CN112941098B - Arabidopsis thaliana anther tapetum promoter expression vector and construction method and application thereof - Google Patents
Arabidopsis thaliana anther tapetum promoter expression vector and construction method and application thereof Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/823—Reproductive tissue-specific promoters
- C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
Abstract
The construction method of the arabidopsis thaliana anther tapetum promoter expression vector comprises the following steps: defining about 1380bp sequence before the AT1G75050 protein coding region as the gene promoter, cloning the promoter, connecting the promoter into an expression vector containing a GUS reporter gene, transforming an arabidopsis thaliana plant, screening and identifying to finally obtain a transgenic plant. The invention clones the arabidopsis gene promoter AT1G75050 promoter, starts the construction of GUS expression vector, transforms the GUS expression vector into plants for research, analyzes the screened plants to find that GUS gene is specifically expressed in anther tapetum, and the transgenic material can be used for the separation and research of tapetum cells. On the other hand, the promoter can promote the specific expression of related genes in a tapetum and create more male sterile materials related to pollen development.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to an arabidopsis thaliana anther tapetum promoter expression vector, and a construction method and application thereof.
Background
Fertilization of plants is crucial to fructification, the function of pollen is particularly critical in the double fertilization process, and many breeding techniques are developed by using male sterility, such as three-line hybrid rice, photosensitive sterile rice, and the like. Mature pollen comes from anthers, of which tapetum cells, which are in direct contact with pollen and located in the innermost layer, play an important role in the development of pollen. There are many unknown functions of the tapetum, and it is an effective research means to directly obtain tapetum cells for omics analysis, but how to label and separate the tapetum is the first difficulty to overcome. In addition, the specific expression of related genes in the tapetum is also a strategy for creating male sterile materials. In conclusion, the search for tapetum-specific gene promoters becomes particularly critical.
Disclosure of Invention
The invention aims to construct an arabidopsis thaliana anther tapetum promoter expression vector as a marker of anther tapetum tissues for separating, researching or creating a male sterile material of tapetum cells.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the construction method of the arabidopsis thaliana anther tapetum promoter expression vector comprises the following steps: cloning an arabidopsis gene AT1G75050 promoter sequence, wherein the sequence is shown as SEQ ID No.1, or a nucleotide sequence which is formed by replacing, deleting or adding one or more bases and has the same function; and (3) connecting the AT1G75050 promoter with a Gateway expression vector of a GUS reporter gene.
The invention also provides the vector and engineering bacteria, transgenic plants or cells containing the vector.
The invention also provides the application of the vector in marking tapetum cells and the application in preparing male sterile materials.
Compared with the prior art, the invention has the beneficial effects that:
the invention clones the arabidopsis gene promoter AT1G75050 promoter, starts the construction of GUS expression vector, transforms the GUS expression vector into plants for research, analyzes the screened plants to find that GUS gene is specifically expressed in anther tapetum, and the transgenic material can be used for the separation and research of tapetum cells. In addition, the promoter can be connected with other genes to create more genetic materials related to pollen development. These are of great significance in production.
Drawings
FIG. 1 is a vector map of pMDC163 in example 1 of the present invention;
FIG. 2 is a GUS vector map of AT1G75050p in example 1 of the present invention;
FIG. 3 is a picture of GUS staining of anthers of transgenic Arabidopsis thaliana observed in example 3 of the present invention;
FIG. 4 is a picture of anther internal structure observed in anther paraffin section at different developmental stages.
The specific implementation mode is as follows:
the project embodiments provided by the invention are used for illustrating the invention, but are not limited to the research scope of the invention. The technical means in the practice of the invention are, unless otherwise indicated, techniques commonly employed in the research field, and the starting materials used are commercially available.
Example 1 obtaining of arabidopsis AT1G75050 gene promoter sequence and construction of plant expression vector.
Logging in arabidopsis thaliana information resource database website
https:// SEQ viewer.arabidopsis.org/servlets/svactionum ═ cycle & pid ═ 0& option2 ═ 0& on ═ 0, found the sequence of AT1G75050 gene, the sequence 1386bp before the start codon was selected to be defined as the promoter region (SEQ ID No.1) of the gene, according to the design of PCR amplification primers,
AT1G75050p-F:5'-CACCgaagcttcgagttagatacagtc-3'
AT1G75050p-R:5'-tcgccatctttgttttaacgagttttgtgtg-3'
culturing wild arabidopsis Col-0, extracting arabidopsis leaf DNA, and amplifying a target sequence by taking the DNA as a template.
The specific steps for extracting the plant DNA are as follows:
(1) will cultivate to the plant number about 3 weeks, cut tender blade one slice with the scissors, place in writing the 1.5mL centrifuging tube of corresponding serial number, directly fully grind with the grinding rod in the centrifuging tube.
