KR101554209B1 - Microspore or pollen specific expression promoter from Oryza sativa Os05g0543000 gene and uses thereof - Google Patents

Abstract

The present invention is rice (Oryza sativa ) Os05g0543000 gene-derived microparticle or pollen-specific gene expression promoter, a recombinant plant expression vector containing the promoter, and a transformant plant in which a foreign gene including a step of transforming the vector into a plant is expressed in a microparticle or pollen- , A transgenic plant in which the foreign gene produced by the above method is specifically expressed in a microspore or pollen, and a seed thereof, and the promoter of the present invention has excellent expression specificity in the stage of development of the microspore and mature pollen, It can be very useful for biotechnologically cultivating male sterile plants.

Description

[0002] The present invention relates to a microspore or pollen-specific expression promoter from Oryza sativa Os05g0543000 gene and uses thereof,

The present invention relates to a rice bran- specific microbial or pollen-specific expression promoter derived from the rice OsO5g0543000 gene and its use, and more particularly to rice bran sativa ) Os05g0543000 gene-specific microparticle or pollen-specific expression promoter, a recombinant plant expression vector containing the promoter, and a step of transforming the vector into a plant, wherein the foreign gene is expressed in a microspore or pollen-specific expression The present invention also relates to a method for producing the transgenic plant, a method for producing the transgenic plant, and a method for producing the transgenic plant.

The development of mature pollen from angiosperm spore forming cells requires highly complex and elaborate gene expression programs in haploid gametophytes and sporozoic tissues. In particular, it is known that the developmental process of male gametes, represented by plant pollen, involves a large number of genes (about 20,000 or more) that require elaborate expression control. In order to search genes expressing in microspore and pollen, genetic methods using mutants that can not normally produce male gametophytes or pollen were traditionally used, but now microarrays and next generation sequencing NGS, next generation sequencing) to produce a large amount of data in a short time.

The development process from the microspores to the mature pollen belongs to the germplasm of a plant that produces male spouses. It is an important biological process based on the existence of a relatively short cycle or seed plant, and the genes involved in this development are agricultural utilization The field of crop breeding using biotechnological methods is very large. In particular, a promoter that is expressed specifically in the microspores and pollen tissues can be used for cultivating a transformant that expresses a specific gene locally to the pollen tissue of a plant by producing a transgenic vector that binds to a foreign gene capable of inhibiting pollen development . Male sterile plants grown in this manner can be used directly as fertility seeds to produce one hybrid seed of major crops, including rice, and thus agricultural utilization is very high.

The heterozygosity of the crops can be explained by the fact that the F1 generation hybrid plants obtained through crossing between lines or cultivars of different genetic backgrounds in the cross-pollination or self-pollination plants are more vigorous than the parents used for crossing, And environmental stress, and it is widely used as a method of crop breeding. In addition, since hybridity is significantly weakened at the second generation of hybrids, most of the seeds currently marketed by seed companies are hybrid seeds of the F1 generation.

Since the reproductive structure producing plant seeds is so diverse and complex, it is not easy to produce hybrid seeds. In the case of rice, which is a typical self-fertilization crop, the one hybrid breeding technique has the potential to greatly increase the yield, but there are many breeding problems that have yet to be solved, such as the difficulty of seed production. In particular, Indica x Japonica first generation hybrids have a great effect of hybrids on nutritional growth, but they have not been put to practical use due to problems such as delayed heading and infertility. Currently, in China, the three systems of infertility, maintenance, and recovery are converted to two systems using environmentally sensitive male sterility systems, but due to various problems, they are going back to three systems.

Korean Patent No. 1185845 discloses a pLim3 promoter expressed in a pollen of rice, an expression vector containing the promoter, a transformant transformed with the expression vector, and a method for producing the transformant Korean Patent No. 0527285 discloses a method of producing male sterile-induced rice by using a rice-specific expression cysteine protease gene, a syringe-specific expression promoter of the gene, and suppression of expression of the gene, Os05g0543000 gene-based vesicle or pollen-specific expression promoters and their uses have not been described.

