CN112251435B - Plant pollen specific expression promoter POsPTD1 and application thereof - Google Patents
Plant pollen specific expression promoter POsPTD1 and application thereof Download PDFInfo
- Publication number
- CN112251435B CN112251435B CN202010878167.3A CN202010878167A CN112251435B CN 112251435 B CN112251435 B CN 112251435B CN 202010878167 A CN202010878167 A CN 202010878167A CN 112251435 B CN112251435 B CN 112251435B
- Authority
- CN
- China
- Prior art keywords
- plant
- pollen
- posptd1
- promoter
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pregnancy & Childbirth (AREA)
- Reproductive Health (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a plant pollen specific expression promoter POsPTD1 and application thereof, wherein the nucleotide of the promoter POsPTD1 is shown as SEQ ID NO. 1; the pollen specific promoter POsPTD1 is derived from a promoter of a rice endogenous gene, and can specifically drive a target gene or a DNA fragment to express in pollen; the promoter is used for driving the gene or DNA fragment with the function of controlling the pollen fertility to be specifically expressed in the pollen, so that the gametophyte male sterility can be prepared, and the promoter has important effects on preventing transgene from drifting along with the pollen, breeding sporophyte nuclear sterility and the like.
Description
Technical Field
The invention belongs to the field of genetic engineering, and relates to a plant pollen specific promoter POsPTD1 and application thereof, wherein the promoter can drive a target gene or a DNA fragment to be specifically expressed in pollen, so that the pollen fertility is changed, transgene drift is controlled, male gametophyte sterility is prepared, sporophyte nuclear male sterility is propagated, and the like.
Background
Promoters are generally located upstream of the coding region of a gene, contain cis-acting elements, are specific DNA sequences, and determine the binding of specific transcription factors, thereby achieving transcriptional regulation of the gene. Promoters also provide information on the location of RNA polymerase, which recognizes and binds to the promoter-associated region to initiate transcription of the RNA. Thus, a promoter is essential for gene transcription, and a particular promoter determines the particular expression pattern of the gene to which it is linked. Screening and separating some promoters with high tissue or organ expression level and analyzing the functions of related important specific regulatory elements, and the method has wide application in gene engineering research.
Pollen is one of the two major participants in sexual reproduction of higher plants. Pollen can move with multiple vectors between plant individuals and even species relative to an immobile female gametophyte, which is of exceptional importance for the genetic communication and the accumulation of variation in plants. The development regulation of the pollen needs a plurality of genes to participate, and the genes are specifically expressed in the anther wall, are specifically expressed in the pollen and are expressed in the anther wall and the pollen. The promoter of the gene specifically expressed in pollen is a pollen-specific promoter.
In the utilization of plant heterosis, a male sterile line is an important genetic material, and is especially necessary in the utilization of heterosis of self-flowering propagation crops. The male sterility includes sporophyte sterility and gametophyte sterility, wherein the sporophyte sterility includes sporophyte sterility and sporophyte sterility. Sporophyte genic sterility has greater heterosis utilization potential than sporophyte sterility, but the inherent genetic characteristics make it difficult to propagate a complete sterile population, and a corresponding propagation method of the sporophyte genic sterility complete sterile population must be developed.
The pollen specific promoter can drive the expression of some genes which influence the pollen development in the pollen, influence the normal development of the pollen and can be used for breeding sporophyte male nuclear sterile lines (Wu et al, 2015; Chang et al 2016). The currently found pollen specific promoters are few, and mainly comprise a tomato pollen specific promoter LAT52, a rice pollen specific RA8 gene promoter, a corn ZmC5 gene promoter, a ZM13 promoter, a PG47 promoter and the like.
With the completion of the sequencing of various plant genomes and the disclosure of various gene expression profile data, functional genes which are specifically and efficiently expressed in rice pollen can be screened by means of a public database. The upstream DNA sequences of these functional genes are candidates for pollen-specific promoters.
