CN114561387A - Peanut promoter and application thereof - Google Patents
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- CN114561387A CN114561387A CN202210193478.5A CN202210193478A CN114561387A CN 114561387 A CN114561387 A CN 114561387A CN 202210193478 A CN202210193478 A CN 202210193478A CN 114561387 A CN114561387 A CN 114561387A
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Images
Classifications
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a peanut promoter and application thereof. Wherein the peanut promoter comprises: (a) the peanut endogenous promoters pahbq 4, pahbq 4 have SEQ ID NO: 1; or (b) a peanut promoter comprising the nucleotide sequence of (a); or (c) a peanut promoter which has more than 90% of homology with the nucleotide sequence in the (a) and has the same function. Solves the problem that the peanut transgenic application in the prior art lacks an endogenous promoter with broad-spectrum expression, and is suitable for the field of biotechnology.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a peanut promoter and application thereof.
Background
In gene expression, a promoter is one of important elements, and is classified into an exogenous promoter derived from outside a host and an endogenous promoter derived from the host itself, depending on the origin. Promoters currently used in peanut transgenesis are primarily exogenous promoters, such as the cauliflower mosaic virus promoter 35S promoter. However, since the exogenous promoter does not belong to the host itself, it is often inefficient to stabilize the transgene, and it is easy to lose or be methylated after a long passage time.
Commonly used promoters in plant genetic engineering are nos, ocs, mas derived from T-DNA; viral-derived CaMV19S, CaMV 35S; ubi-1, Act-1, PHAL-1 and the like derived from plants. Among them, ubiquitin promoters are favored because of their high promoter efficiency, low methylation level, stable genetic traits, and the like. At present, promoter sequences have been isolated from many ubiquitin genes, including Ubi-1 promoter, rice ubiquitin RUBQ2 promoter, arabidopsis ubiquitin promoter, sunflower ubiquitin UbB1 promoter, tobacco ubiquitin Ubi-U4 promoter, potato ubiquitin Ubi7 promoter, tomato ubiquitin Ubi1-1 promoter, barley ubiquitin Mub1 promoter in maize genome. The ubiquitin Ubi-1 promoter of maize has been widely applied to monocotyledons such as maize, wheat and rice. Secondly, the rice ubiquitin RUBQ2 promoter is also applied to rice and sugarcane.
At present, endogenous strong promoters suitable for peanuts have not been reported. Therefore, the development of endogenous strong promoters suitable for peanut transgenic is the basis for further development of peanut transgenic technology, and has great significance for peanut gene editing and breeding. Most of the existing peanut endogenous promoters are tissue-specific promoters, and promoters with broad-spectrum expression like ubiquitin promoters are not reported yet.
Disclosure of Invention
The invention mainly aims to provide a peanut promoter and application thereof, and aims to solve the problem that an endogenous promoter with broad-spectrum expression is lacked in peanut transgenic application in the prior art.
In order to achieve the above object, according to a first aspect of the present invention, there is provided a peanut promoter. The peanut promoter comprises (a) an endogenous peanut promoter pAhUBQ4, and pAhUBQ4 has the nucleotide sequence shown in SEQ ID NO: 1; or (b) a peanut promoter comprising the nucleotide sequence of (a); or (c) a peanut promoter which has more than 90% of homology with the nucleotide sequence in the (a) and has the same function.
In order to achieve the above object, according to a second aspect of the present invention, there is provided a recombinant vector comprising the above peanut promoter.
In order to achieve the above object, according to a third aspect of the present invention, there is provided a host cell transformed with the above recombinant vector.
Further, the host cell includes Escherichia coli or Agrobacterium.
In order to achieve the above object, according to a fourth aspect of the present invention, there is provided a transformation method comprising transforming a foreign gene into a target plant using the above host cell, the foreign gene being located on a recombinant vector.
Further, the target plant includes peanut or nicotiana benthamiana.