The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a recombinant plant expression vector containing a microorganism or pollen-specific expression promoter derived from rice Os05g0543000 gene into rice and Arabidopsis thaliana, The present inventors have completed the present invention by confirming that they are specifically expressed in microspheres or pollen.

In order to solve the above-mentioned problems, the present invention provides a rice plant comprising the nucleotide sequence of SEQ ID NO: 1 ( Oryza sativa ) Os05g0543000 gene-specific vesicle or pollen-specific expression promoter.

The present invention also provides a microspore or pollen-specific plant expression vector comprising the promoter.

The present invention also provides a plant transformed with said vector.

Also, the present invention relates to a method for producing a recombinant vector, comprising: recombining a foreign gene into the vector; And a step of transforming the recombinant plant expression vector into a plant, wherein the foreign gene is expressed in a microspore or pollen-specific expression.

The present invention also provides a transgenic plant wherein the foreign gene produced by the above method is expressed in a microspore or pollen-specific manner.

The present invention also provides a seed of the transgenic plant.

The present invention relates to a rice-derived promoter capable of inducing limited expression in the stage of development of a microspore and in mature pollen during the development of rice pollen comprising the nucleotide sequence of SEQ ID NO: 1. In particular, the rice-derived promoter of the present invention is a promoter derived from a rice gene expressing a pollen-specific expression and induces a specific expression only in a micropore or flowerpot of rice and Arabidopsis developing in a mature pollen from a 2 cell sporangium . Therefore, this promoter shows that it can be used not only in monocotyledonous plants but also in dicotyledonous plants, and also in the case of cruciferous plants such as Arabidopsis, Chinese cabbage, rapeseed and tobacco. The promoter of the present invention can induce gene expression specifically in the development stage of the microspore and mature pollen, and has excellent expression specificity in the stage of pollen formation and development, so that it is very useful for biotechnologically cultivating male sterile plants .

Brief Description of the Drawings Fig. 1 is a graph showing the relationship between the total nucleotide sequence of rice OsO5g0543000 gene (Fig. 1A) derived from the microspore or pollen-specific promoter of the present invention and the recombinant vector obtained by fusing the GUS :: GFP gene to a microspore or pollen-specific promoter derived from the gene (Fig. 1B). (A) is the rice-derived microparticle or pollen-specific promoter region of the present invention.
FIG. 2 shows the results of cultivating a rice plant transformant using a recombinant vector obtained by fusing a GUS :: GFP gene to a rice-derived microspore or pollen-specific promoter, and comparing the number of microspots and pollen development stages of the rice transgenic plants in the T0 and T1 generations GUS gene expression was observed by fluorescence and optical microscopy. anther; About, UC (Uninucleate micrpore); 1 Bicellular microspore; BC; 2 nucleus microspheres, TC (Tricellular microspore); 3 nucleus micropore, MP (Mature pollen); Maturing pots.
FIG. 3 shows the expression pattern of the OsO5g0543000 gene derived from the promoter used in the present invention in a rice plant transformant transformed with a recombinant vector pSK815 in which a GUS :: GFP gene was fused to a rice-derived microspore or pollen-specific promoter The results of RT-PCR analysis (A) and RT-PCR analysis of GUS gene in leaves, stems and root tissues of transgenic rice plants of T1 generation (rice, leaves, stem and root tissues) )to be. MP; Drug tissues containing microparticles and mature pollen, L1, L2 and L3; Leaf tissues grown at 2 weeks, 3 weeks and 4 weeks after germination, S1, S2 and S3; R1, R2 and R3; stem tissues grown at 2 weeks, 3 weeks and 4 weeks after germination; Root tissue that grew 2, 3 and 4 weeks after germination.
4 is a microarray analysis result of the rice Os05g0543000 gene. In heatmap, yellow means high expression and blue means low expression.
FIG. 5 shows the GUS gene expression pattern in the flower peaks, microspheres and pollen tissues of the T1 generation Arabidopsis transformants transformed with the recombinant vector pSK815 in which the GUS :: GFP gene was fused to the rice-derived microspore or pollen-specific promoter . (A) shows the expression pattern of GUS gene in T1 genera, (B) shows fluorescence and optical microscope (GUS) gene expression pattern of transgenic plants in stages of pollen development and pollen development (UN, BC, TC and MP are the same as those described in Fig. 2), (C) is a flowering flour (Fl) of the T2 generation Arabidopsis transgenic plants, a flower bud (Bd) The expression of GUS gene in stem (S), root (R) and seedling (Sd) tissues was analyzed by RT-PCR.