The invention provides a male gametophyte specific promoter, which is used for preventing transgene from drifting along with pollen and providing genetic resources for male sterile plant genetic engineering and hybrid rice research.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a promoter specifically expressed in plant pollen; the second object of the present invention is to provide a complementary nucleic acid sequence of a plant pollen specific promoter, POsPTD 1; the third object of the present invention is to provide a recombinant expression vector containing the plant pollen specific promoter POsPTD1 or a complementary nucleotide sequence; the fourth object of the present invention is to provide a host cell containing said plant pollen specific promoter POsPTD1 or a complementary nucleotide sequence; the fifth purpose of the invention is to provide the application of the plant pollen specific expression promoter POsPTD1 in the specific driving of the expression of target genes in plant pollen; it is a further object of the present invention to provide a method for driving a specific target fragment in plant pollen; the seventh purpose of the invention is to provide the application of the plant pollen specific expression promoter POsPTD 1.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a plant pollen specific expression promoter, POsPTD1, said plant pollen specific expression promoter POsPTD1 comprising at least:
(a) the nucleotide sequence shown in SEQ ID NO. 1;
(b) or a nucleotide sequence which is derived from the nucleotide sequence shown in SEQ ID NO.1 by substituting, deleting or adding at least one nucleotide and has the same pollen specificity starting function.
2. The complementary nucleic acid sequence of said plant pollen specific promoter POsPTD 1.
3. A recombinant expression vector comprising said plant pollen specific promoter, POsPTD1, or a complementary nucleotide sequence.
4. A host cell comprising said plant pollen specific promoter, POsPTD1, or a complementary nucleotide sequence.
5. The plant pollen specific expression promoter POsPTD1 is applied to the specific driving of the expression of target genes in plant pollen.
In the invention, the plant is a flowering plant, such as a gramineae plant, and can also be other flowering plants; preferably rice, maize, sorghum, wheat, barley, oats, rye or oilseed rape.
6. The method for driving the expression of the specific target segment in the plant pollen comprises the following specific steps:
(1) linking the plant pollen specific expression promoter POsPTD1 of claim 1 to the fragment of interest such that POsPTD1 is upstream of the fragment of interest, and then loading into a plant expression vector;
(2) and (2) transforming the plant expression vector obtained in the step (1) into a plant through agrobacterium tumefaciens mediation, and screening a transgenic plant to obtain a plant which drives the expression of a specific target segment in the pollen.
In the invention, the plant is a flowering plant, such as a gramineae plant, and can also be other flowering plants; preferably rice, maize, sorghum, wheat, barley, oats, rye or oilseed rape.
Preferably, the target fragment is a structural gene or a regulatory gene, or a mutant gene of a structural gene or a regulatory gene, or a downregulated expression element of a structural gene or a regulatory gene.
7. The application of the plant pollen specific expression promoter POsPTD1 is any one of the following:
(1) cultivating a plant variety of which transgenic components do not drift along with pollen;
(2) cultivating male gametophyte sterile plant material;
(3) breeding male sporophyte genic sterile line.
The invention has the beneficial effects that: the promoter and the interference fragment are from rice, so that exogenous transgenic components can be reduced, transgenic risk is reduced, and worry of people about transgenic products is reduced; the rice promoter POsPTD1 obtained by the invention can regulate the specific expression of genes in the male gametophyte of the plant, and the promoter drives the expression of genes or DNA fragments which can influence the pollen fertility, so as to destroy the fertility of the pollen containing transgenic components, and can prepare male gametophyte sterility which is used for propagating a sporophyte genic sterile line; the invention has important value for researching male gametophyte sterility mechanism, blocking pollen-mediated gene drift, breeding sporophyte genic sterility and other aspects.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows the analysis of gene expression profiles related to male gametophyte development (A is qPCR analysis of Loc _ Os05g40740 gene expression in various wild organs and flower development stages and in the late stage of interference homozygous progeny flower development; B is GUS staining detection of promoter POsPTD1 driving GUS gene expression specifically in pollen to show GUS staining of transgenic heterozygote pollen; C is GUS staining detection of promoter POsPTD1 driving GUS gene expression specifically in pollen to show GUS staining of transgenic homozygote pollen; black arrow indicates GUS positive pollen, white arrow indicates GUS negative pollen).