(2) Add 400. mu.L of the extract to the centrifuge tube, grind it further, then take out the grinding rod, mix the extract well with a shaker, centrifuge it for 5 minutes at 13000rpm at room temperature.
(3) Numbering a large number of 1.5mL centrifuge tubes, adding 300 μ L of isopropanol corresponding to the number of the previous extractive solution, pouring the supernatant of the extractive solution into the centrifuge tubes, slightly inverting and mixing, and standing at 4 deg.C or-20 deg.C for at least 30 min.
(4) Centrifuge at 13000rpm for 10 minutes at room temperature and pour off the supernatant.
(5) Add 300. mu.L 70% ethanol and mix by inversion.
(6) Centrifuge at 13000rpm for 3 minutes at room temperature and decant the ethanol.
(7) And opening the centrifugal tube, standing at room temperature for a period of time until the ethanol is completely volatilized, and adding 60 mu L of double distilled water for redissolving.
The formula of the plant DNA extracting solution comprises the following steps:
obtaining a promoter sequence of arabidopsis AT1G 75050: PCR was performed according to Phusion high fidelity cloning enzyme amplification system and reaction program. Cloning the PCR product to pENTR/D/TOPO vector entry vector, and sequencing to obtain the complete same sequence as the target gene. The AT1G75050 promoter was constructed by LR reaction on the plant expression vector pMDC163 (FIG. 1), pMDC163 was purchased from a company, and finally the vector AT1G75050p:: GUS (FIG. 2) was obtained, the complete sequence of which is shown in SEQ ID No. 2.
Example 2 acquisition of transgenic Arabidopsis plants
Firstly, culturing the plant to the flowering period, then transforming by using an agrobacterium dipping method, and screening the transgenic plant after collecting seeds.
The method for sterilizing and cultivating the surfaces of the arabidopsis seeds comprises the following steps:
(1) and (4) screening excessive impurities by using a sieve, only reserving seeds, and subpackaging the seeds into 1.5mL centrifuge tubes. The seeds were treated with 70% ethanol (in sterile tween water) for 5 minutes in a clean bench, then 2.6% NaClO (in sterile tween water) for 10 minutes, then rinsed four times with sterile tween water, and then spread evenly on 1/2MS solid medium.
(2) Vernalizing in a refrigerator at 4 deg.C for 3 days, culturing in a long-day illumination incubator for more than 4 days until the seeds germinate and grow larger true leaves, and transplanting the plantlets into a greenhouse.
(3) Vermiculite was filled with pots and then the pots were placed in large pots, one for each 20. And pouring a proper amount of nutrient solution containing a lot of fertilizers for flowers into the culture pot, and transplanting the seedlings when the vermiculite is completely soaked by the nutrient solution.
(4) Pricking several small holes on the surface of vermiculite by using a pair of tweezers, then selecting seedlings with good growth conditions, slightly supporting the seedling leaves, putting the seedling leaves into the small holes, covering the roots and hypocotyls of the seedlings with the vermiculite, and only leaving two cotyledons exposed outside the vermiculite. Five seedlings are transplanted in each nutrition pot. And covering the whole nutrition pot with a transparent preservative film after transplanting so as to preserve moisture. Several holes are cut on the plastic wrap to facilitate air permeability.
(5) After the seedlings survive, the preservative film is removed when the seedlings grow to about three weeks. Watering and deinsectization are needed in time in the whole growth process of the plants.
The genetic transformation of arabidopsis adopts an inflorescence dip-dyeing method, and the method comprises the following steps:
(1) the arabidopsis thaliana can be used for infection after flowering, and infected plants preferably grow to be strong. Before infection, the fruit pods and the flowers which are already opened need to be cut off, and a proper amount of nutrient solution needs to be poured in advance.
(2) Agrobacterium infection used: the agrobacterium is cultured by about 4mL of YEP liquid culture medium one day in advance, 1-2 mL of cultured turbid bacterial liquid is added into 100mL of YEP culture medium, shaking culture is carried out on a table at 28 ℃ overnight, and the bacterial liquid can be used for infection when the color of the bacterial liquid is turbid orange.
(3) The bacterial solution was dispensed into a large centrifuge tube, centrifuged at 4000rpm for 10 minutes at room temperature, the supernatant was removed, and 100mL of infection was used to suspend the bacteria.
(4) And (3) fully soaking the arabidopsis inflorescence into the staining solution, and slightly shaking the inflorescence up and down to facilitate full contact between the staining solution and the inflorescence. The infection time is generally 1 minute. Infected Arabidopsis plants were placed flat in a clean culture pot. After infection, black plastic bags were covered outside the culture pots to protect from light. After the plants are placed in the greenhouse for 24 hours, the plants are taken out and put back into the original culture pot for continuous culture, and the plants are waited for harvest. If the infected plant grows many buds again after one week of growth, it can be infected a second time.