In order to achieve the object of the present invention, the present invention provides a rice comprising the nucleotide sequence of SEQ ID NO: 1 ( Oryza sativa ) Os05g0543000 gene-specific vesicle or pollen-specific expression promoter.

The gene introduced with the CaMV35S promoter derived from the commonly used cauliflower mosaic virus is expressed in whole tissues, whereas the microparticle or pollen-specific expression promoter of the present invention expresses the gene introduced from the transgenic plant into the microparticle or pollen-specific . ≪ / RTI >

Also, homologues of the promoter sequences are included within the scope of the present invention. The homologue is a base sequence having a functional characteristic similar to that of SEQ ID NO: 1, although the base sequence is changed. Specifically, the promoter sequence has a nucleotide sequence that has at least 70% homology, more preferably at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The present invention also provides a vesicle or pollen-specific plant expression vector comprising the sporadic or pollen-specific expression promoter of the onset.

The microparticle or pollen-specific expression vector of the present invention can be used as a transient expression vector capable of transient expression in a plant into which a foreign gene has been introduced and a plant expression vector capable of permanently expressing the foreign gene in the introduced plant .

Binary vectors that can be used in the present invention include the RB (right border) and LB (left border) of T-DNA, which can transform a plant when present together with the Ti plasmid of Agrobacterium tumefaciens (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121, pCAMBIA vector, etc., which are frequently used in the art, may be used good.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

A preferred example of a plant expression vector is a Ti-plasmid vector that is capable of transferring a so-called T-region into a plant cell when it is present in a suitable host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce a gene according to the invention into a plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include antibiotic resistance genes such as herbicide resistance genes such as glyphosate or phosphinotricin, kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, It is not.

In a plant expression vector according to an embodiment of the present invention, the terminator can be a conventional terminator, such as nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agro And the terminator of the Octopine gene of Agrobacterium tumefaciens , but is not limited thereto.

The present invention also provides E. coli or A. tumefaciens transformed with the microparticle or pollen-specific plant expression vector of the present invention.

The present invention also provides a plant transformed with the microparticle or pollen-specific plant expression vector of the present invention.

In a transgenic plant according to an embodiment of the present invention, the plant may be selected from the group consisting of Arabidopsis thaliana, eggplant, tobacco, pepper, tomato, burdock, ciliaceae, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, Such as rice, barley, wheat, rye, corn, sugarcane, oats, onion, etc., such as Chinese cabbage, cabbage, gangbu, watermelon, melon, cucumber, amber, pak, strawberry, soybean, mung bean, Lt; / RTI >

