CN112481268B - Cotton promoter P GhPGF And recombinant vector and application thereof - Google Patents
Cotton promoter P GhPGF And recombinant 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 cotton promoters and construction and application of recombinant vectors, and in particular relates to a cotton promoter P GhPGF And recombinant vectors and uses thereof. The nucleotide sequence is shown as SEQ ID NO.1. P according to the invention GhPGF The promoter is proved to be a promoter for regulating the specific expression of genes in cotton pigment gland cells, has important application value in the field of plant genetic engineering, can promote the development of cotton breeding industry and improves the comprehensive utilization value of cotton.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, relates to cloning of cotton promoters and construction and application of recombinant vectors, and in particular relates to a cotton promoter P GhPGF And recombinant vectors and uses thereof.
Background
A promoter is a DNA sequence that recognizes, binds to, and initiates transcription by RNA polymerase and contains conserved sequences required for specific binding and transcription initiation by RNA polymerase, mostly upstream of the transcription initiation site of the gene. The structure of the promoter directly influences its binding capacity to RNA polymerase and thus the expression level of the gene (Jiao Yong et al, 2019). Therefore, research on the structure, function and expression pattern of a promoter is of great importance for research on the regulatory mechanism of genes at the transcription level.
Pigment glands, also known as black glands or gossypol glands, are cavity structures specific to cotton and its related plants, and are distributed on the surface of each tissue of the plant, i.e. macroscopic black or brown spot-like structures. It is generally believed that the mature pigment gland is formed by 1-3 layers of secretory cells surrounding a cavity containing secretions. Later on, team Liu Wenzhe experiments demonstrated that cotton pigment gland body cavity formation was typical of lytic development and was formed by certain primitive cells belonging to meristem after undergoing cell lytic and apoptosis processes (Liuet al, 2010). These glands confine a large number of secondary metabolites in their cavities and are important defensive substances of cotton against pest and germ infestation. The research shows that gossypol is the most studied secondary metabolite in cotton pigment glands, is also an important metabolite for resisting diseases and insect pests, and has medicinal value. Gossypol is a sesquiterpene compound and distributed in all tissues of cotton, wherein the content of cotton seeds is the highest. The cotton seeds contain a large amount of protein, fat, carbohydrate and other nutrient substances, so that the cotton seeds have great application prospects in aspects of eating, health care, feeding and industrial application. However, gossypol has deleterious effects on certain monogastric animals and humans, and multiple processes are required to remove gossypol, which severely limits comprehensive utilization of cotton seed by humans (Qian Yuyuan et al, zhang et al, 2007). It is generally believed that gossypol and its derivatives are synthesized at the root tip of plants and then transported to the aerial parts of the plants and stored in pigment glands, the gossypol content being in positive correlation with the number of glands (Smith, f.h., 1962). Later, as research was advanced, it was found that in some new cotton seeds, there was not an absolute positive correlation between gossypol content and glands. This makes the relationship between gossypol and glands more complex. Therefore, research on the regulatory mechanism of cotton pigment gland development and gossypol metabolism has important reference and application value for low-phenol cotton molecular breeding. To date, little research has been done on cotton pigment gland development and metabolism, goPGF is the first gene cloned to regulate gland development, and the expression pattern and regulatory mechanism of this gene is unknown (Ma et al, 2016).
Disclosure of Invention
The invention provides a cotton promoter P GhPGF And the recombinant vector and the application thereof provide reference for the related researches of cotton pigment gland development regulation mechanism, substance metabolism and the like.
The technical scheme of the invention is realized as follows:
cotton promoter P GhPGF The nucleotide sequence is shown as SEQ ID NO.1.
Or the nucleotide sequence thereof has more than 70% homology with the sequence shown in SEQ ID NO.1.
Or a subfragment whose nucleotide sequence is similar to the sequence shown in SEQ ID NO.1.
Cotton promoter P containing any of the above GhPGF Including plant expression vectors and RNAi vectors.
The recombinant vector is applied to regulating and controlling the expression of a target gene in cotton gland cells.
A strain containing the recombinant vector.
The application of the strain in preparing transgenic plants.
The invention provides two P GhPGF Driven plant expression vector P GhPGF GUS and P GhPGF RNAi. The two vectors comprise P GhPGF Sequences, wherein P GhPGF The amplification primer sequences are: forward primer P GhPGF -F1:5'-GAAGCTGCCCTTCGCTGCAG-3' and reverse primer P GhPGF -R1：5’-TCGTCTAGATATTGAATATG-3’。
Taking two recombinant strains of the recombinant vector provided by the inventor of the present application as an example, wherein P GhPGF GUS transformed with LBA4404 Agrobacterium, P GhPGF RNAi transformation is of E.coli. P (P) GhPGF GUS is applied as in example 4. Can be at P GhPGF And (3) connecting any gene of interest on the basis of RNAi vectors by using a Gateway technology, and transferring the gene into cotton plants by using a genetic transformation method to obtain a transgenic line for specifically silencing the gene in cotton glandular cells.
The invention has the following beneficial effects:
1. p according to the invention GhPGF The promoter proved to be a promoter for regulating the specific expression of genes in cotton pigment gland cells, has important application value in the field of plant genetic engineering, can promote the development of cotton breeding industry and improves the comprehensive utilization value of cottonValues.
2. The invention provides a tissue-specific expression promoter, which provides a novel method for utilizing molecular breeding. This method can be realized by the following steps, P GhPGF The recombinant vector with the promoter connected with the target gene can drive the gene to express in gland cells after being transferred into cotton, thereby improving the expression of the gene in pigment gland cells. The recombinant vector of the promoter can also be utilized to specifically silence the expression of a target gene in gland cells. The invention can provide or utilize the transgenic plant and the corresponding seed obtained by the recombinant vector, and then utilize the hybridization technology to transfer the promoter of the invention into other plants, thereby avoiding the complicated genetic transformation process.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows P in an embodiment of the invention GhPGF Agarose gel electrophoresis detection diagram of promoter PCR amplification.
FIG. 2 is P GhPGF Schematic representation of recombinant plasmid with promoter linked to GUS (beta-glucuronidase) reporter gene.
FIG. 3 is a diagram of the P GhPGF Schematic representation of RNAi recombinant plasmid.
FIG. 4 is a schematic representation P of GUS expression pattern in genetically transformed cotton plants GhPGF The promoter promotes the specific expression of the gene in pigment gland cells.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.
Taq DNA Polymerase, phanta Max Super-Fidelity DNA Polymerase fidelity enzyme and ClonExpress II One Step Cloning Kit seamless cloning enzyme used in the invention are purchased from Nanjinouzan biotechnology Co., ltd; the Cycle-Pure Kit DNA purification Kit is purchased from Beijing plakom Biotechnology Co., ltd; pEASY-Blunt Simple Cloning Kit is available from Beijing full gold Biotechnology Co., ltd; BP, LR kit, gateway intermediate cloning vector PDNOR-221, PKGWWFS 7.0 vector, pK7 GWIGG 2 (II), 0 vector purchased from Invitrogen corporation, USA; plant genome DNA extraction kit and rapid plasmid small extraction kit are purchased from Tiangen Biochemical technology (Beijing) limited company; plant transformation and tissue culture related culture medium and X-Gluc staining fluid are purchased from Beijing Cool Lei Bo technology Co., ltd; the primers used and sequencing work were done by Shanghai Biotechnology Co.
Example 1: clone P GhPGF Promoters
1. Cotton DNA extraction
DNA of upland cotton true leaf tissues is extracted according to the instruction of a plant genome DNA extraction kit. The concentration of the extracted DNA was then determined with a Nanodrop 2000c ultraviolet-visible spectrophotometer (Tbermo Fisber, USA), and finally the purity of the DNA was detected with 1.0% agarose gel electrophoresis.
2. Polymerase chain reaction amplification of P GhPGF Promoters
Amplification of P Using DNA as template GhPGF The 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 products were detected by agarose gel electrophoresis (FIG. 1), followed by purification and recovery of the amplified products using the Cycle-Pure Kit, followed by attachment of the PCR products to the vector of the pEASY-Blunt Simple Cloning Kit Kit, followed by enzymatic ligationThe product was transformed into E.coli DH 5. Alpha. And tested by PCR using Taq DNA Polymerase and the positive clones were sent to Shanghai Biotechnology Co.Ltd for sequencing. Clones with correct sequencing were used to extract plasmids using the rapid plasmid miniprep kit, designated P GhPGF The sequence information is shown in SEQ ID NO.1.
Example 2: p (P) GhPGF Construction of GUS recombinant expression vector
The adaptor-carrying promoter fragment is obtained through PCR amplification, and a PCR reaction system is as follows:
the amplified template was the one containing P in example one GhPGF The pEASY-Blunt Simple vector of the sequence, the amplification primers are as follows: p (P) GhPGF -F2：
5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGAAGCTGCCCTTCGCTGCAG-3’，
P GhPGF -R2：
5’-GGGGACCACTTTGTACAAGAAAGCTGGGTTCGTCTAGATATTGAATATG-3', wherein the underlined nucleotide sequence is the linker sequence of the BP reaction.
The procedure for PCR was the same as that used in example one. The amplified PCR product was recovered by purification and then P was purified by Gateway method GhPGF The specific procedures on the entry cloning vector pDOR 221 were: PCR product 0.5. Mu.L; pDOR 221 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 enzyme-linked product was then transformed into E.coli DH 5. Alpha. By heat shock method, PCR was performed using Taq DNA Polymerase and the positive clones were sent to Shanghai Biotechnology Co.Ltd for sequencing. The plasmid was extracted with the rapid plasmid miniprep kit for cloning with correct sequencing, and was designated as pDOR 221-P GhPGF . Subsequently, an LR reaction was performed, the LR reaction system was: pDOR 221-P GhPGF Plasmid 0.5. Mu.L; PKGWFS7.0 vector 0.5 μl; LR enzyme 0.5 μl;10 XTE (8.0) Buffer 1. Mu.L, reaction 2h at 25 ℃. The ligation product is transformed into escherichia coli DH5 alpha, and positive clone plasmids are selected and marked as P after PCR detection GhPGF GUS, vectorThe specific information is shown in fig. 2. Finally, the LR plasmid is transferred into the agrobacterium LBA4404 through electric shock transformation, and the correct positive clone is detected and stored in a refrigerator at the temperature of minus 80 ℃ for standby.
Example 3: p (P) GhPGF Construction of a driven interferometric Carrier
P GhPGF The RNAi recombinant vector is a specific interference vector modified on the basis of the pK7 GWIGG 2 (II), 0 interference vector. The specific method comprises the following steps: first, pK7 GWIGG 2 (II) was excised from the p35S promoter on the 0 vector and the adjacent attR1, CCDB, attR2 sites by two cleavage sites of SacI and MluI to form a linear vector for use. Then, a primer with a joint is designed by utilizing a one-step cloning technology, and the sequence of the primer is as follows: p (P) GhPGF -F3 forward primer: 5'GCTCAAGCTAAGCTTGAGCTCGAAGCTGCCCTTCGCTGCAGTAGAAG 3', P GhPGF -R3 reverse primer: 5'TAGTGCGGCCGCCTGCAGGGAGCTCTCGTCTAGATATTGAATATGATAGTG 3'. CCDB-F forward primer: 5'TATCATATTCAATATCTAGACGAGAGCTCCCTGCAGGCGGCCGCACTAGTG 3', CCDB-R reverse primer: 5 'TGGCAGGGCGGGGCGTAAACGCGTGGATCAGGCTTAATTAATGACTCTCC 3'. P obtained by PCR amplification GhPGF The fragments and the fragments containing attR1, CCDB and attR2 are purified and recovered, and then are subjected to enzyme linkage and transformation by utilizing one-step cloning enzyme and the linear vectors subjected to SacI and MluI digestion, positive clones with correct sequencing are finally obtained, and the plasmids are extracted and stored for later use, and the structure of the recombinant vectors is shown in a schematic diagram in FIG. 3. The recombinant vector replaces the original P35S promoter with P GhPGF Promoters, retaining attR1, CCDB and attR2 related sequences required by Gateway methods. Therefore, we can still use Gateway method to join the gene of interest into the vector, thus realizing specific silencing of the target gene in cotton gland cells.
Example 4: p (P) GhPGF Functional verification of promoters
1. Transgenic material creation
P carried by LBA4404 Agrobacterium by using agrobacterium-mediated cotton genetic transformation method GhPGF GUS was introduced into cotton (Jin et al, 2006). The method comprises shelling cotton to be transformed, sterilizing with 0.1% mercuric chloride solution, and collecting sterilized seedThe seed was cultured in sterile shoot germination medium (1/2 MS) for 8 days at room temperature under dark conditions. Then carrying the activated P GhPGF Diluting the LBA4404 agrobacterium of GUS plasmid with MGL culture medium to OD value of 0.6, cutting the hypocotyl of the aseptic seedling growing for about 8 days into 0.6 cm segments, placing the segments into prepared agrobacterium until the hypocotyl tissue is completely submerged and swayed for 7 min, removing bacterial liquid, and finally sucking the excessive bacterial liquid on the tissue surface with sterile filter paper and spreading the bacterial liquid on the MSB culture medium. After light-shielding co-culture of 72 h, embryogenic callus was induced by placing the hypocotyl tissue on an induction medium containing kanamycin. And 2, subculturing for 1 time about 30 days until the callus is subjected to embryo differentiation to form transgenic cotton seedlings. The invention finally obtains 6 independent transgenic lines and obtains offspring.
2. P GhPGF The promoter drives GUS to specifically express in glandular cells
GUS staining experiments were performed on different tissues of the obtained T0-generation positive cotton lines. The sheared cotton tissue is rapidly added with pre-cooled 80% (v/v) acetone for fixing for 15 min-30 min, then washed three times with pre-cooled 100 mM phosphate buffer solution, transferred into a staining solution containing an X-Gluc reaction substrate, subjected to vacuum treatment for 5 min, placed in an incubator at 37 ℃ for overnight, and the staining effect is observed, and the reaction is stopped. Then, 75% alcohol was added at 37℃for decolorization, and after the decolorization was completed, the resultant was observed with a microscope (Leica MZFL III) and photographed. And decolorizing, waxing and embedding the photographed sample according to the paraffin slicing step, and finally cutting the sample into 10 mu m slices. After the wax-removed sealing, the film was observed and photographed by an optical microscope (OLYMPUS IX73, japan). As shown in FIG. 4, in these tissues, GUS staining was concentrated in pigment glandular cells, demonstrating P GhPGF The promoter can promote the specific expression of the gene in the glandular cells and is a plant tissue specific promoter.
Examples of the effects
The invention provides two P GhPGF Driven plant expression vector P GhPGF GUS and P GhPGF RNAi. Two recombinant strains of the above recombinant vector, wherein P GhPGF GUS transformed with LBA4404 Agrobacterium, P GhPGF RNAi transformation is of E.coli. P (P) GhPGF GUS is applied as in example 4. Can be at P GhPGF And (3) connecting any gene of interest on the basis of RNAi vectors by using a Gateway technology, and transferring the gene into cotton plants by using a genetic transformation method to obtain a transgenic line for specifically silencing the gene in cotton glandular cells. P of the invention GhPGF The promoter is proved to be a promoter for regulating the specific expression of genes in cotton pigment gland cells, has important application value in the field of plant genetic engineering, can promote the development of cotton breeding industry and improves the comprehensive utilization value of cotton.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
<110> university of Henan
<120>Cotton promoter P GhPGF And recombinant vector and application thereof
<141> 2021-01-25
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1847
<212> DNA
<213> Cotton (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 (6)
1. Cotton promoter P GhPGF The nucleotide sequence of the polypeptide is shown as SEQ ID NO.1.
2. Comprising the cotton promoter P of claim 1 GhPGF Is characterized in that: the recombinant vector is a plant expression vector.
3. Comprising the cotton promoter P of claim 1 GhPGF Is characterized in that: the recombinant vector is an RNAi vector.
4. Use of the recombinant vector of claim 2 or 3 for regulating expression of a gene of interest in cotton glandular cells.
5. A strain comprising the recombinant vector of claim 2 or 3.
6. Use of the strain according to claim 5 for the preparation of transgenic plants.
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Citations (4)
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|---|---|---|---|---|
| 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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|---|---|---|---|---|
| WO2010108262A1 (en) * | 2009-03-25 | 2010-09-30 | Diamedica Inc. | TISSUE KALLIKREIN FOR THE TREATMENT OF PANCREATIC β-CELL DYSFUNCTION |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
|---|
| The gland localized CGP1 controls gland pigmentation and gossypol accumulation in cotton;Wei Gao et al;《Plant Biotechnology Journal》;第18卷;第1573-1584页 * |
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