CN112481268A - Cotton promoter PGhPGFRecombinant vector and application thereof - Google Patents
Cotton promoter PGhPGFRecombinant vector and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, relates to cloning of a cotton promoter and construction and application of a recombinant vector, and particularly relates to a cotton promoter PGhPGFAnd recombinant vectors and uses thereof. The nucleotide sequence is shown in SEQ ID NO. 1. P of the inventionGhPGFThe promoter is proved to be a promoter for regulating the specific expression of the gene in the cotton pigment gland cells, has important application value in the field of plant genetic engineering, can promote the development of the cotton breeding industry and improve the comprehensive utilization value of cotton.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, relates to cloning of a cotton promoter and construction and application of a recombinant vector, and particularly relates to a cotton promoter PGhPGFAnd recombinant vectors and uses thereof.
Background
A promoter is a DNA sequence recognized, bound and initiated by RNA polymerase and contains conserved sequences required for RNA polymerase specific binding and transcription initiation, mostly upstream of the transcription initiation site of a gene. The structure of the promoter directly affects its ability to bind to RNA polymerase, and thus the level of gene expression (heroic et al, 2019). Therefore, the research on the structure, function and expression mode of the promoter has important significance for the research on the regulation mechanism of the gene at the transcription level.
The pigment gland, also called black gland or gossypol gland, is a cavity structure peculiar to gossypium and its kindred plants, and is distributed on the surface of each tissue of the plant, i.e. a black or brown punctate structure visible to naked eyes. It is believed that the mature pigmented gland is composed of 1-3 layers of secretory cells surrounding a cavity containing secretions. Later the Liuwen philosophy team demonstrated experimentally that the formation of cotton chromoglandular cavities was typical of lytic development and was formed by certain primary cells belonging to meristems after undergoing a process of cytolytic and programmed cell death (Liuet al, 2010). These glands confine a large number of secondary metabolites in their cavities and are important defense substances of cotton against infestation by pests and germs. The research shows that the gossypol is the most studied secondary metabolite in the cotton pigment gland, is also an important metabolite for resisting plant diseases and insect pests, and has medicinal value. Gossypol is a sesquiterpene compound distributed in various tissues of cotton, wherein the content of the cotton seed is the highest. The cottonseed contains a large amount of nutrients such as protein, fat, carbohydrate and the like, so the cottonseed has great application prospect in the aspects of eating, health care, feeding and industrial application. However, gossypol is toxic to some monogastric animals and humans, and needs to be removed by multiple processes, which severely limits the comprehensive utilization of cotton seeds by people (qianyuan, et al, zhang et al, 2007). It is generally believed that gossypol and its derivatives are synthesized at the root tip of plants, transported to the aerial parts of plants and stored in pigmented glands, and that gossypol content is directly related to the number of glands (Smith, f.h., 1962). Later, with the progress of research, it was found that in some new cotton species, the gossypol content is not absolutely in direct correlation with glands. This makes the relationship between gossypol and glands more complex. Therefore, the research on the regulation and control mechanism of cotton pigment gland development and gossypol metabolism has important reference basis and application value for the molecular breeding of the low-gossypol cotton. To date, there have been few studies on the development and metabolism of cotton pigment glands, and GoPGF is the first gene cloned to regulate gland development, and the expression pattern and regulation mechanism of the gene are not clear (Ma et al, 2016).
Disclosure of Invention
The invention provides a cotton promoter PGhPGFAnd a recombinant vector and application thereof, and provides a reference basis for related researches such as cotton pigment gland development regulation mechanism and substance metabolism.
The technical scheme of the invention is realized as follows:
cotton promoter PGhPGFThe nucleotide sequence is shown in SEQ ID NO. 1.
Or the nucleotide sequence has more than 70 percent of homology with the sequence shown in SEQ ID NO. 1.
Or a subfragment with the nucleotide sequence having similar functions with the sequence shown in SEQ ID NO. 1.
Cotton promoter P containing any of the aboveGhPGFThe recombinant vector of (1), which comprises a plant expression vector and an RNAi vector.
The recombinant vector is applied to regulating and controlling the expression of a target gene in a cotton gland cell.
A strain containing the above recombinant vector.
The application of the strain in preparing transgenic plants.