The formula of the arabidopsis staining solution is as follows:
the pH was adjusted to around 5.7 with 1M KOH.
Screening of transgenic plants:
(1) seeds collected by infected plants are T0 generations, and the seeds are spread on 1/2MS solid culture medium added with hygromycin after surface disinfection treatment. The seeds may be suitably spread more densely. And (5) culturing for about 10 days under illumination to distinguish resistant plants. The plants containing the resistance generally have larger cotyledons and longer main roots, and the resistant plants are transplanted into culture soil to continue to grow, namely transgenic T1 generations.
(2) And (3) when the seeds of the T1 generation plants are mature, the seeds collected by the single plants need to be harvested, the seeds collected by the single plants are respectively paved on 1/2MS solid culture media added with corresponding antibiotics, and the T-DNA insertion of the T1 generation plants is determined to be single copy by calculating the resistance segregation ratio of the plants.
(3) Single copy transgenic plants with moderate fluorescence were selected for both analytical photography and seed propagation.
EXAMPLE 3 GUS Observation of transgenic Arabidopsis
(1) Taking Arabidopsis thaliana inflorescence as an example, 3mL of 80% acetone fixing solution is put in a vial and is placed on ice for precooling. Taking down the arabidopsis inflorescence by using forceps, directly putting the arabidopsis inflorescence into the fixing solution, and exhausting air in a vacuum pump for 5 minutes to ensure that the fixing solution completely enters plant tissues.
(2) Pouring out the fixing solution, adding GUS staining Buffer, oscillating, pouring out the Buffer after 5 minutes, and washing out the residual acetone fixing solution. This step is repeated.
(3) Adding GUS dye solution, and allowing the dye solution to pass through the material. The cells were stained in the dark at 37 ℃ for several hours. During the period, the dyeing condition should be observed continuously to avoid the observation being affected by too deep dyeing.
(4) After the staining was completed, the GUS staining solution was poured off, and 70% ethanol was added to decolorize the material, mainly to remove chlorophyll. The material can be stored in ethanol for a period of time.
(5) The microscope used for photographing is OLYMPUS stereoscope and common fluorescence microscope, and is observed by tabletting on 70% ethanol, and brightness and contrast are adjusted during photographing. FIG. 3 shows pictures of GUS staining of anthers of the whole inflorescence of transgenic Arabidopsis, where there is no GUS signal in the opened flowers and a strong GUS signal is detected in the developing buds.
The GUS dye solution formula is as follows:
example 4 Observation of Paraffin section of plant anther
The material used in this experiment was preserved in 70% ethanol by GUS staining.
1. Embedding in paraffin wax
(1) The material was dehydrated through gradient ethanol (70%, 80%, 90%, 95%, 100%) for 1 hour per gradient.
(2) Xylene for ethanol, 50% ethanol-50% xylene for 30 minutes, 100% xylene for 30 minutes.
(3) Xylene was replaced with paraffin, 50% xylene-50% paraffin, overnight at 42 ℃, 100% paraffin, 60 ℃ for three days, 2 new waxes were replaced daily, and embedded on the third day.
(4) Slicing with Leica hand paraffin slicer to obtain slices with thickness of 8-10 μm.
(5) And (3) spreading the slices by a 42 ℃ exhibition platform, transferring the slices into a 42 ℃ oven after 30 minutes, and standing for 48-72 hours.
2. Dewaxing and ruthenium red dyeing
Dewaxing, dyeing and mounting were carried out as follows.
Xylene for 5 minutes, 50% xylene-50% absolute ethanol for 5 minutes, absolute ethanol for 3 minutes, 95%, 90%, 80%, 70%, 50% gradient ethanol rehydration, 2 minutes per stage, distilled water for 5 minutes, 0.2g/L ruthenium red staining for 1 minute, distilled water for 5 minutes, 50%, 70%, 80%, 90%, 95%, 1 minute per stage, absolute ethanol for 5 minutes, xylene for 5 minutes, and neutral gum sealing.
The paraffin section thus obtained was observed and photographed with a microscope (Olypmus BH-2). As shown in FIG. 4, the paraffin sections can show the internal structures of the buds and the anthers, A-I in the figure respectively show the internal structures and GUS signal intensities of the buds with different sizes, namely the anthers in different development stages, and the positions shown by arrows are tapetum cells, and strong GUS signals are detected at the positions.