Preferably, the dicotyledonous plants may be selected from the group consisting of radish, Chinese cabbage, rapeseed, poles, Arabidopsis thaliana, mustard, horseradish, mustard, broadleaf, pole tree, flower bud, And may be Brassicaceae plants such as broccoli, cabbage, cauliflower, kale, and the like. Monocotyledonous plants include butyral, reed, , Barley, sorghum, sorghum, turfgrass, oats, and the like, and more preferably Arabidopsis thaliana or rice, but is not limited thereto.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. The method is based on the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202,179-185 (Klein et al., 1987, Nature 327, 70), infiltration of plants, or Agrobacterium tumefaciens by transformation of mature pollen or microparticles of various plant elements (DNA or RNA-coated) Infection by viruses (non-integrative) in virus-mediated gene transfer (EP 0 301 316), and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Particularly preferred is the use of so-called binary vector techniques as described in EP A 120 516 and U.S. Pat. No. 4,940,838.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cell may be any of a cultured cell, a cultured tissue, a culture or whole plant, preferably a cultured cell, a cultured tissue or culture medium, and more preferably a cultured cell.

"Plant tissue" refers to cells of differentiated or undifferentiated plants, such as, but not limited to, various types of cells used in fruit, stem, leaf, pollen, seed, protoplasts, shoots and callus tissue. The plant tissue may be in planta or may be in an organ culture, tissue culture or cell culture.

The present invention also relates to a method for producing a plant-specific plant expression vector, comprising: recombining a foreign gene into the microspore or pollen-specific plant expression vector of the present invention; And

And a step of transforming the recombinant plant expression vector into a plant, wherein the foreign gene is specifically expressed in a microparticle or a flowerpot.

The foreign gene may be any gene that desires expression in plant microspheres or pollen, and may be located behind the promoter in the microparticle or pollen-specific expression vector of the present invention, and may be expressed by fusion with a reporter gene, if necessary. Methods for transforming the recombinant microspheres and pollen-specific expression vectors into plants can be carried out as described above. The plant according to one embodiment of the present invention is as described above.

The present invention also provides a transgenic plant and a seed thereof, wherein the foreign gene produced by the method of the present invention is expressed in a microspore or pollen-specific manner. The transgenic plants are capable of expressing the foreign gene in a microspore or pollen-specific manner by a microspore or pollen-specific expression promoter.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. Promoter of pollen-specific expression gene from rice and sequencing

In order to search for genes specifically expressed in the developing pollen of rice, the rice Os05g0543000 gene was selected through microarray data analysis of rice anther and microspore development stages. From the NCBI database (http://www.ncbi.nlm.nih.gov/), a nucleotide sequence of about 2 Kbp including the promoter of the gene and the 5'-UTR was obtained. To amplify the promoter fragment 1,784 bp, primers Os05g0543000P- (Blue color in FIG. 1A) were prepared and the primers of AttB1 and AttB2 (SEQ ID NO: 2) were added to the above primers, respectively, and OsO5g0543000P-R (5'-TTGGCTTCTCCGACAAGCTGAGTACGGTT- (5'-AAAAAGCAGGCT-3 '; SEQ ID NO: 4, 5'-AGAAAGCTGGGT-3'; SEQ ID NO: 5) Genomic DNA was extracted from leaf tissues of the seedlings after 3 weeks of germination by using the CTAB method (Ronald PC and Chen DH, 1999, Plant Mol. Biol. Rep. 17: 53-57). The PCR reaction for the amplification of the promoter fragment proceeded in two steps. The first reaction was a reaction at 94 ° C for 5 minutes, followed by 10 cycles of reaction at 94 ° C for 30 seconds, 58 ° C for 30 seconds and 72 ° C for 2 minutes, Followed by reaction at 72 ° C for 5 minutes. The secondary reaction was carried out by using 10 μl of the first reaction product, adjusting the annealing temperature to 55 ° C., and performing the reaction 20 times. The other conditions were the same as the first reaction conditions, and the total amount was adjusted to 50 μl. The PCR reaction products were extracted from 1% agarose gel after electrophoresis and used for vector preparation.