FIG. 2 is a schematic diagram of the restriction enzyme cutting sites of the vector pOsMYB76R after the multiple cloning sites are modified;
FIG. 3 shows transgene T0Generation positive strain hybrid F1Transmission of the medium purple line: the vast majority of plants without purple line and with few purple line shown by arrows
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1 cloning of Rice Male gametophyte-specific promoter POsPTD1
The male gametophyte development-associated gene is often expressed in flowers/pollen in the middle and late stages. In order to find some gametophyte development related genes, a BAR rice gene expression database (http:// BAR. utontono. ca/efprice/cgi-bin/efpWeb. cgi) is analyzed, a plurality of genes expressed in flowers at the middle and later stages are found, and a batch of genes possibly related to the development of male gametophytes are selected through qPCR verification. The rice Loc _ Os05g40740 gene is specifically and highly expressed in late flowers, the arabidopsis thaliana homologous gene is specifically expressed in pollen, the Loc _ Os05g40740 gene is listed as a male gametophyte development related gene preliminarily, and the promoter thereof is listed as a pollen specific promoter. Subsequently, the spatiotemporal expression pattern of the gene was analyzed by qPCR using qPCR primers for Loc _ Os05g40740 gene: upstream primer MQF 2: 5'-ggcgtcttcaggtacaaccag-3' (SEQ ID NO.4), and downstream primer MQR25'-ggtacacctggaccgagtgc-3' (SEQ ID NO. 5). The internal reference primer is an upstream primer OsActin 2F: 5'-ctctgtatgccagtggtcgt-3' (SEQ ID NO.6), downstream primer OsActin 2R: 5'-ccgttgtggtgaatgagtaac-3' (SEQ ID NO. 7). The qPCR results show that: the Loc _ Os05g40740 gene was not expressed in roots, stems, leaves, leaf sheaths and seeds, and was highly expressed in late mid-development flowers (fig. 1). Further analyzing the expression of the gene at the later development stage of the interference homozygous plant flowers, and finding that the interference causes the serious reduction of the expression of the gene.
Subsequently, in order to determine whether the gene is specifically expressed in pollen, the promoter of the gene was cloned and placed in front of the GUS gene of pCAMBIA 1301. The specific operation is as follows:
with promoter upstream primer P5F 1: 5'-gcgtcgacgtcccatgtcaccgacagtact-3' (SEQ ID NO.8 with a cleavage site SalI) and the reverse primer P5R 1: 5'-catgccatggcgtggaaatgtgatcgctaggct-3' (SEQ ID NO.9, with restriction site NcoI) the promoter of Loc _ Os05g40740 was amplified from Zhonghua 11, the nucleotide sequence of which is shown in SEQ ID NO. 1. The vector pCAMBIA1301 was preceded by GUS gene using SalI and NcoI (incomplete digestion) to obtain p POsPTD 1-GUS.
Example 2 identification of pollen-specific GUS staining of Rice pollen-specific promoter POsPTD1
POsPTD1-GUS was transferred into Agrobacterium LBA4404, and positive Agrobacterium was transfected into middle flower 11. GUS staining observation is carried out on roots, leaves and flowers of transgenic positive offspring in the flowering period, and the result shows that: the roots, leaves, glumes, filaments, pistils, etc. are not dyed, while the anthers are dyed blue. The anthers were further mashed and observed under a microscope to see: the transgenic heterozygous plants had only about 50% of GUS-stained pollen, and the homozygous plants had almost all pollen stained by GUS (FIG. 1). The above results indicate that the promoter POsPTD1 is indeed a pollen-specific promoter.