Sequence listing
<110> university of Jiangsu profession
<120> arabidopsis thaliana anther tapetum promoter expression vector and construction method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1386
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 1
gaagcttcga gttagataca gtcgggttta attatctctc acgtttcttt tgcttattat 60
gtacggaaag ataaataagg aaagctgatg actatgaatc atcgtcttcc acttttaagc 120
ttttttagtg agtattagtc agttgttaca ctcagctgat ttgtttacaa agaaataaaa 180
caaaatgatt gatctacgga tacttatcta ttgcattcca tgctgtgtaa gtgagagatc 240
atgtatcttt ctgttcgaaa cggaattttg aacgttcttt accaaagaaa aagtctatag 300
atacattttt catcaactca cttaaaagaa aacttaacag ctattaatac ctgataaaac 360
catggccgca tatatagatt aaaaccgtgt ggacgatgta aactctgatt taagaaacaa 420
atagaattct gattagttaa cattgagcga gccattaatg aaatggccct aagataatga 480
aaagttcaag gaaattaatc gtcacgaccg tttacaaata ataaaaacca tttactactc 540
cgacacacag ttaagtaaat ttatgaaact tgattctatg tttaaggtac tcttctcacc 600
ttttaaattt caaagtctcc cctctgtact atttctgaac ccattcgtgg aatggtgtac 660
acgaatttta gacgacatgt ttgtttttgt tttagagatt aaaacaaaaa cgtattttag 720
agattagacg acatgaaacg tatttttgta aagaaaaaag gaatgctaaa agaaactacc 780
agggaataaa aataatgaaa atagaaagtc caaaacaaaa taataaagga aaatacaaga 840
cacaattgat ggaatcatga gattggaaga ccatcacacg attgtgggat gttcttgcgc 900
aggatcatag ttgtgggttg ttttttttaa cggaacctca cacgttcgtc tggaaaatag 960
tacacgtgcg gggatctctt cacgtctctt tgattttttg gtttttaagt taagaaaaat 1020
attaactatt gggcatgctc tttcaccatt actgttaaaa aaagaaaaat attaactatg 1080
acccacgata tttcctttta accgtcaact ttttaaaaaa ctttagtaaa ataacgcaat 1140
aaataccatt actgccaata aacttcacag agccaaagaa catgggttaa caataggcac 1200
aactacagta tatttttact gtaaactgaa aatatttacc aaaattaaaa atgataacac 1260
tacctcggaa aagcacttat aaatagagga ttggtgcgag taactgacat tcatcatcat 1320
catcatcatc atcatcatca ttcacagaac acacacacac aaaactcgtt aaaacaaaga 1380
tggcga 1386
<210> 2
<211> 12530
<212> DNA
<213> vector (AT1G75050p:: GUS)
<400> 2
aattcccgat ctagtaacat agatgacacc gcgcgcgata atttatccta gtttgcgcgc 60
tatattttgt tttctatcgc gtattaaatg tataattgcg ggactctaat cataaaaacc 120
catctcataa ataacgtcat gcattacatg ttaattatta catgcttaac gtaattcaac 180
agaaattata tgataatcat cgcaagaccg gcaacaggat tcaatcttaa gaaactttat 240
tgccaaatgt ttgaacgatc ggggaaattc gagctcggta gcaattcccg aggctgtagc 300
cgacgatggt gcgccaggag agttgttgat tcattgtttg cctccctgct gcggtttttc 360
accgaagttc atgccagtcc agcgtttttg cagcagaaaa gccgccgact tcggtttgcg 420
gtcgcgagtg aagatccctt tcttgttacc gccaacgcgc aatatgcctt gcgaggtcgc 480
aaaatcggcg aaattccata cctgttcacc gacgacggcg ctgacgcgat caaagacgcg 540
gtgatacata tccagccatg cacactgata ctcttcactc cacatgtcgg tgtacattga 600
gtgcagcccg gctaacgtat ccacgccgta ttcggtgatg ataatcggct gatgcagttt 660
ctcctgccag gccagaagtt ctttttccag taccttctct gccgtttcca aatcgccgct 720
ttggacatac catccgtaat aacggttcag gcacagcaca tcaaagagat cgctgatggt 780
atcggtgtga gcgtcgcaga acattacatt gacgcaggtg atcggacgcg tcgggtcgag 840
tttacgcgtt gcttccgcca gtggcgcgaa atattcccgt gcaccttgcg gacgggtatc 900
cggttcgttg gcaatactcc acatcaccac gcttgggtgg tttttgtcac gcgctatcag 960
ctctttaatc gcctgtaagt gcgcttgctg agtttccccg ttgactgcct cttcgctgta 1020
cagttctttc ggcttgttgc ccgcttcgaa accaatgcct aaagagaggt taaagccgac 1080
agcagcagtt tcatcaatca ccacgatgcc atgttcatct gcccagtcga gcatctcttc 1140
agcgtaaggg taatgcgagg tacggtagga gttggcccca atccagtcca ttaatgcgtg 1200
gtcgtgcacc atcagcacgt tatcgaatcc tttgccacgc aagtccgcat cttcatgacg 1260
accaaagcca gtaaagtaga acggtttgtg gttaatcagg aactgttcgc ccttcactgc 1320