Example  2. Plant expression vector production using the promoter of the pollen-specific expression gene derived from rice

GUS-GFP-tagged gene fusion vector was constructed through the gateway cloning method (Invitrogen, USA) using the 1,784 bp promoter fragment described above in order to test specific expression of rice flower pollen in the following manner. The amplified promoter region was firstly cloned into pGEM-T Easy vector (Promega, USA) and inserted into a gateway pDONR201 entry cloning vector (Invitrogen) containing two recombination sites (AttB1 and AttB2) for BR clonase reaction Secondary cloning. After extracting the pDONR201 plasmid DNA having the 1,784 bp promoter region, the base sequence was analyzed using a primer complementary to the promoter region and the pDONR201 vector region. Using the BIOEDIT program, no nucleotide sequence abnormality was found (Fig. 1A). Through the LR recombination reaction, the 1,784 bp promoter region was finally cloned into the destination vector pKGWFS7 and named pSK815 (Fig. 1B). pKGWFS7 contains the GFP-GUS fused marker gene lacking the promoter region and also has the recombination sites AttB1 and AttB2 for the LR reaction. The final plant expression vector pSK815 was Agrobacterium tumefaciens), the freeze-thaw method (Freeze-thaw) in the strain LBA4404 (An G, 1987, Methods in Enzymology, 153: 292-293 was transformed by using a), was used for transformation of rice plants using tissue culture methods.

Example  3. The plant expression vector pSK815 Transformation of rice plants and Arabidopsis plants using

Agrobacterium strain containing the plant expression vector pSK815 prepared as described above was shake cultured at 28 ° C for 3 days and then used for transformation of rice. (Toki et al, 2006, Plant J. 47: 969-976) reported by Toki et al. In 2006 were used. The seeds of the Japanese rice seedlings, Dongjin seed, cultured in continuous light for 5 days at 32 ° C Lt; / RTI > (Clough SJ and Bent AF, 1998, Plant J. 16: 735-743) using an ecological Colombian type of flowering period to transform Arabidopsis thaliana.

Example  4. Transgenic rice plants GUS  Gene expression analysis

More than 20 T0 generations of rice plants were obtained using the plant expression vector pSK815. As a result of PCR amplification using a primer for amplifying a part of the inserted T-DNA, one copy or more of T- DNA was inserted. Each tissue of the plant was treated with 1 mM X-glu (5-bromo-4-chloro-3-indolyl-β-glucuronide), 100 mM sodium phosphate (pH 7.0), 10 mM EDTA , 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% Triton X-100 for 12 hours at 37 ° C and then reacted with 100% ethanol Chlorophyll was removed. As a result of GUS-tagged gene analysis, strong GUS expression was observed only in the microspheres and mature pollen tissues of T0 generation transformants (Fig. 2). The expression of the GUS-tagged gene begins at a two-cell stage where the nutrient and germ cells divide and is divided into three cellular stages in which the germ cells divide and a mature pollen stage consisting of one feeding cell and two sperm cells And the activity gradually increased (Fig. 2). RNAs of the rice tissues (drug, lobe, stem and root tissue) were extracted and the expression pattern of the OsO5g0543000 gene derived from the promoter used in the present invention was examined. (5'-CATCATCGACAATGGCCGCAAGATCACC-3 '; SEQ ID NO: 6) and Os05g0543000-R primer (5'-TGTCGTACCCTTCCTTGCAGGTGTCCA-3') for RT-PCR analysis using the protein translation partial nucleotide sequence of the rice Os05g0543000 gene and the Os05g0543000-F primer 3 '; SEQ ID NO: 7) was prepared and analyzed (the primer position in the gene is shown in red in FIG. 1A). As a result of the analysis, Os05g0543000 gene was specifically expressed only in the diseased tissues including the developing microsomes , and no expression was observed in the leaves, stem and root tissues grown at 2 weeks, 3 weeks and 4 weeks (FIG. 3A). In addition, leaf, stem and root tissues were collected and the expression of GUS-tagged gene was analyzed. As a result, it was confirmed that the GUS-tagged gene was not expressed in the above tissues (FIG. 3B). Therefore, the promoter used in the plant expression vector pSK815 was found to induce a specific expression only in the rice micropowder / flower pollen developing mature pollen from the two cellular epidermis of rice.