Further, the transformation method is a transient transformation method, and the transient transformation method for peanuts comprises the following steps: culturing peanut seeds in a dark place for 15 days to obtain yellow seedlings; after the agrobacterium is cultured, resuspending in an infection solution, and standing for 0.5-2 h at 28 ℃ to form an agrobacterium liquid; cutting the upper and lower embryonic axes of the yellow flower seedlings into stem disks respectively, and placing the stem disks into agrobacterium liquid for infection; placing the stem disc in a culture dish for culturing to obtain the peanut with the gene instantaneously transformed; preferably, the thickness of the stem disc is 0.5-2 mm; preferably, the infection time is 0.5-2 h; preferably, the stem disc is placed in a petri dish containing wet filter paper for culturing.
Further, the infection liquid comprises MS liquid culture liquid containing 20 mu g/L acetosyringone; preferably, the agrobacterium comprises GV3101, LBA4404, EHA105, or AGL 1.
Further, the transformation method is a transient transformation method, and the transient transformation method for nicotiana benthamiana comprises the following steps: after the agrobacterium is cultured, resuspending in an infection solution, and standing for 0.5-2 h at 28 ℃ to form an agrobacterium liquid; injecting agrobacterium liquid into the leaf back of the Nicotiana benthamiana to obtain the Nicotiana benthamiana with gene transient transformation; preferably, the infestation liquid comprises MS liquid culture medium containing 20 μ g/L acetosyringone; preferably, the agrobacterium comprises GV3101, LBA4404, EHA105, or AGL 1.
In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a use of the above peanut promoter, recombinant vector, host cell, or transformation method in genetic transformation and/or breeding of a plant.
By applying the technical scheme of the invention, the peanut endogenous promoter pAhUBQ4 or homologous genes thereof, or a recombinant vector and a host cell containing the promoter are utilized, so that a large amount of exogenous genes can be expressed in peanuts.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 shows a schematic diagram of the expression level of 18 peanut ubiquitin genes in 15 tissues in the peanut development process according to the embodiment 1 of the invention.
FIG. 2 shows an agarose gel electrophoresis of pAhUBQ4 promoter and surrounding sequences according to example 1 of the present invention.
FIG. 3 shows the results of GUS histochemical staining according to example 2 and comparative example 1 of the present invention.
FIG. 4 is a graph showing the expression amount of GUS gene in tobacco leaves according to example 2 and comparative example 1 of the present invention.
FIG. 5 is a diagram showing the expression level of GUS gene in a stem disk of peanut according to example 2 and comparative example 1 of the present invention.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
Interpretation of terms:
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, most of which are located upstream of the transcription initiation point of a structural gene, and is not transcribed per se. However, promoters, such as tRNA promoters, are located downstream of the transcription initiation point and these DNA sequences can be transcribed. The nature of the promoter was originally identified by mutations that increase or decrease the transcription rate of the gene. Promoters are generally located upstream of the transcription start site.
As mentioned in the background art, no endogenous strong promoter suitable for peanuts has been reported at present. Therefore, the development of endogenous strong promoters suitable for peanut transgenic is the basis for further development of peanut transgenic technology, and has great significance for peanut gene editing and breeding.
Therefore, in the present application, the inventors tried to identify peanut ubiquitin genes by taking the peanut tifmunner genome data as a reference, further analyze the expression amount of the peanut ubiquitin genes in a plurality of tissues, and find out ubiquitin genes which are constitutively and strongly expressed in the plurality of tissues. Further, a promoter fragment pAhUBQ4 of 971bp upstream of the ATG was cloned and ligated to a vector for promoting GUS marker for transgene. Comparing the promoter strength with that of 2X35S, pAhUBQ4 is proved to be an endogenous strong promoter from peanut and can be used for late transgenosis. Thus a series of protection schemes of the application are proposed.
In a first exemplary embodiment of the present application, a peanut promoter is provided, comprising (a) a peanut endogenous promoter pahbuq 4, pahbuq 4 having the sequence of SEQ ID NO: 1; or (b) a peanut promoter comprising the nucleotide sequence of (a); or (c) a peanut promoter which has more than 90% of homology with the nucleotide sequence in the (a) and has the same function.