Example 3 preparation of Male gametophyte sterility and identification by POsPTD 1-driven RNA interference
In order to determine whether the POsPTD1 promoter can drive a target gene or a DNA fragment to destroy pollen fertility, a male gametophyte sterile material is prepared and identified. The creation and identification of male gametophytic sterility were carried out by targeting Loc _ Os05g40740 and driving the interference fragment of Loc _ Os05g40740 gene with the potsttd 1 promoter:
1. in order to facilitate loading of related target genes or DNA fragments and accurately and directly identify transgenic positive strains and male gametophyte sterility, a GUS gene is replaced by a modified gene OsMYB76R of a rice anthocyanin synthesis related gene, and a multiple cloning site of a vector pCAMBIA1301 is modified, and the specific operations are as follows:
(1) OsMYB76 gene was modified and replaced with GUS: the OsMYB76 gene is an essential gene for anthocyanin synthesis in rice and can be used as an endogenous reporter gene; in order to avoid the influence of enzyme cutting sites carried by the OsMYB76 gene on loading of a target fragment, common enzyme cutting sites in the OsMYB76 gene are removed through base substitution, a CDS sequence of the OsMYB76R gene after the OsMYB76 gene is modified is obtained and artificially synthesized, and the sequence is shown as SEQ ID No. 10. Taking the synthesized OsMYB76R gene as a template, and carrying out reaction by using a primer OsMYB76 RF: 5'-ggactcttgaccatgatgggacgcagggcttgct-3' (SEQ ID NO.11, BglII-near homologous fragment with pCAMBIA 1301) and OsMYB76 RR: 5'-attcgagctggtcactcacgcacacaggttccaag-3' (SEQ ID NO.12, BstEII near homologous fragment with pCAMBIA 1301) to obtain the target fragment, and ligating the target fragment into pCAMBIA1301 by homologous recombination to obtain vector pOsMYB 76R.
(2) And (3) multi-cloning site modification: the kind and sequence of the enzyme cutting sites of the multiple cloning sites of the vector pOsMYB76R are modified. Firstly, a new multiple cloning site sequence is artificially synthesized, and the nucleotide sequence is shown as SEQ ID NO. 13. The synthetic sequence was then replaced by homologous recombination with the original sequence of the multiple cloning site, and the new multiple cloning site was shown in FIG. 2. The vector after the multi-cloning site modification is named as pOsMYB76 RM.
2. Using stem-loop structure upstream primer LF 2: 5'-gaagatctacgagctggtgagctagcta-3' (SEQ ID NO.14, with restriction site BglII) and the downstream primer LR 2: 5'-ctggtcacccctcaaacctgaaaattcag-3' (SEQ ID NO.15, with restriction site BstEII) the first intron of the OsMYB76 gene was amplified from R25 as an interference stem-loop (SEQ ID NO.2) and loaded into pOsMYB76RM to obtain vector pOsMYB76 RL.
3. With the interference left arm forward primer I5F 1: 5'-cggaattcgcagaaggtgatcctgatcaac-3' (SEQ ID NO.16, with restriction site EcoRI) and the reverse primer I5R 1: 5'-gaagatctgcaggcggctgacgacgctgat-3' (SEQ ID NO.17, with restriction site BglII) from Zhonghua 11 expanded the interference left arm of Loc _ Os05g 40740; with the interfering right arm forward primer I5F 2: 5'-gacctgcagggcagaaggtgatcctgatcaac-3' (SEQ ID NO.18 with an enzyme cleavage site SbfI) and the reverse primer I5R 2: 5'-ctggtcaccgcaggcggctgacgacgctgat-3' (SEQ ID NO.19, with restriction site BstEII) the interfering right arm of Loc _ Os05g40740 was amplified from middle flower 11. The left arm and the right arm are respectively loaded at two sides of a stem loop in a vector pOsMYB76RL, and a Tnos terminator is loaded behind the interference right arm by using SbfI and AscI to obtain the vector pOsMYB76Rli, wherein the nucleotide sequence of the interference fragment is shown as SEQ ID NO. 3.