It was confirmed that these expression characteristics were similar to the rice microarray data used for selecting the pollen-specific promoter (Fig. 4). Analyzed microarray data includes developmental and pollen array results in both Indica and Japonica varieties. Sixteen surgical arrays and six pollen arrays were used for the Indica varieties, and 27 surgical arrays and 15 pollen arrays for the Japonica varieties. From the above results, it was confirmed that the rice strain Os05g0543000 used in the present invention was specifically expressed only in the rice microspore and flowerpot, which develop in the micropowder to mature pollen in both rice varieties.

Example  5. Transgenic Arabidopsis thaliana plants GUS  Gene expression analysis

Twenty or more T1 generation Arabidopsis thaliana transformants were obtained using the plant expression vector pSK815. As a result of PCR amplification using a primer that amplifies a part of the inserted T-DNA, a T -DNA was inserted. The tissues of each T1 generation transgenic plant were stained by the same method as the method for staining the rice transformants, and the expression pattern of GUS-labeled gene was examined by each tissue and developmental stage. As a result of analysis of the expression of the GUS gene by collecting buds containing all of the flowering flowers from the young glume, the activity of the GUS enzyme was specifically observed only in the drug tissues containing the microparticles and pollen (FIG. 5A ). In order to investigate the expression patterns of GUS - tagged genes in the stages of development of the microspores, the microspheres collected by size were dissected and the released microspheres were classified into 1 cell, 2 cell and 3 cell sporulation and mature pollen by DAPI staining. The Arabidopsis thaliana transformant showed the highest level of GUS enzyme activity in the three cellular stages and the highest level in the mature pollen stage as the GUS gene was expressed in the two cell stage, (Fig. 5B). RNA was extracted from Arabidopsis thaliana (flowering flowers, pre-flowering buds, leaves, stems, roots and seedling tissues) and the expression of GUS gene introduced into Arabidopsis thaliana was examined. GUS-F primer (5'-CGTCCTGTAGAAACCCCAACCC-3 '; SEQ ID NO: 8) and GUS-R primer (5'-GCATTACGCTGCGATGGATTCCG-3') for the RT-PCR analysis using the GUS gene sequence, 9) were constructed and analyzed. As a result, it was confirmed that the GUS gene was strongly expressed in flowering buds containing mature pollen and pre-flowering buds, and not expressed in other tissues (FIG. 5C). The results showed that the promoter used in the plant expression vector pSK815 induces the expression specifically in the Arabidopsis vesicles and the pollen of the Arabidopsis developing in the mature seedlings at the two cellular epidermal stage.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Microspore or pollen specific expression promoter from Oryza          sativa Os05g0543000 gene and uses thereof <130> PN13237 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 1784 <212> DNA <213> Oryza sativa <400> 1 caaggactgt aaaattgctc tttcatttta gttcaaactt atccttgaac caacaactct 60 cttcataaat gacacttata tccctcatca ttcacatata taagagaaaa attgcatgct 120 tgaatttttt atagataata catagtatat tatgtaaaac gaaaaaaaat tatgcactaa 180 caagtgtata cgatagccat tgcttaagtt tcatgtgcac atattgaaat aatgggaagt 240 acatctataa tgttgtttat tcatgttggc tttctctttt ctagtatata gatgtataca 300 aattaagggc aaaaatgata ttttctaata gaaattattga ctagaaatag ccatatggca 360 tattaagtgg acccaaggat gattttaagc caaaacaaat atttgaagga cctatgtgga 420 caaaatgaaa cgtgaaggac