsG had been successfully transformed into Agrobacterium LBA4404. The Agrobacterium LBA4404 clone containing the plant expression vector pRDBofsG identified by enzyme digestion and sequencing was named pRDBofsG/LBA4404.

2. Obtaining of Bofs fusion gene (transformation pRDBofsG) tobacco

1) Preparation of Agrobacterium

Pick the single colony of Agrobacterium LBA4404 (pRDBofsG/LBA4404) carrying the plant expression vector pRDBofsG, inoculate in 5ml of YEB liquid medium containing streptomycin (Str) 100mg/L and kanamycin (Kan) 50mg/L, Shake culture overnight at 28°C (about 12h) for activation. Take the activated Agrobacterium liquid, add it into the YEB liquid medium containing Str 100mg/L and Kan 50mg/L at a ratio of 1:100, and continue to cultivate until the _OD600 value is 0.4-0.6; centrifuge at 5000rpm for 5min, and collect the bacteria ; Wash the thalline once with 1/2MSO liquid medium (MS inorganic salt+B5 vitamin+inositol 0.1g/L+sucrose 30g/L, pH 5.80), and dilute it to 1/3 of the volume of 3 times the volume of the bacterial solution before centrifugation 2MS liquid medium, ready for infection.

2) Transformation of tobacco and regeneration of transformed plants

Transformation of tobacco was performed according to the leaf disk method (Horsch RB et al., 1985, Science, 227: 1229-1231). Select the aseptic seedlings of tobacco (variety "NC89", the seeds are purchased from the Chinese Academy of Agricultural Sciences) about 30 days old, cut off the leaves of fresh and tender dark green, and use a puncher with a diameter of 9 mm to prepare leaf disc explants; Put the prepared explants into the prepared Agrobacterium (LBA4404/pRDBofsG) bacterial solution, and infect for 15 minutes; take out the leaf disc, absorb the remaining Agrobacterium bacterial solution on the surface of the leaf disc with high-pressure sterilized absorbent paper, and place in Solid regeneration medium (MS inorganic salt + B5 vitamin + inositol 0.1g/L + BA 2.0mg/L + IAA 0.5mg/L + sucrose 30g/L + agar powder 0.7%, pH 5.8) co-cultured in the dark for 2 days; Then transfer the co-cultured leaf discs to the solid regeneration medium containing carbenicillin (Carb) 500mg/L and Kan 50mg/L under light (1500-2500Lx, light 16h dark 8h) to carry out selection culture, every 2- The medium was replaced every 3 weeks, and Carb was gradually reduced to 200mg/L. When the Kan-resistant shoot grows to 1-1.5cm, it is cut off and replaced with a solid rooting medium containing Carb 200mg/L and Kan 50mg/L (MS inorganic salt+B5 vitamin+inositol 0.1g/L+IAA 0.5mg/L+sucrose 30g/L+agar powder 0.7%, pH 5.80) to induce rooting. The obtained 58 complete kanamycin-resistant transgenic pRDBofsG tobacco plants were used for molecular identification.

3) Molecular identification of tobacco transformed with pRDBofsG

Firstly, the Kan resistance transgenic pRDBofsG tobacco plants obtained in step 2) were identified by PCR. According to the above-mentioned CTAB method, the leaf genome DNA of the transgenic pRDBofsG tobacco (Kan resistant strain) and the non-transgenic tobacco control strain was extracted, and the specific primer pair at both ends of the floral organ-specific promoter Bofs (primer 1: 5'-AAG CTT AGC AGC ACG AAT GAA GTT C-3'; Primer 2: 5'-GGA TCC TTG TAG5'-AAG CTT AGC AGC ACG AAT GAA GTT C-3'; Primer 2: 5'-GGA TCC TTG TAGTGA GAA AAC TCG GGG AA -3') carry out PCR amplification, and the target band of size 1200bp is amplified in the transformed pRDBofsG tobacco, while the untransformed control tobacco does not amplify this target fragment at the corresponding position (partial PCR amplification results are shown in Figure 5 Show, among Fig. 5, swimming lane 1-7 is different transgenic pRDBofsG tobacco (Kan resistance strain) T0 generation plant; Swimming lane NC89 is non-transgenic plant control (negative control); Swimming lane Bofs is vector plasmid control (positive control); M is DNA size molecular standard (marker)). This result preliminarily indicated that the target gene (Bofs::GUS) had been integrated into the tobacco genome.

Example 4, Histochemical detection of GUS gene expression in transpRDBofsG tobacco

The transgenic pRDBofsG tobacco T0 generation lines (lines 2, 6, 7, 9, 11, 12, 14) and non-transgenic tobacco NC89 control plants (CK) test tubes with both kanamycin resistance and PCR positive The seedlings were opened in the triangular bottle for one week, then moved into flowerpots filled with common flower nutrient soil, cultivated in the greenhouse, bagged and kept moist for a week, then removed from the bag for routine management.