SEQ ID NO：1：tcagctgccgattaatggacagtggatcccaccgtgataataaaatctataaaagcccacttgtctatttttttattttgatttttgatttggtataccgtatacccctttctcagcccacttgccaattactacaacttgtgtgggcccaaatattatgttaaggatccacttataataagataacatctattaccacaaaaaaagagataataacaccgtggcggtcaatacgagaagagcgtttgcgtggtttgttgtttgtttgtgatggggaggtctgaggtgccttaactccaaccaacgccttccaatttccaacccttcacgtctggccaaacattgccgtgtctatagtgtcaattgacacctcaaccgtacacgtgtcgcaaactgataaggtaaatggtcaccctctttctgttgcgtaaataaggcggacacctctttatgctataaaggaacctccagaccccattgtttcttcacaattctcattttcattctctctttgttgtccgaaatccttcaaggtaccttcctcctctttctctttgcttttgattctggttccctttagttacatctgtagggttttggcatttcttcttaatcaccaatattatcggttttagattcgttatattatgtcttttgttgcctcgttccttggaaattttctctagatctgtttatactgcaatgatttatgatatttaattgttaaatctgatcggttttgatcacgcttttaaactgcaatttttagttgctttctgaaagatcgaaggcctttgtttcggatcgtaaattttctgaaaaaatctgagatcaattgtaccaattttgattataggttttttttttttttccgttgttgatcctgattcatctgtttatgacgatgcgaattttaatgtacgtttccttaactagtttcttatttaattgttgttaacgatgatgcagat。
The peanut endogenous promoter pAhUBQ4 has the sequence shown in SEQ ID NO: 1, including key elements such as a core promoter, upstream promoter elements, conserved sequences required for specific binding of RNA polymerase and transcription initiation, and the like. Adding nucleotides to the 5 'end and/or 3' end of the nucleotide sequence of pahbq 4 to obtain a new nucleotide sequence comprising SEQ ID NO: 1, and the nucleotide sequence has the same or similar promoter activity, and can be used as a peanut promoter. For the peanut promoter, the nucleotide sequence of pAhUBQ4 is subjected to nucleotide substitution, insertion, deletion and other changes at non-critical element positions, and the obtained nucleotide sequence still has the same or similar promoter activity and can be used as the peanut promoter. That is, a nucleotide sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 99.99% or more homology with the nucleotide sequence of pAhUBQ4 and having the same function, and a nucleotide sequence such as a key element thereof is substantially identical to pAhUBQ4 provided in (a), and can be used as a peanut promoter.
According to the application, all ubiquitin genes are identified in peanut genomes, and the expression quantity difference of all ubiquitin genes in peanuts at 15 different tissue parts, namely the difference of the starting capacities of corresponding ubiquitin promoters, is researched by utilizing a public transcriptome database. The promoter sequence of the highly expressed ubiquitin gene, pAhUBQ4, was cloned and applied to peanuts.
In actual research, although the genome of peanut is published, most of the ubiquitin genes of peanut are only predicted to be peanut ubiquitin genes. According to the method, the Arabidopsis ubiquitin genes are compared and found by blast, so that the accuracy is higher, omission is not easy, all the ubiquitin genes are cloned and verified, all the ubiquitin genes in the peanut are really identified, and the problem that a large number of introns exist in the genome of the eukaryote, the gene prediction result is easily influenced, and the deviation is generated in the follow-up test is solved.
Eukaryotic promoters have a longer, more complex structure than prokaryotic promoters, and may even be present downstream of the target gene. Therefore, for eukaryotic gene promoters, it is difficult to obtain the promoter position by simple prediction, and actual cloning and subsequent transcription verification must be performed to determine the promoter function.
In a second exemplary embodiment of the present application, a recombinant vector is provided, which comprises the peanut promoter described above.