caaattgagc cttcgctaaa acttgaggga ctaaattagc 480 tattcaccca tattttttca cattcgtgtg tttctaaaat cctgcattcc aaaaaagctg 540 ggattttgtg cacatatagt tacatggaca aatttccctc tactttgaga gaggggtagg 600 agttggtact cttagttagg agcatttact tctatagagt aaaacatgta gtaagccttc 660 aaatttatta ataaaaagat aactgtcgct cgatcaagaa tctttaccca ttttgcacat 720 agcaccatgc ttttccttcc taagtcctca aatataatgc taacttatct ttttttaata 780 cgattccaat tattgtttaa gatgtgagct ttgttttatt atgaataatt agatagcata 840 taattattgg gagtcactaa cccaaaccca ttttcagatc aaacttgaga ttgtagtaat 900 tacaacgata tctagattac atatgaacaa taacattcat atatcaatac ttgtattgta 960 atattgaagg tacatatgaa gctacattag tattttgtat atacataggc tgcgttcggt 1020 tgttggtgtt gggaactaaa tccgtcacgc acgtaaaaca gaaagattta ttagcatata 1080 gctaattaag tattaactat tttttaaaaa atgcattaac atgattttct aaagcaactt 1140 tcgtatagat ttttttaaga acatactgtt tagtagtttg ggaagcgtgt acatggaaaa 1200 tgagggagat gagttaggca gatagggtaa ataacacaac catagagttg catctaatcc 1260 tttagaactt tgattttggt tgacgataga actaatgaat cccactgtaa ggcgcacaca 1320 tggtgccaac tagtttaatt aaaatgttat ggcgggcaag cattgcggaa atcatggtat 1380 agatttttga catgttgccc ttctgtactc ttataaaaaa ataaataaac tagctggatg 1440 gtggtgcaaa ttatacggct agatctaatt tatttttaga aaacaaatca tacatcgcta 1500 attttggcgt tgtcgaaaca ttcataaaaa aaaaataagt tacaagttta attttaaaaa 1560 atatgtacaa agttcttata aacaataaaa gaaaaaaaaa aggcaacacc aagtttagcc 1620 aagacagcct gaaaccaagg agagcccatg gtatgcaacc accaaaatct gaaaataacc 1680 atgacccatg gcatgcaacc acccaacctg aaagaaacca tggccattca tttaccttta 1740 cctctcccct atataaaccg tactcagctt gtcggagaag ccaa 1784 <210> 2 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 caaggactgt aaaattgctc tttcatttta gttcaaac 38 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ttggcttctc cgacaagctg agtacggtt 29 <210> 4 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aaaaagcagg ct 12 <210> 5 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 agaaagctgg gt 12 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 catcatcgac aatggccgca agatcacc 28 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tgtcgtaccc ttccttgcag gtgtcca 27 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cgtcctgtag aaaccccaac cc 22 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gcattacgct gcgatggatt ccg 23

Claims (11)

Rice consisting of the nucleotide sequence of SEQ ID NO: 1 ( Oryza sativa ) Os05g0543000 gene-based vesicle or pollen-specific expression promoter. A vesicle or pollen-specific plant expression vector comprising the vesicle or pollen-specific expression promoter of claim 1. Plant of Gramineae transformed with the microspheres or pollen-specific plant expression vectors of claim 2. Recombining the exogenous gene into the vesicle or pollen-specific plant expression vector of claim 2; And
Wherein the recombinant plant expression vector is transformed into a Gramineae plant or a Brassicaceae plant, wherein the exogenous gene is expressed specifically in a microspore or flowerpot, and a method for producing a plant or a crucifer plant. delete delete A transgenic plant (Gramineae) in which the foreign gene produced by the method of claim 4 is expressed in a microspore or pollen-specific expression. A transformed seed of a transgenic plant (Gramineae) of claim 7. A Brassicaceae plant transformed with the vesicle or pollen-specific plant expression vector of claim 2. A transformed Brassicaceae plant wherein the foreign gene produced by the method of claim 4 is expressed in a microspore or pollen-specific expression. A transformed seed of a transgenic Brassicaceae plant of claim 10.

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