Histochemical detection of GUS gene expression in pRDBofsG tobacco was carried out according to the method of Jefferson RA [1987, PlantMol Biol Rep, 5(4):387-405]. In different development stages of floral organs (according to literature (Cao Kehao, China Agricultural University master's degree thesis, sequence and function analysis of rice Osg6B promoter and cloning of E. coli argE gene and transformation of tobacco, 2003) method to divide floral organs development period), the fresh roots, stems, leaves, calyx, petals, filaments, ovary, stigma and anthers of transgenic pRDBofsG tobacco and its control (tobacco variety "NC89", seeds purchased from the Chinese Academy of Agricultural Sciences) were placed in X-GLuc Incubate in the solution for 24-48 hours for dyeing, then rinse the dyed plant material thoroughly with 70% and 100% ethanol, decolorize and fix it, observe the sample under a microscope and take pictures.

The results of GUS staining of floral organs are shown in Table 1. The results showed that each organ of the non-transgenic tobacco NC89 control plant (CK) could not be stained, while in the 7 PCR-positive T0 generation lines of transgenic pRDBofsG tobacco detected , the anthers of all lines can be stained to varying degrees (Fig. 8, A in Fig. 8 is the non-transgenic tobacco NC89 control plant, B is the transgenic pRDBofsG tobacco T0 plant). 5 lines (except 2 and 12) were stained in varying degrees in petals and stigmas (Fig. 6, Fig. 7, A in Fig. 6 and Fig. 7 are all non-transgenic tobacco NC89 control plants, B are transgenic pRDBofsG tobacco T0 plants). The pollen of lines 6 and 11 were stained in different degrees (Fig. 9, A in Fig. 9 is the non-transgenic tobacco NC89 control plant, B is the pRDBofsG transfected tobacco T0 plant). Filament, ovary, etc. were not dyed blue in all the lines. These results showed the floral organ specificity of GUS staining, which indicated that the promoter Bofs had floral organ-specific driving activity. Fig. 6 is GUS staining of petals of non-transgenic control (A) and transgenic pRDBofsG tobacco (B); Fig. 7 is GUS staining of stigma of control (A) and transgenic pRDBofsG tobacco (B). Fig. 8 shows GUS staining of anthers of non-transgenic control (A) and transgenic pRDBofsG tobacco (B). Figure 9 shows pollen GUS staining of non-transgenic control (A) and transgenic pRDBofsG tobacco (B). The vegetative organs of tobacco transformed with pRDBofsG, such as roots, stems and leaves, had no GUS activity.

Table 1. GUS staining of T0 generation of transgenic pRDBofsG tobacco

Note: "+" in the table indicates positive GUS staining; "+++" indicates the strongest staining; "-" indicates negative GUS staining,

Example 5: Expression of GUS in the T1 generation of tobacco transfected with pRDBofsG

The seeds of transgenic pRDBofsG tobacco T0 generation lines 6, 11 and 14 (T1 generation) and their non-transgenic control seeds (NC89) were surface-sterilized and inoculated into seed germination medium (1/2MS) with and without Kan. ), cultured in the dark for 7 days, and then cultured in the light at 25±1°C. On the medium without Kan, both the transgenic pRDBofsG tobacco and the non-transgenic control seeds could germinate (the germination rate was over 95%), and the seedlings were green. On the medium containing 100mg/L Kan, although some of the non-transgenic control seeds could germinate, the seedlings were all albino and died; but nearly 70% of the seedlings of the transgenic pRDBofsG tobacco were green and grew normally. After culturing for about 30 days, randomly select one of the Kan-resistant seedlings of the T1 generation of the T0 generation lines 6, 11 and 14 of pRDBofsG transfected tobacco for test tube culture. After obtaining vigorously growing large seedlings, the vegetative organs were selected for GUS staining. When the T1 seedlings grow to have 2-3 true leaves, take the leaf discs for in vitro anti-Kan test, and randomly select 3 plants from each line to develop to maturity. During the development process, take different organs and tissues according to the method described above. GUS staining was performed to determine the stability and specificity of Bofs::GUS expression at T1 in pRDBofsG tobacco. The results are shown in Table 2.

Table 2. GUS staining of Kan-resistant seedlings of the T1 generation of transgenic pRDBofsG tobacco

strain root stem leaf anther pollen filament ovary Stigma petals sepal 6 - - - +++ + - - ++ ++ - 11 - - - +++ + - - +++ ++ - 14 - - - +++ ++ - - ++ ++ - control - - - - - - - - - -

Note: "+" in the table indicates positive GUS staining; "+++" indicates the strongest staining; "-" indicates negative GUS staining,

It can be seen from Table 2 that the GUS gene driven by Bofs can be stably transmitted to the offspring in the transgenic pRDBofsG tobacco, and the GUS gene is not expressed in the vegetative organs of all tested progeny plant test-tube plantlets and greenhouse plantlets, only specifically Expressed in floral organs.

The results of the above examples show and confirm that the Bofs of the cabbage (Brassica oleracea L) promoter provided by the present invention not only has strong drive, but also has good flower organ specificity, and this strong drive and specificity can stabilize passed on to future generations. Therefore, the promoter Bofs isolated from cabbage (Brassica oleracea L) provided by the present invention is a flower organ-specific promoter with stability, strong driving force and high specificity.

sequence listing

<160>1

<210>1

<211>1244

<212>DNA

<213> Brassicia oleracea L.