cactgaccgg atgccgacgc gaagcgggta gatatcacac tctgtctggc ttttggctgt 1380
gacgcacagt tcatagagat aaccttcacc cggttgccag aggtgcggat tcaccacttg 1440
caaagtcccg ctagtgcctt gtccagttgc aaccacctgt tgatccgcat cacgcagttc 1500
aacgctgaca tcaccattgg ccaccacctg ccagtcaaca gacgcgtggt tacagtcttg 1560
cgcgacatgc gtcaccacgg tgatatcgtc cacccaggtg ttcggcgtgg tgtagagcat 1620
tacgctgcga tggattccgg catagttaaa gaaatcatgg aagtaagact gctttttctt 1680
gccgttttcg tcggtaatca ccattcccgg cgggatagtc tgccagttca gttcgttgtt 1740
cacacaaacg gtgatacgta cacttttccc ggcaataaca tacggcgtga catcggcttc 1800
aaatggcgta tagccgccct gatgctccat cacttcctga ttattgaccc acactttgcc 1860
gtaatgagtg accgcatcga aacgcagcac gatacgctgg cctgcccaac ctttcggtat 1920
aaagacttcg cgctgatacc agacgttgcc cgcataatta cgaatatctg catcggcgaa 1980
ctgatcgtta aaactgcctg gcacagcaat tgcccggctt tcttgtaacg cgctttccca 2040
ccaacgctga tcaattccac agttttcgcg atccagactg aatgcccaca ggccgtcgag 2100
ttttttgatt tcacgggttg gggtttctac aggacgtaac ataagggact gaccgtaccc 2160
ggggatcgat cctctagagt cgaggcgcgc caagctatca accacttttc gccatctttg 2220
ttttaacgag ttttgtgtgt gtgtgttctg tgaatgatga tgatgatgat gatgatgatg 2280
atgaatgtca gttactcgca ccaatcctct atttataagt gcttttccga ggtagtgtta 2340
tcatttttaa ttttggtaaa tattttcagt ttacagtaaa aatatactgt agttgtgcct 2400
attgttaacc catgttcttt ggctctgtga agtttattgg cagtaatggt atttattgcg 2460
ttattttact aaagtttttt aaaaagttga cggttaaaag gaaatatcgt gggtcatagt 2520
taatattttt ctttttttaa cagtaatggt gaaagagcat gcccaatagt taatattttt 2580
cttaacttaa aaaccaaaaa atcaaagaga cgtgaagaga tccccgcacg tgtactattt 2640
tccagacgaa cgtgtgaggt tccgttaaaa aaaacaaccc acaactatga tcctgcgcaa 2700
gaacatccca caatcgtgtg atggtcttcc aatctcatga ttccatcaat tgtgtcttgt 2760
attttccttt attattttgt tttggacttt ctattttcat tatttttatt ccctggtagt 2820
ttcttttagc attccttttt tctttacaaa aatacgtttc atgtcgtcta atctctaaaa 2880
tacgtttttg ttttaatctc taaaacaaaa acaaacatgt cgtctaaaat tcgtgtacac 2940
cattccacga atgggttcag aaatagtaca gaggggagac tttgaaattt aaaaggtgag 3000
aagagtacct taaacataga atcaagtttc ataaatttac ttaactgtgt gtcggagtag 3060
taaatggttt ttattatttg taaacggtcg tgacgattaa tttccttgaa cttttcatta 3120
tcttagggcc atttcattaa tggctcgctc aatgttaact aatcagaatt ctatttgttt 3180
cttaaatcag agtttacatc gtccacacgg ttttaatcta tatatgcggc catggtttta 3240
tcaggtatta atagctgtta agttttcttt taagtgagtt gatgaaaaat gtatctatag 3300
actttttctt tggtaaagaa cgttcaaaat tccgtttcga acagaaagat acatgatctc 3360
tcacttacac agcatggaat gcaatagata agtatccgta gatcaatcat tttgttttat 3420
ttctttgtaa acaaatcagc tgagtgtaac aactgactaa tactcactaa aaaagcttaa 3480
aagtggaaga cgatgattca tagtcatcag ctttccttat ttatctttcc gtacataata 3540
agcaaaagaa acgtgagaga taattaaacc cgactgtatc taactcgaag cttcaaactt 3600
gttgataatt cttaattaac tagagcttgg cactggccgt cgttttacaa cgtcgtgact 3660
gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 3720
ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 3780
gcgaatgcta gagcagcttg agcttggatc agattgtcgt ttcccgcctt cagtttaaac 3840
tatcagtgtt tgacaggata tattggcggg taaacctaag agaaaagagc gtttattaga 3900
ataacggata tttaaaaggg cgtgaaaagg tttatccgtt cgtccatttg tatgtgcatg 3960
ccaaccacag ggttcccctc gggatcaaag tactttgatc caacccctcc gctgctatag 4020
tgcagtcggc ttctgacgtt cagtgcagcc gtcttctgaa aacgacatgt cgcacaagtc 4080
ctaagttacg cgacaggctg ccgccctgcc cttttcctgg cgttttcttg tcgcgtgttt 4140
tagtcgcata aagtagaata cttgcgacta gaaccggaga cattacgcca tgaacaagag 4200
cgccgccgct ggcctgctgg gctatgcccg cgtcagcacc gacgaccagg acttgaccaa 4260
ccaacgggcc gaactgcacg cggccggctg caccaagctg ttttccgaga agatcaccgg 4320
caccaggcgc gaccgcccgg agctggccag gatgcttgac cacctacgcc ctggcgacgt 4380
tgtgacagtg accaggctag accgcctggc ccgcagcacc cgcgacctac tggacattgc 4440
cgagcgcatc caggaggccg gcgcgggcct gcgtagcctg gcagagccgt gggccgacac 4500
caccacgccg gccggccgca tggtgttgac cgtgttcgcc ggcattgccg agttcgagcg 4560
ttccctaatc atcgaccgca cccggagcgg gcgcgaggcc gccaaggccc gaggcgtgaa 4620
gtttggcccc cgccctaccc tcaccccggc acagatcgcg cacgcccgcg agctgatcga 4680
ccaggaaggc cgcaccgtga aagaggcggc tgcactgctt ggcgtgcatc gctcgaccct 4740
gtaccgcgca cttgagcgca gcgaggaagt gacgcccacc gaggccaggc ggcgcggtgc 4800
cttccgtgag gacgcattga ccgaggccga cgccctggcg gccgccgaga atgaacgcca 4860
agaggaacaa gcatgaaacc gcaccaggac ggccaggacg aaccgttttt cattaccgaa 4920
gagatcgagg cggagatgat cgcggccggg tacgtgttcg agccgcccgc gcacgtctca 4980
accgtgcggc tgcatgaaat cctggccggt ttgtctgatg ccaagctggc ggcctggccg 5040
gccagcttgg ccgctgaaga aaccgagcgc cgccgtctaa aaaggtgatg tgtatttgag 5100
taaaacagct tgcgtcatgc ggtcgctgcg tatatgatgc gatgagtaaa taaacaaata 5160
cgcaagggga acgcatgaag gttatcgctg tacttaacca gaaaggcggg tcaggcaaga 5220
cgaccatcgc aacccatcta gcccgcgccc tgcaactcgc cggggccgat gttctgttag 5280
tcgattccga tccccagggc agtgcccgcg attgggcggc cgtgcgggaa gatcaaccgc 5340
taaccgttgt cggcatcgac cgcccgacga ttgaccgcga cgtgaaggcc atcggccggc 5400
gcgacttcgt agtgatcgac ggagcgcccc aggcggcgga cttggctgtg tccgcgatca 5460
aggcagccga cttcgtgctg attccggtgc agccaagccc ttacgacata tgggccaccg 5520
ccgacctggt ggagctggtt aagcagcgca ttgaggtcac ggatggaagg ctacaagcgg 5580
cctttgtcgt gtcgcgggcg atcaaaggca cgcgcatcgg cggtgaggtt gccgaggcgc 5640
tggccgggta cgagctgccc attcttgagt cccgtatcac gcagcgcgtg agctacccag 5700
gcactgccgc cgccggcaca accgttcttg aatcagaacc cgagggcgac gctgcccgcg 5760
aggtccaggc gctggccgct gaaattaaat caaaactcat ttgagttaat gaggtaaaga 5820
gaaaatgagc aaaagcacaa acacgctaag tgccggccgt ccgagcgcac gcagcagcaa 5880
ggctgcaacg ttggccagcc tggcagacac gccagccatg aagcgggtca actttcagtt 5940
gccggcggag gatcacacca agctgaagat gtacgcggta cgccaaggca agaccattac 6000
cgagctgcta tctgaataca tcgcgcagct accagagtaa atgagcaaat gaataaatga 6060
gtagatgaat tttagcggct aaaggaggcg gcatggaaaa tcaagaacaa ccaggcaccg 6120
acgccgtgga atgccccatg tgtggaggaa cgggcggttg gccaggcgta agcggctggg 6180
ttgtctgccg gccctgcaat ggcactggaa cccccaagcc cgaggaatcg gcgtgacggt 6240
cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg gtgatgacct ggtggagaag 6300
ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg ccccggtgaa 6360
tcgtggcaag cggccgctga tcgaatccgc aaagaatccc ggcaaccgcc ggcagccggt 6420
gcgccgtcga ttaggaagcc gcccaagggc gacgagcaac cagatttttt cgttccgatg 6480
ctctatgacg tgggcacccg cgatagtcgc agcatcatgg acgtggccgt tttccgtctg 6540
tcgaagcgtg accgacgagc tggcgaggtg atccgctacg agcttccaga cgggcacgta 6600
gaggtttccg cagggccggc cggcatggcc agtgtgtggg attacgacct ggtactgatg 6660
gcggtttccc atctaaccga atccatgaac cgataccggg aagggaaggg agacaagccc 6720
ggccgcgtgt tccgtccaca cgttgcggac gtactcaagt tctgccggcg agccgatggc 6780
ggaaagcaga aagacgacct ggtagaaacc tgcattcggt taaacaccac gcacgttgcc 6840
atgcagcgta cgaagaaggc caagaacggc cgcctggtga cggtatccga gggtgaagcc 6900
ttgattagcc gctacaagat cgtaaagagc gaaaccgggc ggccggagta catcgagatc 6960
gagctagctg attggatgta ccgcgagatc acagaaggca agaacccgga cgtgctgacg 7020
gttcaccccg attacttttt gatcgatccc ggcatcggcc gttttctcta ccgcctggca 7080
cgccgcgccg caggcaaggc agaagccaga tggttgttca agacgatcta cgaacgcagt 7140
ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat cgggtcaaat 7200
gacctgccgg agtacgattt gaaggaggag gcggggcagg ctggcccgat cctagtcatg 7260
cgctaccgca acctgatcga gggcgaagca tccgccggtt cctaatgtac ggagcagatg 7320
ctagggcaaa ttgccctagc aggggaaaaa ggtcgaaaag gtctctttcc tgtggatagc 7380
acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat tgggaaccca 7440
aagccgtaca ttgggaaccg gtcacacatg taagtgactg atataaaaga gaaaaaaggc 7500
gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg cctggcctgt 7560
gcataactgt ctggccagcg cacagccgaa gagctgcaaa aagcgcctac ccttcggtcg 7620
ctgcgctccc tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg ccgctcaaaa 7680
atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc gtcgccactc 7740
gaccgccggc gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg acggtgaaaa 7800
cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag 7860
cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac 7920
ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt 7980
gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac 8040
cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 8100
cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 8160
aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 8220
gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 8280
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 8340
agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 8400
ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 8460
taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 8520
gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 8580
gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 8640
ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 8700
ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 8760
gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 8820
caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 8880
taagggattt tggtcatgca ttctaggtac taaaacaatt catccagtaa aatataatat 8940
tttattttct cccaatcagg cttgatcccc agtaagtcaa aaaatagctc gacatactgt 9000
tcttccccga tatcctccct gatcgaccgg acgcagaagg caatgtcata ccacttgtcc 9060
gccctgccgc ttctcccaag atcaataaag ccacttactt tgccatcttt cacaaagatg 9120
ttgctgtctc ccaggtcgcc gtgggaaaag acaagttcct cttcgggctt ttccgtcttt 9180
aaaaaatcat acagctcgcg cggatcttta aatggagtgt cttcttccca gttttcgcaa 9240
tccacatcgg ccagatcgtt attcagtaag taatccaatt cggctaagcg gctgtctaag 9300
ctattcgtat agggacaatc cgatatgtcg atggagtgaa agagcctgat gcactccgca 9360
tacagctcga taatcttttc agggctttgt tcatcttcat actcttccga gcaaaggacg 9420
ccatcggcct cactcatgag cagattgctc cagccatcat gccgttcaaa gtgcaggacc 9480
tttggaacag gcagctttcc ttccagccat agcatcatgt ccttttcccg ttccacatca 9540
taggtggtcc ctttataccg gctgtccgtc atttttaaat ataggttttc attttctccc 9600
accagcttat ataccttagc aggagacatt ccttccgtat cttttacgca gcggtatttt 9660
tcgatcagtt ttttcaattc cggtgatatt ctcattttag ccatttatta tttccttcct 9720
cttttctaca gtatttaaag ataccccaag aagctaatta taacaagacg aactccaatt 9780
cactgttcct tgcattctaa aaccttaaat accagaaaac agctttttca aagttgtttt 9840
caaagttggc gtataacata gtatcgacgg agccgatttt gaaaccgcgg tgatcacagg 9900
cagcaacgct ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc atccgtgttt 9960
caaacccggc agcttagttg ccgttcttcc gaatagcatc ggtaacatga gcaaagtctg 10020
ccgccttaca acggctctcc cgctgacgcc gtcccggact gatgggctgc ctgtatcgag 10080
tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggctggtg gcaggatata 10140
ttgtggtgta aacaaattga cgcttagaca acttaataac acattgcgga cgtttttaat 10200
gtactgaatt aacgccgaat taattcgggg gatctggatt ttagtactgg attttggttt 10260
taggaattag aaattttatt gatagaagta ttttacaaat acaaatacat actaagggtt 10320
tcttatatgc tcaacacatg agcgaaaccc tataggaacc ctaattccct tatctgggaa 10380
ctactcacac attattatgg agaaactcga gcttgtcgat cgacagatcc ggtcggcatc 10440
tactctattt ctttgccctc ggacgagtgc tggggcgtcg gtttccacta tcggcgagta 10500
cttctacaca gccatcggtc cagacggccg cgcttctgcg ggcgatttgt gtacgcccga 10560
cagtcccggc tccggatcgg acgattgcgt cgcatcgacc ctgcgcccaa gctgcatcat 10620
cgaaattgcc gtcaaccaag ctctgataga gttggtcaag accaatgcgg agcatatacg 10680
cccggagtcg tggcgatcct gcaagctccg gatgcctccg ctcgaagtag cgcgtctgct 10740
gctccataca agccaaccac ggcctccaga agaagatgtt ggcgacctcg tattgggaat 10800
ccccgaacat cgcctcgctc cagtcaatga ccgctgttat gcggccattg tccgtcagga 10860
cattgttgga gccgaaatcc gcgtgcacga ggtgccggac ttcggggcag tcctcggccc 10920
aaagcatcag ctcatcgaga gcctgcgcga cggacgcact gacggtgtcg tccatcacag 10980
tttgccagtg atacacatgg ggatcagcaa tcgcgcatat gaaatcacgc catgtagtgt 11040
attgaccgat tccttgcggt ccgaatgggc cgaacccgct cgtctggcta agatcggccg 11100
cagcgatcgc atccatagcc tccgcgaccg gttgtagaac agcgggcagt tcggtttcag 11160
gcaggtcttg caacgtgaca ccctgtgcac ggcgggagat gcaataggtc aggctctcgc 11220
taaactcccc aatgtcaagc acttccggaa tcgggagcgc ggccgatgca aagtgccgat 11280
aaacataacg atctttgtag aaaccatcgg cgcagctatt tacccgcagg acatatccac 11340
gccctcctac atcgaagctg aaagcacgag attcttcgcc ctccgagagc tgcatcaggt 11400
cggagacgct gtcgaacttt tcgatcagaa acttctcgac agacgtcgcg gtgagttcag 11460
gctttttcat atctcattgc cccccgggat ctgcgaaagc tcgagagaga tagatttgta 11520
gagagagact ggtgatttca gcgtgtcctc tccaaatgaa atgaacttcc ttatatagag 11580
gaaggtcttg cgaaggatag tgggattgtg cgtcatccct tacgtcagtg gagatatcac 11640
atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg atgctcctcg 11700
tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg atagcctttc 11760
ctttatcgca atgatggcat ttgtaggtgc caccttcctt ttctactgtc cttttgatga 11820
agtgacagat agctgggcaa tggaatccga ggaggtttcc cgatattacc ctttgttgaa 11880
aagtctcaat agccctttgg tcttctgaga ctgtatcttt gatattcttg gagtagacga 11940
gagtgtcgtg ctccaccatg ttatcacatc aatccacttg ctttgaagac gtggttggaa 12000
cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg 12060
cagaggcatc ttgaacgata gcctttcctt tatcgcaatg atggcatttg taggtgccac 12120
cttccttttc tactgtcctt ttgatgaagt gacagatagc tgggcaatgg aatccgagga 12180
ggtttcccga tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg 12240
tatctttgat attcttggag tagacgagag tgtcgtgctc caccatgttg gcaagctgct 12300
ctagccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 12360
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 12420
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 12480
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 12530
Claims (5)
1. The construction method of the arabidopsis thaliana anther tapetum promoter expression vector is characterized by comprising the following steps of: cloning a promoter of an arabidopsis gene AT1G75050, wherein the sequence of the promoter is shown as SEQ ID No. 1; the AT1G75050 promoter was ligated to a Gateway expression vector containing the GUS reporter gene.
2. The arabidopsis thaliana anther tapetum promoter expression vector constructed by the construction method of claim 1.
3. An engineered bacterium comprising the vector of claim 2.
4. Use of a vector according to claim 2 for labelling tapetum cells.
5. Use of a vector according to claim 2 for the preparation of a male sterile material.
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