The invention provides two PGhPGFDriven plant expression vector PGhPGFGUS and PGhPGF-RNAi. The two vectors contain PGhPGFSequence of which PGhPGFThe sequence of the amplification primer is as follows: forward primer PGhPGF-F1: 5'-GAAGCTGCCCTTCGCTGCAG-3' and reverse primer PGhPGF-R1：5’-TCGTCTAGATATTGAATATG-3’。
Provided by the inventors of the present applicationTwo recombinant strains of the above recombinant vector are exemplified, wherein PGhPGFGUS transformed by LBA4404 Agrobacterium, PGhPGFRNAi transformation is E.coli. PGhPGFGUS was used as in example 4. Can be at PGhPGFOn the basis of RNAi vector, Gateway technology is adopted to connect any gene of interest, and then the gene is transferred into cotton plant by genetic transformation method, thus obtaining transgenic line for specifically silencing the gene in cotton gland cell.
The invention has the following beneficial effects:
1. p of the inventionGhPGFThe promoter is proved to be a promoter for regulating the specific expression of the gene in the cotton pigment gland cells, has important application value in the field of plant genetic engineering, can promote the development of the cotton breeding industry and improve the comprehensive utilization value of cotton.
2. The invention provides a tissue-specific expression promoter, which provides a new method for utilizing molecular breeding. This process can be carried out by reacting PGhPGFThe recombinant vector with the promoter connected with the target gene is transferred into cotton to drive the gene to express in gland cells, so that the expression of the gene in pigment gland cells is improved. The expression of a target gene in gland cells can also be specifically silenced by using a recombinant vector of the promoter. The invention can provide or utilize the transgenic plant and the corresponding seed obtained by the recombinant vector, and then the promoter of the invention is transferred into other plants by utilizing a hybridization technology, thereby avoiding a fussy genetic transformation process.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows an embodiment of the present invention PGhPGFAgarose gel electrophoresis detection of promoter PCR amplification is presented.
FIG. 2 is PGhPGFSchematic representation of a recombinant plasmid with promoter linked to GUS (β -glucuronidase) reporter gene.
FIG. 3 shows P according to the present inventionGhPGFSchematic representation of the RNAi recombinant plasmid.
FIG. 4 is an illustration of the expression pattern of GUS in genetically transformed cotton plantsGhPGFThe promoter promotes the specific expression of the gene in the pigment gland cells.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Taq DNA Polymerase, Phanta Max Super-Fidelity DNA Polymerase Fidelity enzyme and Clon express II One Step Cloning Kit seamless Cloning enzyme used in the invention are purchased from Nanjing Nodezac Kinza Biotech Co., Ltd; the Cycle-Pure Kit DNA purification Kit is purchased from Beijing Promega biotechnology limited; pEASY-Blunt Simple Cloning Kit available from Beijing Quanjin Biotechnology Ltd; BP, LR kit, Gateway intermediate cloning vector PDNOR-221, PKGWFS7.0 vector, pK7GWIWG2(II),0 vector purchased from Invitrogen, usa; the plant genome DNA extraction kit and the rapid plasmid small-scale extraction kit are purchased from Tiangen Biotechnology (Beijing) Co., Ltd; the culture medium and the X-Gluc staining solution related to plant transformation and tissue culture are purchased from Beijing Kulyobo science and technology Limited; the primers and sequencing work are completed by Shanghai biological engineering Co.
Example 1: clone PGhPGFPromoters
1. Cotton DNA extraction
And extracting DNA of true leaf tissue of upland cotton according to the instruction of the plant genome DNA extraction kit. The extracted DNA concentration was then determined using a NanoDrop 2000c UV-visible spectrophotometer (Tbermo Fisber, USA) and the DNA purity was finally checked by electrophoresis on a 1.0% agarose gel.
2. Polymerase chain reaction amplification of PGhPGFPromoters
Amplification of P Using DNA as templateGhPGFThe full-length sequence of the promoter and the PCR reaction system are as follows:
wherein the template is the cotton true leaf DNA extracted in the first embodiment. The PCR product was detected by agarose gel electrophoresis (FIG. 1), after which the amplification product was recovered by purification using the Cycle-Pure Kit, and then the PCR product was ligated to a vector of the pEASY-Blunt Single Cloning Kit, followed by transformation of the ligation product into E.coli DH 5. alpha. using Taq DNA Polymerase for PCR detection, and the positive clone was sequenced by Shanghai Biometrics. Cloning with correct sequencing plasmid was extracted using a rapid plasmid miniprep kit and denoted as PGhPGFThe sequence information is shown in SEQ ID NO. 1.
Example 2: pGhPGFConstruction of GUS recombinant expression vector
Obtaining a promoter fragment with a joint by PCR amplification, wherein the PCR reaction system is shown as follows:
amplification template as example one containing PGhPGFpEASY-Blunt Simple vector of sequence, the amplification primers are as follows: pGhPGF-F2：
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGAAGCTGCCCTTCGCTGCAG-3’，
PGhPGF-R2：
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTCGTCTAGATATTGAATATG-3', wherein the underlined nucleotide sequence is the linker sequence for the BP reaction.