<400>1

aagcttagca gcacgaatga agttcgtcaa gtttttaatt aggcttcgct tcttgtgatt 60

catcgaaaat ttatatcatt tcatacgttc attcttgttt tcatgtgact ttcctcttct 120

ctaccgtgag tctcatcaat ttcgtagatc gctatgttaa cgatccacgt atcatatata 180

caccttcttt ctatagccgt acgtatacca cacattacct catcccactt cctaacttat 240

ataattttac tactcagatc acaagtgtac gtatatcatg aagtcatttc ttctccttgt 300

cctactcctc tctttctttg tcggctctat cttcgctagt aggaattttc cgacgcaccc 360

ctatccaagt atgtatgctc ttcaattctc tctcactctc cttaatttta cccacctctt 420

tcactatctt caacgtcttt taacttgttc aattatgttc gtgtgggtgg gcaggtcata 480

atcatcatca tgtcggaatg atgggtagga aaatgaagcg tcagaggagg ccggacacgg 540

tgcaggtggc agggtctagg ctgccggact gctcacacgc gtgtggctca tgctccccat 600

gccgtcttgt gatggttagc ttcgtgtgtg catcgctaga ggaggctgag acttgtccca 660

tggcttataa gtgcatgtgc aagaacaaat cctaccccgt cccatgatga attagcctct 720

ctcacactta actctgtgca ttcagacgtt ttgtttcttt ccttttgctt cttcggataa 780

atgaccatgt gtatgtataa aatgcatctt ttcctttttt taattctgtt tgtctttttg 840

atatcttaaa cacagtttta cgaaacaaga ataagattag ttgagccact caaaagcgtg 900

gtcgactaaa ttgaaacaga aagccacaca actcattggg ctcttgttta tggcccatga 960

caccgcattt cagactgcaa caaccaaagt tgtagaaaga ataatattta aagggcacgt 1020

acatacgttg ttggcttcca ccaaactttg gaggctctct aataattagc acactccat 1080

ctatgcattt gttacacacc ttctattttc aaccatttca tctcaccttt tttaaatgtt 1140

tccacagtta gctcagtaaa ttcactatat acagacatac accttccctc cacaagacca 1200

aacaaccaca ctaccttccc cgagttttct cactacaagg atcc 1244

<210>2

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>2

aagcttagca gcacgaatga agttc 25

<210>3

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>3

ggatccttgt agtgagaaaa ctcggggaa 29

Claims (15)

1, a kind of plant flower organ specificity promoter, it is characterized in that: its nucleotide sequence is:

(1), sequence 1 described nucleotide sequence in the sequence table; Or

(2), with (1) described nucleotide sequence complementary nucleotide sequence.

2, the expression cassette that contains the described promotor of claim 1.

3, expression cassette according to claim 2 is characterized in that: the inverted defined gene of the downstream syndeton gene of described flower organ specificity promoter, regulatory gene, structure gene, the inverted defined gene of regulatory gene or the little RNA that can disturb native gene to express.

4, the recombinant expression vector that contains the described promotor of claim 1.

5, the recombinant expression vector that contains claim 2 or 3 described expression cassettes.

6, recombinant expression vector according to claim 5 is characterized in that: described recombinant expression vector is by claim 2 or 3 described expression cassettes and the constructed recombinant vectors of vehicle expression vector.

7, recombinant expression vector according to claim 6 is characterized in that: described vehicle expression vector is plasmid or virus.

8, recombinant expression vector according to claim 6 is characterized in that: described recombinant expression vector is binary vector or closes carrier altogether.

9, the described flower organ specificity promoter of claim 1 cultivate do not suit shape, pattern, the fragrance of a flower and/or long shelf-life flower plant strain, cultivation " willow catkins flying in the air " is eliminated or the willow catkins flying in the air garden plants strain that reduces significantly, cultivate the plant lines that the pollination fertility strengthens or weaken or cultivate application in the transgenic plant strain of polluting its wild species and sibling species that can prevent to drift about by pollen.

10, application according to claim 9 is characterized in that: described plant is a crop.

11, the described flower organ specificity promoter of claim 1 cultivate do not suit shape, pattern, the fragrance of a flower and/or long shelf-life flower plant kind, cultivation " willow catkins flying in the air " is eliminated or the willow catkins flying in the air garden plants kind that reduces significantly, cultivate the plant variety that the pollination fertility strengthens or weaken or cultivate application in the transgenic plant kind of polluting its wild species and sibling species that can prevent to drift about by pollen.

12, application according to claim 11 is characterized in that: described plant is a crop.

13, according to arbitrary described application among the claim 9-12, it is characterized in that: described plant is a polycarpeae.

14, application according to claim 13 is characterized in that: described plant is a terrestrial plant.

15, the preparation method of the described flower organ specificity promoter of a kind of claim 1, being that genomic dna with wild cabbage is a template, is that sequence 2 and nucleotide sequence are that a pair of primer of sequence 3 carries out pcr amplification and obtains promotor in the sequence table in the sequence table with nucleotide sequence.

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