4. With promoter upstream primer P5F 1: 5'-gcgtcgacgtcccatgtcaccgacagtact-3' (SEQ ID NO.8 with a cleavage site SalI) and the reverse primer P5R 2: 5'-cggaattccgtggaaatgtgatcgctaggct-3' (SEQ ID NO.20, EcoRI with the restriction site) the promoter of Loc _ Os05g40740 was amplified from Zhonghua 11, the nucleotide sequence of which is shown in SEQ ID NO. 1. Loading it into vector pOsMYB76RLi to obtain male gametophyte sterile vector pOsMYB 76R-GM.
5. pOsMYB76R-GM was transferred into Agrobacterium LBA4404, and positive Agrobacterium was transfected into colorless ZhongjiuB in all organs.
6. At T0And selecting positive plants according to the existence of purple stem base, selfing, and pollinating the middle nine B which is not transgenic by using the positive plants as male parents. The purple line investigation is carried out on the inbred and hybrid seeds, and the inbred F is found2The separation ratio of the seed purple line is 1:1, and is not 3:1, and the cross F of 96.4 percent1The seeds showed no purple line (FIG. 3), indicating that the transgene components are difficult to transfer with pollen between generations, but with female gametes, which is characteristic of male gametophyte sterility. Thus, Loc _ Os05g40740 was involved in male gametophyte pollen development, and by interfering with this, male gametophyte sterile material was obtained and successfully genetically identified by genetic investigation of linked purple line traits.
F used for genetic analysis of the above-described created male gametophyte sterile material1The seed can also realize the identification of the probability of the transgenic components transmitted by the pollen by investigating the proportion of purple line individuals, and can screen out the transgenic pollution transmitted by the pollen by eliminating individuals with purple lines.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of Yunnan
<120> plant pollen specific expression promoter POsPTD1 and application
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1887
<212> DNA
<213> Rice (oryza. sativa L.)
<400> 1
gtcccatgtc accgacagta ctaaatgggt aaagattgga taaagtatat ggggtatttg 60
tgaggtatta ttagaaaact tcgtgtggtt ttgatggacc tgttttatgt gttgaaaata 120
tgaatggtta tagggtgtgt ttgcaagtgc aggatgggaa ctcatccctc ctgcacgcaa 180
aacggagcgg ctttttaaca catgattaat taaatattag ctaatttttt taaaaaaaat 240
ggattaattt gattttttta agcaactttc atatagaaat tttttgcaaa aaacacaccg 300
tttaatagtt taaaaacgtg cgcgcgaaaa acgagggaga ggggttggga acatgggttt 360
gcaaacacaa ccatagtatt ggcgattcct tttcgtttga gtaaatttta caaaactaca 420
ggtattttga ccaaattatc acaaaactac agatttaagg agttgtatca taaaactaca 480
catttagcat caaatttatc acaaaactgc agattttagg ttaagtatca caaaaataca 540
tatttaatat tgaacttatc acaaaactat aacttttgga gtttaaatcc ctagcaccat 600
tgttatggtg gagctataaa cattattact ttgtgattaa attggttcta aacctttagt 660
tttatgataa tttagtaact aaacgtgtag ttttgtaaca cttcatcttt aatatgtagt 720
tttgtgctaa atttggtgct aaatgtgtaa ttttgtgata taattcctta aatatgtagt 780
tttgtgatag tttggttata atatctgtag ttttatgaaa tttactcttt tcgttttcac 840
tgcaatttgg aatgatggaa ttgactagat ccggcattac cgatgggctg ccgaacgctg 900
tgatgcggtt gatcttgagc gatccgggac gccacaagca ccgatgggtt ctgggagttc 960
atacggctgg tgcagcagtg tgtcaatagc agccgggatg tgcgcccaac catggtcgcc 1020
gtcgagagga ggatcgaaga catcctgaac tcggttgtca ggtcatccac caccgggttc 1080
atgactgccg gaggcgacac acccagcaac gagccaaatc gtgaagataa cggaaacgag 1140
ccaaatccca gcaacgagat cgccagggac tagtagtacg tacagcagtg gtgatttgtc 1200
atataggtgt atatcggctg ttttcgcatc tcaaggcctc aagcagtgtg tgcaatctgg 1260
agtagtatat aaatatgtaa aatgttcatt tcgatatact gtcaaatgcg tgtaaattaa 1320
ccaatgctaa aacaacacac tgtgactaaa tttactgagt tggatgatga ggatgattat 1380