PCR procedure as in example oneThe procedure is the same. Purifying and recovering the amplified PCR product, and then using Gateway method to separate PGhPGFThe gene is connected to an entry cloning vector pDNOR221, and the specific operation is as follows: 0.5 mu L of PCR product; pDNOR221 vector 0.5. mu.L; BP enzyme 0.5. mu.L; TE (8.0) Buffer (1. mu.L) was reacted at 25 ℃ for 2 hours. The ligation products were subsequently transformed into E.coli DH 5. alpha. by heat shock method, PCR detection was performed using Taq DNA Polymerase, and positive clones were sequenced by Shanghai Biotech Ltd. The correctly sequenced clone was extracted with a rapid plasmid miniprep kit, denoted pDNOR221-PGhPGF. Then LR reaction is carried out, wherein the LR reaction system is as follows: pDNOR221-PGhPGFPlasmid 0.5 μ L; PKGWFS7.0 vector 0.5. mu.L; LR enzyme 0.5 μ L; 10 × TE (8.0) Buffer 1 μ L, reacted at 25 ℃ for 2 h. The ligation product is transformed into Escherichia coli DH5 alpha, and through PCR detection, positive clone is selected and extracted to obtain plasmid, which is marked as PGhPGFGUS, the specific information of the vector is shown in figure 2. Finally, LR plasmid was transferred to Agrobacterium LBA4404 by electric shock transformation, and positive clones detected correctly were stored in a-80 ℃ freezer with glycerol.
Example 3: pGhPGFConstruction of driven interference vectors
PGhPGFThe RNAi recombinant vector is a specific interference vector obtained by modifying the pK7GWIWG2(II) and 0 interference vector. The specific method comprises the following steps: first, pK7GWIWG2(II), the p35S promoter on the 0 vector and the adjacent attR1, CCDB and attR2 are cut off through two enzyme cleavage sites of SacI and MluI to form a linear vector for standby. Then, a primer with a joint is designed by utilizing a one-step cloning technology, and the sequence of the primer is as follows: pGhPGF-F3 forward primer: 5 'GCTCAAGCTAAGCTTGAGCTCGAAGCTGCCCTTCGCTGCAGTAGAAG 3', PGhPGF-R3 reverse primer: 5 'TAGTGCGGCCGCCTGCAGGGAGCTCTCGTCTAGATATTGAATATGATAGTG 3'. CCDB-F forward primer: 5 'TATCATATTCAATATCTAGACGAGAGCTCCCTGCAGGCGGCCGCACTAGTG 3', CCDB-R reverse primer: 5 'TGGCAGGGCGGGGCGTAAACGCGTGGATCAGCTTAATATGACTCTC 3'. Obtaining P by PCR amplificationGhPGFPurifying and recovering the fragment and the fragment containing attR1, CCDB and attR2, performing enzyme ligation on the fragment and the linear vector subjected to enzyme digestion by SacI and MluI by using one-step cloning enzyme, converting, and finally obtaining the correct sequencingThe positive clone of (2) is stored for later use after plasmid extraction, and the schematic structural diagram of the recombinant vector is shown in figure 3. The recombinant vector replaces the original P35S promoter with PGhPGFThe promoters, retain the attR1, CCDB and attR2 related sequences required for the Gateway procedure. Therefore, we can still use Gateway method to connect the gene of interest into the vector, and can achieve the specific silencing of the target gene in cotton gland cells.
Example 4: pGhPGFFunctional verification of promoters
1. Creation of transgenic Material
P carried by LBA4404 agrobacterium tumefaciens by agrobacterium-mediated cotton genetic transformation methodGhPGFGUS was introduced into cotton (Jin et al, 2006). Specifically, cotton to be transformed is first dehulled and then sterilized with 0.1% mercuric chloride solution, and the sterilized seeds are cultured in sterile seedling germination medium (1/2 MS) for 8 days at room temperature in the dark. Then the activated carrier PGhPGFDiluting LBA4404 Agrobacterium of GUS plasmid with MGL culture medium to OD value of 0.6, cutting hypocotyl of aseptic seedling growing for about 8 days into 0.6 cm sections, placing into prepared Agrobacterium until hypocotyl tissue is completely submerged and lightly shaking for 7 min, removing bacterial liquid, and finally sucking excess bacterial liquid on tissue surface with aseptic filter paper and spreading on MSB culture medium. After culturing for 72 h in the dark, the hypocotyl tissue is placed on an induction culture medium containing kanamycin to induce and generate embryonic callus. Subculturing for 1 time about 30 days later until callus embryogenic differentiation occurs to form transgenic cotton seedling. The invention finally obtains 6 independent transgenic lines and all the independent transgenic lines obtain offspring.
2. PGhPGFThe promoter drives GUS to be specifically expressed in glandular cells
GUS staining experiments were performed on different tissues of the obtained T0 generation positive cotton lines. The cut cotton tissue is rapidly added into pre-cooled 80% (v/v) acetone for fixation for 15 min-30 min, then washed three times by pre-cooled 100 mM phosphate buffer solution, transferred into a staining solution containing an X-Gluc reaction substrate, treated for 5 min in vacuum, placed in an incubator at 37 ℃ overnight to see the staining effect, and the reaction is stopped. Followed by addition at 37 deg.CDecolorization was performed with 75% ethanol, and after the end of decolorization, the resultant was observed with a stereomicroscope (Leica MZFL III) and photographed. And (3) decoloring, waxing and embedding the photographed sample according to the paraffin slicing step, and finally cutting the sample into 10-micron slices. The photographs were taken by observation with an optical microscope (OLYMPUS IX73, Japan) after dewaxing and mounting. As shown in FIG. 4, GUS staining was concentrated in pigmented glandular cells in these tissues, indicating PGhPGFThe promoter can promote the specific expression of the gene in the cells of the re-glandular organ and is a plant tissue specific promoter.
Examples of the effects of the invention
The invention provides two PGhPGFDriven plant expression vector PGhPGFGUS and PGhPGF-RNAi. Two recombinant strains of the above recombinant vector, wherein PGhPGFGUS transformed by LBA4404 Agrobacterium, PGhPGFRNAi transformation is E.coli. PGhPGFGUS was used as in example 4. Can be at PGhPGFOn the basis of RNAi vector, Gateway technology is adopted to connect any gene of interest, and then the gene is transferred into cotton plant by genetic transformation method, thus obtaining transgenic line for specifically silencing the gene in cotton gland cell. P of the inventionGhPGFThe promoter is proved to be a promoter for regulating the specific expression of the gene in the cotton pigment gland cells, has important application value in the field of plant genetic engineering, can promote the development of the cotton breeding industry and improve the comprehensive utilization value of cotton.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
<110> university of Henan
<120>Cotton promoter PGhPGFRecombinant vector and application thereof
<141> 2021-01-25
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1847
<212> DNA
<213> Cotton genus (Gossypium spp)
<400> 1
gaagctgccc ttcgctgcag tagaagatgt gctgtatgag tatccaaaat ttaagatgct 60
ttaatcttgt tatgcttggc aaatagagtt ggtggattat aaacaatccc aatcctcttc 120
agagtcaaat attcaaagct aaatagttcc cgaattgtgg gtttcttgaa tctagtgaag 180
gattgattgt tgaaaactta gaccatgttt taatgatgta tatcaaagct caatttgtat 240
tgcatattgt tgggttaagg ctattgcaag attcaacctt ggagtttctc tattaccacc 300
ttaatatgca agatgataat gagctgagtt ggtggttagg tgtagcatgg agtatatgga 360
cacataggaa ccagtttatt tagaataaaa aaagatcata atgtagtcca aattgttcaa 420
tttgaatatt cttacatgga ggcttggtag ggagctcaca cgacgatgac tcgtattcaa 480
ccttccaggt taagttgttc tacttctaag tagagttggt gtaaatcgct aggtaatggg 540
ttcaaattta acatcgatat ggttattttt ataagaatgt tgccacgagc tgatcgacta 600
tcattcataa tgaaaatggt gattttgaca aaggtcgcac aggttttatc caagcaacta 660
tggctccaaa agttacacat gattgttatt tgaaaggcat tgctttagtt gaagtcgttg 720
caaattgaga gagtgatcat tgagacggat tgtttgattg taccgcaaga tttaacgagt 780
aaaaaaaggt tatctctatg atgggtttga ttattgaagg ctgtctcatg ttgaaagctt 840
tttttttttt caatttatgt ccttatgttg aatgagtaga aacattgata agatcgctca 900
tgctcttgca agggtagctt tatgtcatgc aaatccttat gtttagaatt cttctcctag 960
ttgcatgtct tccattctta tttaagatgt tcgtactctt ttatctaatg aggagtcgag 1020
tgtataggat aagcccatta gcttaagaaa ataattttcc aatccttatt ttgggtaatt 1080