The recombinant vector may be an empty vector not containing the target gene or a recombinant vector into which the target gene to be expressed is inserted. On the recombinant vector, downstream of the peanut promoter, a gene fragment to be expressed may be present. The peanut promoter can activate RNA polymerase and promote the mass transcription and expression of downstream gene segments. By utilizing the common methods of enzyme digestion connection, homologous recombination and the like in the prior art, the target gene can be inserted into the empty vector, and the recombinant vector capable of transcribing and expressing the target gene in a large quantity is constructed. On the recombinant vector (including an empty vector), various elements such as a replication initiation site, a multiple cloning site, a translation control signal, a resistance gene or a selection marker can be flexibly selected according to requirements.
In a third exemplary embodiment of the present application, a host cell transformed with the above-described recombinant vector is provided. In a preferred embodiment, the host cell comprises E.coli or Agrobacterium.
The host cell is transformed with a recombinant vector, and can carry the recombinant vector to perform various functions such as recombinant vector copy, gene expression, gene integration into chromosome and the like. The host cell may be various strains of Escherichia coli, which may be commonly used DH 5. alpha. and Agrobacterium tumefaciens, which may be commonly used GV3101 used in the examples.
In a fourth exemplary embodiment of the present application, there is provided a transformation method comprising transforming a foreign gene into a target plant using the above-described host cell, the foreign gene being located on a recombinant vector.
By using the transformation method, a recombinant vector carrying the exogenous gene can be transformed into a target plant, and the recombinant vector is provided with a peanut promoter and can promote the transcription and expression of the exogenous gene.
In a preferred embodiment, the target plant comprises peanut or nicotiana benthamiana.
Exogenous genes can be expressed in plants such as peanut, Nicotiana benthamiana and the like by transforming a recombinant vector carrying the exogenous genes into a target plant. Different recombinant vectors and transformation methods in the prior art are selected, so that the exogenous gene target plant exists and is expressed in the form of an episome plasmid, and the target gene can be integrated on the chromosome of the target plant by methods such as homologous recombination, and the transgenic plant of which the exogenous gene can be stably inherited is obtained.
In a preferred embodiment, the transformation process is a transient transformation process, comprising for peanuts: culturing peanut seeds in a dark place for 15 days to obtain yellow seedlings; after culturing the agrobacterium, resuspending the agrobacterium in an infection solution, and standing the agrobacterium at 28 ℃ for 0.5-2 hours to form an agrobacterium liquid; cutting the upper and lower embryonic axes of the yellow flower seedlings into stem disks respectively, and placing the stem disks into agrobacterium liquid for infection; placing the stem disc in a culture dish for culturing to obtain the peanut with the gene instantaneously transformed; preferably, the thickness of the stem wafer is 0.5-2 mm; preferably, the infection time is 0.5-2 h; preferably, the stem disc is placed in a petri dish containing wet filter paper for culturing.
In a preferred embodiment, the infestation liquid comprises MS liquid culture medium containing 20. mu.g/L acetosyringone; preferably, the agrobacterium comprises GV3101, LBA4404, EHA105, or AGL 1.
By utilizing the transient transformation method, a recombinant vector carrying a foreign gene can be transformed into the peanut, and the peanut with the gene transiently transformed is obtained. Because the used promoter is an endogenous promoter derived from peanuts or a promoter with higher homology with the endogenous promoter, the problems of low efficiency, unstable heredity after long passage time, easy loss or easy methylation of the exogenous promoter can be solved. The peanut promoter has high starting strength and can express genes in multiple tissues with high efficiency.
In a preferred embodiment, the transformation process is a transient transformation process, comprising, for Nicotiana benthamiana: after the agrobacterium is cultured, resuspending in an infection solution, and standing for 0.5-2 h at 28 ℃ to form an agrobacterium liquid; injecting agrobacterium liquid into the leaf back of the Nicotiana benthamiana to obtain the Nicotiana benthamiana with instantaneously transformed genes; preferably, the infestation liquid comprises MS liquid culture medium containing 20 μ g/L acetosyringone; preferably, the agrobacterium comprises GV3101, LBA4404, EHA105, or AGL 1.