gttgcgtgca cacctgatca ggaggacata taatataggc catttgggcc gtcttggaca 1440
ccaccgtttg atttgtatga agttgggccg aactatgcaa gcccagaggc gctgcctctg 1500
tgccacggcc cacgggcatc gctggatggt caagcaggtg atcggtggag cgccaatggc 1560
ggcggcgaga cacacagcgc ggcgcgcgcg cgaacgtgcg gacgcgcgcg ccccggccac 1620
ggccgccgcg ctcgtctcct ggcctcccgc gcccgctaca aatggcggcc ccggcgtccc 1680
ctcctcactc cgaagcttcc cggttgacga cctctccggt ctcccccctc accccaccgc 1740
aacccgggac gtcttccatg gccgccgccg ccgccgcccc cgcctactaa accaccctac 1800
ccaccccctc caaactccca cacattacat ccttcaaaga gagcatcaca cacacacaca 1860
caccagccta gcgatcacat ttccacg 1887
<210> 2
<211> 150
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
acgagctggt gagctagcta ttacctaatc gatcgatggt catcgatcat gagatgatga 60
tgatgagatt tgtacttaat tgtgatctgt atggatgctg ttgttgatca agttcttgcg 120
atcgatcgat ctgaattttc aggtttgagg 150
<210> 3
<211> 311
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcagaaggtg atcctgatca acggcgagtt cccgggcccg cggatcaact gctcgtccaa 60
caacaacatc gtggtgaacg tgttcaacca gctggacgag ccgctgctct tcacctggaa 120
cgggatgcag caccgcaaga actcgtggca ggacggcctc gccgggacgc agtgccccat 180
cgcgccgggc accaactaca cgtacaagtg gcagcccaag gaccagatcg gcagcttctt 240
ctacttcccg tcgctgggga tgcaccgcgc cgccggcggc tacggcggga tcagcgtcgt 300
cagccgcctg c 311
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggcgtcttca ggtacaacca g 21
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggtacacctg gaccgagtgc 20
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ctctgtatgc cagtggtcgt 20
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ccgttgtggt gaatgagtaa c 21
<210> 8
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gcgtcgacgt cccatgtcac cgacagtact 30
<210> 9
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
catgccatgg cgtggaaatg tgatcgctag gct 33
<210> 10
<211> 819
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atgggacgca gggcttgctg tgcaaaggaa ggaatgaagc ggggtgcctg gacgtccaag 60
gaggacgatg tcctggcgag ctacattaag tctcacggag agggtaagtg gcgggaggtt 120
ccgcagagag ccggacttcg gagatgcggc aagtcctgtc gccttaggtg gttgaactat 180
ctccgcccaa atatcaagag gggcaacatt gacgatgacg aggaagagct gatcgtgcgg 240
cttcatacgc tcctggggaa tagatggtcc ctgatcgcag gccgccttcc gggaaggacc 300
gataacgaga ttaagaacta ctggaatagt acactctcca ggaagatcgg aacggccgcg 360
accgccgccg ccggctcacg cggcggctcc acccctgata cagccagggc gactgacgcg 420
gcttccagct cttcagtggt cccaccagga cagcaacagc aaccagcaag ccgggcggat 480
actgacaccg caaccgccgc cgccgccgcc gccgccacta caacgactgt ttgggctcct 540
aaggcagtgc ggtgcacgag aggcttcttt ttccacgata gagaaactgc accattggct 600
gctgcggcgc ctgctcccgc aggagagctg ggagacggag atgacgtgga ttgcgactac 660
tattgttctg gttcgagttc cgctgcaacc acaacctcct cctcctccct cccggttgtg 720
gtcgagccat gctttagtgc cggcgatgac tggatggatg acgtccgcgc cttggcgtca 780
ttcctcgaca cagatgacgc ttggaacctg tgtgcgtga 819
<210> 11
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ggactcttga ccatgatggg acgcagggct tgct 34
<210> 12
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
attcgagctg gtcactcacg cacacaggtt ccaag 35
<210> 13
<211> 227
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
acagctatga ccatgattac aagcttcata cctctagaca cccatggagt gtatgagtaa 60
ggtacacgtg cacgagctct ttttagcggg gtcgacacaa ccgaattcca cagatctatg 120
gaatgagtat gtgaggtgac ccaccctgca ggtttttgaa gggggcgcgc cacaaccact 180
agtcaccacg gtaccagcgg gggatccggc actggccgtc gttttac 227
<210> 14
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gaagatctac gagctggtga gctagcta 28
<210> 15
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ctggtcaccc ctcaaacctg aaaattcag 29
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
cggaattcgc agaaggtgat cctgatcaac 30
<210> 17