ttgtttgttt cattccgatt tgataataat ctattttctt ctttccttaa aaaaaaaggc 1140
taggaagagt tatttgaata aattaacaaa gttgaagagc taatttaata gattaacatt 1200
tgtatcaaat attaaaacaa caaggtagtt aattgggagt tgagttgggg ttaaaggtga 1260
cacgataagc tgaataatta acaaaatatg gatgggaatg gtcctaatat ctaaaaatag 1320
ttaaaaatca tgggaaatag aaaaaagaca gataaaagat gatcacgaaa tcgccccata 1380
ataactggtg tgaattaagt taagatcatg agtggagggg ttaagacgcc aacagaagca 1440
ccgcaactcc acatcaaata gagtaaacca tccatgccat aaagaacaaa aaagaagaag 1500
aaaattcaga ccggtcatca tccttatttg gagagaatct ggaacaggag atttccacgt 1560
gataatgatg tcaagatttg actgacaata ggaccatgag gttgatggtc ctttatttcc 1620
ccggaaaatt atactttgtt ttctccaaag cacttcactt aataattata gttttcctct 1680
tttttctcat ctgctccatg gggtttatag ccacctagtg acgactttga tcatcactct 1740
ttttcctctt tatttattct tcttgctgct gagtccgctt tggttagttt ttcttttttc 1800
tctttgaaaa acggtagtac acactatcat attcaatatc tagacga 1847
Claims (7)
1. Cotton promoter PGhPGFThe nucleotide sequence is shown as SEQ ID NO. 1.
2. The cotton promoter P of claim 1GhPGFCharacterized in that the nucleotide sequence has more than 70 percent of homology with the sequence shown in SEQ ID NO. 1.
3. The cotton promoter P of claim 1GhPGFThe nucleotide sequence is a subfragment with similar function with the sequence shown in SEQ ID NO. 1.
4. Comprising the cotton promoter P of any one of claims 1 to 3GhPGFThe recombinant vector of (1), characterized in that: including plant expression vectors and RNAi vectors.
5. The use of the recombinant vector of claim 4 to regulate expression of a gene of interest in a cotton glandular cell.
6. A strain comprising the recombinant vector of claim 4.
7. Use of the strain of claim 6 for the preparation of transgenic plants.
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Citations (5)
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|---|---|---|---|---|
| US20120070425A1 (en) * | 2009-03-25 | 2012-03-22 | Diamedica, Inc. | TISSUE KALLIKREIN FOR THE TREATMENT OF PANCREATIC Beta-CELL DYSFUNCTION AND FOR Beta-CELL PROLIFERATION |
| CN104450733A (en) * | 2014-11-12 | 2015-03-25 | 南京农业大学 | Cloning and application of cotton gland formation gene GoPGF |
| CN104630228A (en) * | 2015-02-11 | 2015-05-20 | 上海交通大学 | Promoter of cotton heat shock transcription factor GhHsf 39 genes and application of promoter |
| CN108070598A (en) * | 2018-02-26 | 2018-05-25 | 江苏省农业科学院 | A kind of cotton tip of a root specificity promoter and its application |
| CN109321576A (en) * | 2018-10-29 | 2019-02-12 | 华中农业大学 | A kind of method for creating of the low gossypol Cotton Germplasms of Non-gland body |
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2021
- 2021-01-25 CN CN202110094422.XA patent/CN112481268B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120070425A1 (en) * | 2009-03-25 | 2012-03-22 | Diamedica, Inc. | TISSUE KALLIKREIN FOR THE TREATMENT OF PANCREATIC Beta-CELL DYSFUNCTION AND FOR Beta-CELL PROLIFERATION |
| CN104450733A (en) * | 2014-11-12 | 2015-03-25 | 南京农业大学 | Cloning and application of cotton gland formation gene GoPGF |
| CN104630228A (en) * | 2015-02-11 | 2015-05-20 | 上海交通大学 | Promoter of cotton heat shock transcription factor GhHsf 39 genes and application of promoter |
| CN108070598A (en) * | 2018-02-26 | 2018-05-25 | 江苏省农业科学院 | A kind of cotton tip of a root specificity promoter and its application |
| CN109321576A (en) * | 2018-10-29 | 2019-02-12 | 华中农业大学 | A kind of method for creating of the low gossypol Cotton Germplasms of Non-gland body |
Non-Patent Citations (1)
| Title |
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| WEI GAO ET AL: "The gland localized CGP1 controls gland pigmentation and gossypol accumulation in cotton", 《PLANT BIOTECHNOLOGY JOURNAL》, vol. 18, pages 1573 - 1584 * |
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