By using the promoter, a large amount of exogenous genes can be expressed in the Nicotiana benthamiana, and the sequence of the promoter which can be used in the Nicotiana benthamiana is expanded. In practical application, the molecular biological element can be used universally with a constructed molecular biological element explored in peanuts, and the workload and the working cost are greatly reduced. Also solves the problem that the gene expression quantity is different due to the use of different promoters in different plants.
In a fifth exemplary embodiment of the present application, there is provided a use of the above-described peanut promoter, recombinant vector, host cell, or transformation method for genetic transformation and/or breeding of a plant.
The advantageous effects of the present application will be explained in further detail below with reference to specific examples.
Example 1 obtaining of pAhUBQ4 promoter
1. Identification of peanut ubiquitin gene and analysis of expression level
Genomic data of cultivated peanut Arachis Hypogaea CV. Tifrunner (accession number CV-93, PI 644011) was downloaded from https:// www.peanutbase.org/home. The reported protein sequence of Arabidopsis AtUBQ10(AT4G05320) is downloaded in TAIR (https:// www.arabidopsis.org /) as a reference sequence, a peanut genome protein sequence library is searched by blastp to obtain candidate peanut ubiquitin gene sequences, and the obtained peanut ubiquitin genes are confirmed by using a CDD conserved domain database (https:// www.ncbi.nlm.nih.gov/CDD) to obtain 18 peanut ubiquitin genes in total.
Transcriptome data of 15 tissues (leaf, root, flower, unearthed fruit needle, primary expanded pod, aerial fruit stem of primary expanded pod, stage 3 pod, stage 5 husk, stage 5 seed, stage 6 husk, stage 6 seed, stage 7 seed, stage 8 seed, stage 10 seed) in the development process of the cultivated peanut Arachis hypogaea cv. Tifrunnener were downloaded from https:// www.ncbi.nlm.nih.gov/bioproject/PRJNA 291488. The time period nomenclature for pods was established by Pattern 1974. Expression quantity data of 18 peanut ubiquitin genes in 15 tissues are searched in transcriptome data, and an AhUBQ4 gene is located, which constitutes high expression in 15 tissues (as shown in figure 1).
2. Cloning of promoter of peanut AhUBQ4 Gene
Extracting DNA of the cultivated peanut Arachis hypogaea cv. Tifrunner by adopting a CTAB method, designing a primer pAhUBQ4-F based on the genome sequence of the cultivated peanut Arachis hypogaea cv. Tifrunner and the DNA of the cultivated peanut Arachis hypogaea cv. Tifrunner as a template: tcagctgccgattaatggaca (SEQ ID NO: 3) and primer pAhUBQ 4-R: gggtcttcacaaagatttgcat (SEQ ID NO: 4) to amplify a 991bp total of the promoter sequence of interest and surrounding sequences:
SEQ ID NO：2：tcagctgccgattaatggacagtggatcccaccgtgataataaaatctataaaagcccacttgtctatttttttatt ttgatttttgatttggtataccgtatacccctttctcagcccacttgccaattactacaacttgtgtgggcccaaatattatgttaaggatccacttataataagataacatctattaccacaaaaaaagagataataacaccgtggcggtcaatacgagaagagcgtttgcgtggtttgttgtttgtttgtgatggggaggtctgaggtgccttaactccaaccaacgccttccaatttccaacccttcacgtctggccaaacattgccgtgtctatagtgtcaattgacacctcaaccgtacacgtgtcgcaaactgataaggtaaatggtcaccctctttctgttgcgtaaataaggcggacacctctttatgctataaaggaacctccagaccccattgtttcttcacaattctcattttcattctctctttgttgtccgaaatccttcaaggtaccttcctcctctttctctttgcttttgattctggttccctttagttacatctgtagggttttggcatttcttcttaatcaccaatattatcggttttagattcgttatattatgtcttttgttgcctcgttccttggaaattttctctagatctgtttatactgcaatgatttatgatatttaattgttaaatctgatcggttttgatcacgcttttaaactgcaatttttagttgctttctgaaagatcgaaggcctttgtttcggatcgtaaattttctgaaaaaatctgagatcaattgtaccaattttgattataggttttttttttttttccgttgttgatcctgattcatctgtttatgacgatgcgaattttaatgtacgtttccttaactagtttcttatttaattgttgttaacgatgatgcagatgcaaatctttgtgaagaccc。
PCR amplification was performed using high fidelity enzyme (Takara R045A), 50 μ L system: PrimeSTAR Max Premix 25. mu.L, pAhUBQ4-F (10. mu.M) and pAhUBQ4-R (10. mu.M), each 2. mu.L, DNA template 200ng, make up ddH2O to 50. mu.L. The PCR procedure was: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, and extension at 72 ℃ for 10s, for 35 cycles. The PCR product was electrophoresed on 1% agarose gel, which recovered the band of interest (as shown in FIG. 2).
Example 2
Construction of pCAMBIA1381-pAhUBQ4-GUS vector
And constructing a target vector by adopting a homologous recombination method. Primers pAhUBQ4-42bp-inGUS-F were used: tgggcccggcgcgccgaattccctcagctgccgattaatgga (SEQ ID NO: 5) and the primer pAhUBQ4-42 bp-inGUS-R: tcgacggatccccgggaattcatctgcatcatcgttaacaac (SEQ ID NO: 6), the target band recovered from the above step was used as template amplification, and the PCR system and procedure were identical to those described above. The PCR product was electrophoresed on 1% agarose gel, and the gel recovered the band, which is the PCR product with the homology arm.
By using EcoRI-HFTM(NEB) the plasmid pCAMBIA1381-GUS is cut by an enzyme, and the cutting system is as follows: 3 mu.L of buffer, EcoRI-HFTMEnzyme 1. mu.L, plasmid 500ng, complement ddH2O to 30. mu.L. Electrophoresis was performed on 1% agarose gel at 37 ℃ for 2 hours, and the linear fragment after digestion was recovered from the gel. The pCAMBIA1381-pAhUBQ4-GUS vector is constructed by adopting Novozam homologous recombinase (C112), and the reaction system is as follows: linearized vector 2. mu.L, insert 2. mu.L, 5 × CE II Buffer 2. mu.L, Exnase II 1. mu.L, complement ddH2O to 10. mu.L. React for 30min at 37 ℃ and then cool on ice. Taking 2 μ L of the recombinant product, collectingTransforming Escherichia coli DH5 alpha (full-scale gold, CD201-01) by heat shock method, selecting single clone, expanding propagation, adding glycerol to the positive clone with correct sequencing to final concentration of 25%, and storing at-80 deg.C. In the process of homologous recombination, the raw material of the insert is a target promoter sequence and the surrounding sequence which account for 991bp (SEQ ID NO: 2), part of CDS sequence at the 3' end is removed in the process of constructing plasmid by homologous recombination, and pAhUBQ4(SEQ ID NO: 1) of 971bp is really connected to the vector.
2. Agrobacterium-mediated transient transformation of nicotiana benthamiana and peanut stem discs
The competent cell of Agrobacterium GV3101 was placed on ice, and after thawing, 2. mu.L of pCAMBIA1381-pAhUBQ4-GUS vector was added, and mixed well, ice-cooled for 5min, liquid nitrogen for 5min, and at 37 ℃ for 5 min. 500. mu.L of LB liquid medium was added thereto, and the mixture was cultured at 28 ℃ and 200rpm for 2 hours. After centrifugation, the cells were plated on LB medium containing rifampicin (25mg/L), streptomycin (25mg/L) and kanamycin (50mg/L), and cultured at 28 ℃ for 48 hours. Picking a single clone for propagation, and adopting a primer pAhUBQ 4-F: tcagctgccgattaatggaca (SEQ ID NO: 3) and GUS-SEQ-R: tggcacagcaattgcccg (SEQ ID NO: 7), the colonies were PCR verified, and the positive clones were stored at-80 ℃ after adding glycerol to a final concentration of 25%.