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gaagatctgc aggcggctga cgacgctgat 30
<210> 18
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gacctgcagg gcagaaggtg atcctgatca ac 32
<210> 19
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ctggtcaccg caggcggctg acgacgctga t 31
<210> 20
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cggaattccg tggaaatgtg atcgctaggc t 31
Claims (4)
1. The application of the plant pollen specific expression promoter POsPTD1 in the specific driving of the expression of target genes in plant pollen is characterized in that: the sequence of the plant pollen specific expression promoter POsPTD1 is shown in SEQ ID NO. 1; the plant is rice.
2. The method for driving the expression of the specific target segment in the plant pollen is characterized by comprising the following specific steps of:
(1) connecting a plant pollen specific expression promoter POsPTD1 with the target fragment to ensure that POsPTD1 is at the upstream of the target fragment, and then loading a plant expression vector;
(2) transforming the plant expression vector obtained in the step (1) into a plant through agrobacterium tumefaciens mediation, and screening a transgenic plant to obtain a plant which drives the expression of a specific target segment in pollen;
wherein the plant is rice; the sequence of the plant pollen specific expression promoter POsPTD1 is shown in SEQ ID NO. 1.
3. The method of claim 2, wherein: the target gene is a structural gene or a regulatory gene, or a mutant gene of the structural gene or the regulatory gene, or a down-regulated expression element of the structural gene or the regulatory gene.
4. The application of a plant pollen specific expression promoter POsPTD1 is characterized in that the application is any one of the following:
(1) cultivating a plant variety of which transgenic components do not drift along with pollen;
(2) cultivating male gametophyte sterile plant material;
(3) breeding male sporophyte genic sterile line;
the plant is rice; the sequence of the plant pollen specific expression promoter POsPTD1 is shown in SEQ ID NO. 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010878167.3A CN112251435B (en) | 2020-08-27 | 2020-08-27 | Plant pollen specific expression promoter POsPTD1 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010878167.3A CN112251435B (en) | 2020-08-27 | 2020-08-27 | Plant pollen specific expression promoter POsPTD1 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112251435A CN112251435A (en) | 2021-01-22 |
| CN112251435B true CN112251435B (en) | 2022-04-29 |
Family
ID=74224872
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010878167.3A Active CN112251435B (en) | 2020-08-27 | 2020-08-27 | Plant pollen specific expression promoter POsPTD1 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112251435B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015154689A1 (en) * | 2014-04-11 | 2015-10-15 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Identification and uses of plant anther-specific expression promoter ptaasg027 |
| CN111154756A (en) * | 2020-01-07 | 2020-05-15 | 深圳市作物分子设计育种研究院 | Specific expression promoter for late development stage of plant anther pollen and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100537955B1 (en) * | 2003-10-29 | 2005-12-20 | 학교법인고려중앙학원 | A solely pollen-specific promoter |
| CN106834294B (en) * | 2017-03-31 | 2020-04-28 | 西南大学 | Rice anther and seed efficient promoter POsAOM, recombinant expression vector and application thereof |
| CN110055252B (en) * | 2019-04-26 | 2022-11-01 | 海南波莲水稻基因科技有限公司 | Promoter PCHF45 specifically expressed in rice pollen and application thereof |