The leaves of Nicotiana benthamiana were transiently infected with the GV3101 Agrobacterium strain containing the pCAMBIA1381-pAhUBQ4-GUS expression vector by injection, using pCAMBIA 1381-2X 35s-GUS as a control. GV3101 Agrobacterium 1:200 was inoculated into LB liquid medium (containing rifampicin and kanamycin) and cultured overnight at 28 ℃ and 200 rpm. Centrifuging at 5000rpm for 10min to collect strain, washing with MS liquid culture medium twice, re-suspending thallus in staining solution (MS liquid culture medium containing 20 μ g/L acetosyringone), and standing at 28 deg.C for 1 hr.
Transient transformation of Nicotiana benthamiana: injecting the resuspended agrobacterium tumefaciens liquid into the back of the tobacco leaves, shading for 24h, culturing under the light for 24h, taking the leaves, staining one part of the leaves by GUS, and storing the other part of the leaves at minus 80 ℃.
Transient transformation of peanut stem discs: cutting the hypocotyl and epicotyl of yellow flower seedlings growing for 15 days in the dark of peanuts into stem circular sheets with the thickness of about 1mm, placing the stem circular sheets into heavy-suspended GV3101 agrobacterium liquid containing pCAMBIA1381-pAhUBQ4-GUS expression vectors, infecting for 1h, placing the stem circular sheets into a culture dish containing moist filter paper, performing dark culture for 48h, taking materials, staining GUS on one part of the obtained materials, and storing the obtained material at-80 ℃.
GUS histochemical staining
The infected tobacco leaf slices (about 5mm in width), peanut stem disks and negative control materials are added into GUS staining solution (GUS staining kit, COOLABER, SL7160), and the temperature is kept at 25-37 ℃ for 5-8 hours. Subsequently, 100% ethanol decolorization is transferred into 70% ethanol for 2-3 times of decolorization until the negative control material is white. The staining results are shown in FIG. 3.
qRT-PCR calculation of exogenous gene expression level
The RNA, PrimeScript, of the infected tobacco leaves, peanut stem discs and negative control material was extracted using the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, 9769)TMRT Master Mix (TaKaRa, RR036) was reverse transcribed to cDNA. Primers 3(https:// bioinfo. ut. ee/Primer3-0.4.0/) were used to design exogenous GUS gene specific primers: GUS-QF: ctgatagcgcgtgacaaaaa (SEQ ID NO: 8); GUS-QR: ggcacagcacatcaaagaga (SEQ ID NO: 9). The tobacco reference gene primer is as follows: NtL 25-QF: cccctcaccacagagtctgc (SEQ ID NO: 10); NtL 25-QR: aagggtgttgttgtcctcaatctt (SEQ ID NO: 11). The peanut reference gene primer is as follows: AhELF 1B-QF: aagcttccctggcaaagctcaa (SEQ ID NO: 12); AhELF 1B-QR: ttcctcagctgccttcttatcc (SEQ ID NO: 13). By TB
Fast qPCR Mix (TaKaRa, RR430) was reacted in ABI 7500 real-time PCR system. The reaction system is 20 mu L, and the reaction conditions are as follows: initial denaturation was set at 95 ℃ for 5min, thermal cycling was set at 95 ℃ for 30s, 60 ℃ for 10s, for 40 cycles. And calculating the relative expression quantity of the exogenous GUS gene by using an Excel 2010 and a 2^ -delta delta Ct method, and analyzing the data by using SPSS 2019, wherein the data are shown in figures 4 and 5.
Comparative example 1
pCAMBIA1381-2 x35 s-GUS recombinant vector is constructed, agrobacterium is used for transient transformation into Nicotiana benthamiana and peanut stem circular slices, GUS histochemical staining is carried out, qRT-PCR is used for calculating the expression quantity of exogenous genes, and the experimental method is as in example 2, referring to figures 4 and 5.
In FIG. 4, Nt is a tobacco leaf which does not contain a plasmid (negative control), Nt-48h 2X35s is a tobacco leaf transformed into pCAMBIA 1381-2X 35s-GUS, and Nt-48h pAhUBQ4 is a tobacco leaf transformed into pCAMBIA1381-pAhUBQ 4-GUS.
In FIG. 5, Ah is a peanut stem disc without plasmid (negative control), Ah-48h 2X35s is a peanut stem disc transformed into pCAMBIA 1381-2X 35s-GUS, and Ah48h pAhUBQ4 is a peanut stem disc transformed into pCAMBIA1381-pAhUBQ 4-GUS.
35S is a strong promoter, 2X35S is a stronger promoter. Thus the expression level of the pAhUBQ4 promoter in peanuts was slightly lower than normal as compared to 2X35 s. Furthermore, the statistical analysis showed that the expression levels of the pAhUBQ4 promoter and the 2X35s promoter mediating the GUS gene were not significantly different in both peanut and tobacco (p <0.05), and therefore, the promoter efficiency of the pAhUBQ4 promoter was considered to be equivalent to that of 2X35 s. The pAhUBQ4 promoter is used as a promoter for the first discovery of the endogenous broad-spectrum expression of peanuts, and has important application value in the fields of subsequent plant genetic transformation, breeding and the like.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the invention discovers the endogenous strong promoter of the peanut from the peanut, and solves the problem that the peanut transgenic application in the prior art lacks an endogenous promoter with broad-spectrum expression. The peanut endogenous promoter pAhUBQ4 or homologous gene thereof, or a recombinant vector and a host cell containing the promoter are utilized to express a large amount of exogenous genes in peanuts.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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Claims (10)
1. A peanut promoter, comprising
(a) An endogenous peanut promoter pahbq 4, said pahbq 4 having the sequence of SEQ ID NO: 1; or
(b) A peanut promoter comprising the nucleotide sequence set forth in (a); or
(c) A peanut promoter having more than 90% of homology with the nucleotide sequence in (a) and having the same function.
2. A recombinant vector comprising the peanut promoter of claim 1.
3. A host cell transformed with the recombinant vector of claim 2.
4. The host cell of claim 3, wherein the host cell comprises E.coli or Agrobacterium.
5. A transformation method comprising transforming a foreign gene into a target plant using the host cell of claim 4, wherein the foreign gene is located on the recombinant vector.
6. The transformation method of claim 5, wherein the target plant comprises peanut or Nicotiana benthamiana.
7. The transformation method of claim 6, wherein the transformation method is a transient transformation method, comprising for the peanut:
culturing and growing the peanut seeds in a dark place for 15 days to obtain yellow seedlings;
after the agrobacterium is cultured, resuspending in an infection solution, and standing for 0.5-2 h at 28 ℃ to form an agrobacterium liquid;
respectively cutting the upper embryonic axis and the lower embryonic axis of the yellow flower seedlings into stem wafers, and placing the stem wafers into the agrobacterium liquid for infection;
placing the stem disc in a culture dish for culturing to obtain the peanut with the gene transient transformation;
preferably, the thickness of the stem wafer is 0.5-2 mm;
preferably, the infection time is 0.5-2 h;
preferably, the stem disc is cultured in a petri dish containing wet filter paper.
8. The transformation method according to claim 7, wherein the staining solution comprises MS liquid culture solution containing 20 μ g/L acetosyringone;
preferably, the agrobacterium comprises GV3101, LBA4404, EHA105 or AGL 1.
9. The transformation method according to claim 6, wherein the transformation method is a transient transformation method, and the transient transformation method for Nicotiana benthamiana comprises:
after the agrobacterium is cultured, resuspending the agrobacterium in an infection solution, and standing the agrobacterium at 28 ℃ for 0.5-2 h to form an agrobacterium liquid;
injecting the agrobacterium liquid into the leaf back of the Nicotiana benthamiana to obtain Nicotiana benthamiana with instantaneously transformed genes;
preferably, the infection liquid comprises MS liquid culture liquid containing 20 mu g/L acetosyringone;
preferably, the agrobacterium comprises GV3101, LBA4404, EHA105 or AGL 1.
10. Use of the peanut promoter of claim 1, the recombinant vector of claim 2, the host cell of claim 3 or 4, or the transformation method of any one of claims 5 to 9 for genetic transformation and/or breeding of plants.
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