| CN110305895B (en) * | 2019-07-31 | 2020-07-14 | 湖南杂交水稻研究中心 | Method for preventing transgene pollen drift by using rice RAMy1A gene |
-
2020
- 2020-08-27 CN CN202010878167.3A patent/CN112251435B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015154689A1 (en) * | 2014-04-11 | 2015-10-15 | 未名兴旺系统作物设计前沿实验室（北京）有限公司 | Identification and uses of plant anther-specific expression promoter ptaasg027 |
| CN111154756A (en) * | 2020-01-07 | 2020-05-15 | 深圳市作物分子设计育种研究院 | Specific expression promoter for late development stage of plant anther pollen and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112251435A (en) | 2021-01-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA3031844C (en) | Fertility restoration gene in wheat and uses thereof | |
| CN107630031B (en) | Method and system for regulating and controlling plant fertility | |
| WO2017121411A1 (en) | Plant male fertility-related protein, and coding gene and application thereof | |
| CN108064297B (en) | Wheat fertility-related gene TaMS7 and application method thereof | |
| CN105018475B (en) | Multi-control infertility vector constructed on basis of Ms1 gene and used for mediation of male fertility of corn, and application thereof | |
| WO2015035951A1 (en) | Use of genic male sterility gene and mutation thereof in hybridization | |
| WO2022109764A1 (en) | Fertility-related gene and application thereof in hybrid breeding | |
| CN101525630B (en) | Rapeseed plant recessive cytoblast sterile restoring gene BnCYP704B1 and application | |
| WO2022042619A1 (en) | Plant pollen tube growth gene and application | |
| US20220275383A1 (en) | Sterile genes and related constructs and applications thereof | |
| CN104911191B (en) | A kind of sterility changing gene FG3 and its application | |
| CN108794610B (en) | Corn cross-incompatibility related protein ZmGa1S, and coding gene and application thereof | |
| CN112251435B (en) | Plant pollen specific expression promoter POsPTD1 and application thereof | |
| CN108329383B (en) | Protein related to corn hybridization incompatibility and coding gene and application thereof | |
| CN112251523B (en) | Application and implementation method of rice coleoptile purple line gene OsMYB76 | |
| Akasaka et al. | Development of transgenic male-sterile rice by using anther-specific promoters identified by comprehensive screening of the gene expression profile database ‘RiceXPro’ | |
| CN111575284A (en) | Vector containing oryza sativa promoter and capable of restoring male fertility of rice OsCYP704B2 mutant and application | |
| CN109504703B (en) | Method for creating maize dominant nuclear male sterile line by using p5126-ZmMs1D construct and breeding application method thereof | |
| CN112251459B (en) | Method for preparing and identifying male gametophyte sterility | |
| CN111560372A (en) | Expression cassette and vector for restoring male fertility of rice OsCYP704B2 mutant, and detection method and application thereof | |
| CN111575285B (en) | Carrier containing oryza longistaminata promoter and capable of restoring male fertility of rice OsCYP704B2 mutant and application | |
| US11753650B2 (en) | Wheat fertility-related gene TaMS7 and application method thereof | |
| CN109504701B (en) | Method for creating maize dominant nuclear male sterile line by using p5126-ZmMs1M construct and breeding application method thereof | |
| CN109504702B (en) | Method for creating maize dominant nuclear male sterile line by using p5126-ZmMs7M construct and breeding application method thereof | |
| Salgueiro et al. | Insertional tagging of regulatory sequences in tritordeum; a hexaploid cereal species |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |