CN114561387B - Peanut promoter and application thereof - Google Patents
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- CN114561387B CN114561387B CN202210193478.5A CN202210193478A CN114561387B CN 114561387 B CN114561387 B CN 114561387B CN 202210193478 A CN202210193478 A CN 202210193478A CN 114561387 B CN114561387 B CN 114561387B
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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
-
- 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 application provides a peanut promoter and application thereof. Wherein, the peanut promoter includes: (a) The peanut endogenous promoter pAhUBQ4, pAhUBQ4 has the sequence of SEQ ID NO:1, and a nucleotide sequence shown in the specification; or (b) a peanut promoter comprising the nucleotide sequence of (a); or (c) a peanut promoter which has a nucleotide sequence having a homology of 90% or more with the nucleotide sequence in (a) and has the same function. Solves the problem that the prior art lacks an endogenous promoter with broad-spectrum expression in the application of peanut transgenosis, and is suitable for the biotechnology field.
Description
Technical Field
The application relates to the technical field of biology, in particular to a peanut promoter and application thereof.
Background
In gene expression, a promoter is one of important elements, and depending on the source, the promoter is classified into an exogenous promoter derived from outside of a host and an endogenous promoter derived from the host itself. The promoters currently used in peanut transgenes are primarily exogenous promoters, such as the cauliflower mosaic virus promoter 35S promoter. However, exogenous promoters are not host themselves, and therefore, are often inefficient in their use to stabilize transgenes, and are prone to loss or methylation after long passage times.
Promoters commonly used in plant genetic engineering are nos, ocs, mas derived from T-DNA; virus-derived CaMV19S, caMV S; ubi-1, act-1, PHAL-1, etc. derived from plants. Among them, ubiquitin promoters are favored because of their high efficiency of initiation, 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 the maize genome. The maize ubiquitin Ubi-1 promoter has been widely used in monocots such as maize, wheat, rice, etc. 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 yet. Therefore, the development of an endogenous strong promoter suitable for peanut transgene is the basis for further development of peanut transgene technology, and has great significance for peanut gene editing and breeding. Most of existing endogenous peanut promoters are tissue specific promoters, and promoters with broad-spectrum expression like ubiquitin promoters have not been reported.
Disclosure of Invention
The application mainly aims to provide a peanut promoter and application thereof, so as to solve the problem that an endogenous promoter with broad-spectrum expression is lacking in peanut transgene application in the prior art.
In order to achieve the above object, according to a first aspect of the present application, there is provided a peanut promoter. The peanut promoter comprises (a) a peanut endogenous promoter pAhUBQ4, wherein pAhUBQ4 has a nucleotide sequence shown in SEQ ID NO:1, and a nucleotide sequence shown in the specification; or (b) a peanut promoter comprising the nucleotide sequence of (a); or (c) a peanut promoter which has a nucleotide sequence having a homology of 90% or more with the nucleotide sequence in (a) and has the same function.
In order to achieve the above object, according to a second aspect of the present application, 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 application, there is provided a host cell transformed with the above recombinant vector.
Further, the host cell includes E.coli or Agrobacterium.
In order to achieve the above object, according to a fourth aspect of the present application, there is provided a transformation method comprising transforming an exogenous gene into a target plant, the exogenous gene being located on a recombinant vector, using the above-described host cell.
Further, the target plant comprises peanut or nicotiana benthamiana.
Further, the transformation method is a transient transformation method, and the transient transformation method for peanuts comprises: culturing and growing peanut seeds in dark for 15 days to obtain Huang Huamiao; culturing agrobacterium, re-suspending in the infection liquid, and standing at 28 ℃ for 0.5-2 h to form agrobacterium liquid; cutting the upper and lower hypocotyls of the yellow flower seedlings into stem discs respectively, and placing the stem discs into agrobacterium tumefaciens bacteria solution for infection; placing the stem disc in a culture dish for culture to obtain the peanut subjected to gene transient transformation; preferably, the thickness of the stem disc is 0.5-2 mm; preferably, the time of the infection is 0.5-2 hours; preferably, the stem discs are placed in a petri dish containing moist filter paper for cultivation.
Further, the infection liquid comprises MS liquid culture liquid containing 20 mug/L acetosyringone; preferably, the agrobacterium includes GV3101, LBA4404, EHA105 or AGL1.
Further, the transformation method is a transient transformation method, and the transient transformation method for Nicotiana benthamiana comprises: culturing agrobacterium, re-suspending in the infection liquid, and standing at 28 ℃ for 0.5-2 h to form agrobacterium liquid; injecting agrobacterium tumefaciens bacteria liquid into the leaf backs of the Nicotiana benthamiana to obtain Nicotiana benthamiana subjected to gene transient transformation; preferably, the infestation liquid comprises MS liquid culture liquid containing 20 μg/L acetosyringone; preferably, the agrobacterium includes GV3101, LBA4404, EHA105 or AGL1.
In order to achieve the above object, according to a fifth aspect of the present application, there is provided the use of the above peanut promoter, recombinant vector, host cell, or transformation method in genetic transformation and/or breeding of plants.
By applying the technical scheme of the application, the exogenous gene can be expressed in a large amount in the peanut by utilizing the peanut endogenous promoter pAhUBQ4 or homologous genes thereof, or a recombinant vector and a host cell containing the promoter.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 is a schematic view showing the expression levels of 18 peanut ubiquitin genes in 15 tissues during peanut development according to example 1 of the present application.
FIG. 2 shows agarose gel electrophoresis of pAhUBQ4 promoter and surrounding sequences according to example 1 of the present application.
FIG. 3 shows the results of GUS histochemical staining according to example 2 and comparative example 1 of the present application.
FIG. 4 is a schematic view showing the expression amounts of GUS gene in tobacco leaves according to example 2 and comparative example 1 of the present application.
FIG. 5 is a schematic view showing the expression level of GUS gene in peanut stem discs according to example 2 and comparative example 1 of the present application.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to examples.
Term interpretation:
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 point of structural genes, and is not transcribed by itself. However, some promoters (e.g., tRNA promoters) are located downstream of the transcription initiation point, and these DNA sequences can be transcribed. The nature of promoters was initially identified by mutations that increase or decrease the rate of gene transcription. The promoter is generally located upstream of the transcription initiation site.
As mentioned in the background art, no endogenous strong promoter suitable for peanuts is reported at present. Therefore, the development of an endogenous strong promoter suitable for peanut transgene is the basis for further development of peanut transgene technology, and has great significance for peanut gene editing and breeding.
Thus, the inventors tried to identify the peanut ubiquitin gene with reference to peanut tifrenner genome data, further analyze its expression levels in a plurality of tissues, and find the constitutively strong expressed ubiquitin gene in a plurality of tissues. Further, the 971bp promoter fragment pAhUBQ4 upstream of its ATG was cloned and ligated into a vector that initiates GUS markers for transgene. Comparing the promoter strength with that of 2X35S, pAhUBQ4 is proved to be an endogenous strong promoter derived from peanut and can be used for later transgenic. Thus, a series of protection schemes of the present application are presented.
In a first exemplary embodiment of the application, there is provided a peanut promoter comprising (a) a peanut endogenous promoter pAhUBQ4, pAhUBQ4 having the amino acid sequence of SEQ ID NO:1, and a nucleotide sequence shown in the specification; or (b) a peanut promoter comprising the nucleotide sequence of (a); or (c) a peanut promoter which has a nucleotide sequence having a homology of 90% or more with the nucleotide sequence in (a) and has the same function.
SEQ ID NO：1：tcagctgccgattaatggacagtggatcccaccgtgataataaaatctataaaagcccacttgtctatttttttattttgatttttgatttggtataccgtatacccctttctcagcccacttgccaattactacaacttgtgtgggcccaaatattatgttaaggatccacttataataagataacatctattaccacaaaaaaagagataataacaccgtggcggtcaatacgagaagagcgtttgcgtggtttgttgtttgtttgtgatggggaggtctgaggtgccttaactccaaccaacgccttccaatttccaacccttcacgtctggccaaacattgccgtgtctatagtgtcaattgacacctcaaccgtacacgtgtcgcaaactgataaggtaaatggtcaccctctttctgttgcgtaaataaggcggacacctctttatgctataaaggaacctccagaccccattgtttcttcacaattctcattttcattctctctttgttgtccgaaatccttcaaggtaccttcctcctctttctctttgcttttgattctggttccctttagttacatctgtagggttttggcatttcttcttaatcaccaatattatcggttttagattcgttatattatgtcttttgttgcctcgttccttggaaattttctctagatctgtttatactgcaatgatttatgatatttaattgttaaatctgatcggttttgatcacgcttttaaactgcaatttttagttgctttctgaaagatcgaaggcctttgtttcggatcgtaaattttctgaaaaaatctgagatcaattgtaccaattttgattataggttttttttttttttccgttgttgatcctgattcatctgtttatgacgatgcgaattttaatgtacgtttccttaactagtttcttatttaattgttgttaacgatgatgcagat。
The peanut endogenous promoter pAhUBQ4 has a sequence shown in SEQ ID NO:1, including core promoter, upstream promoter element, conserved sequence required for specific binding of RNA polymerase and transcription initiation, etc. Adding nucleotides at the 5 'end and/or the 3' end of the nucleotide sequence of pAhUBQ4 to obtain a novel nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:1, and also has the same or similar promoter activity, and can be used as a peanut promoter. For peanut promoters, the nucleotide sequence of pAhUBQ4 is subjected to non-key element position nucleotide replacement, insertion, deletion and other changes, the obtained nucleotide sequence still has the same or similar promoter activity, and the obtained nucleotide sequence can be used as a 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 to the nucleotide sequence of pAhUBQ4 and having the same function, and a nucleotide sequence of a key element or the like thereof is the same as pAhUBQ4 provided in (a) with a high probability, can be used as a peanut promoter.
According to the application, all ubiquitin genes are identified in peanut genome, and the expression quantity difference of all ubiquitin genes in the peanut at 15 different tissue positions, namely the difference of the starting capability of corresponding ubiquitin promoters, is explored by utilizing a public transcriptome database. The promoter sequence of the highly expressed ubiquitin gene, pAhUBQ4, was cloned and applied to peanut.
In practical studies, although peanut genome has been disclosed, most of peanut ubiquitin genes remain predicted to be peanut ubiquitin genes. The application compares with the Arabidopsis ubiquitin genes and searches blast, has higher accuracy, is not easy to miss, and performs clone verification on all ubiquitin genes, thus truly identifying all ubiquitin genes in peanuts, and overcoming the problems that a large number of introns exist in eukaryote genome, the gene prediction result is easy to influence, and the follow-up test generates deviation.
Compared with prokaryotes, the promoter region of eukaryotes is longer, has a complex structure, and may even exist downstream of the target gene. Therefore, it is difficult to obtain the position of a promoter by simple prediction for a promoter of a eukaryotic gene, and it is necessary to perform actual cloning and subsequent transcription verification to determine the function of the promoter.
In a second exemplary embodiment of the present application, a recombinant vector comprising the above peanut promoter is provided.
The recombinant vector may include the peanut promoter, and may be an empty vector not including 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, there may be a gene fragment that is desired to be expressed. The peanut promoter can activate RNA polymerase and promote the mass transcription and expression of downstream gene fragments. By utilizing the methods of enzyme cutting connection, homologous recombination and the like which are common in the prior art, a target gene can be inserted into an empty vector, and a recombinant vector which can transcribe and express the target gene in a large quantity can be constructed. On the recombinant vector (including the 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 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 the recombinant vector can be carried 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 such as E.coli, agrobacterium tumefaciens, etc., wherein E.coli may be DH 5. Alpha. Commonly used, and Agrobacterium tumefaciens may be GV3101 commonly used and used in the examples.
In a fourth exemplary embodiment of the present application, there is provided a transformation method comprising transforming an exogenous gene into a target plant, the exogenous gene being located on a recombinant vector, using the above-described host cell.
By using the transformation method, the recombinant vector carrying the exogenous gene can be transformed into the target plant, and the peanut promoter is arranged on the recombinant vector, so that the transcription and expression of the exogenous gene can be promoted.
In a preferred embodiment, the target plant comprises peanut or Nicotiana benthamiana.
The exogenous gene can be expressed in plants such as peanut, nicotiana benthamiana and the like by transforming the recombinant vector carrying the exogenous gene into the target plant. By selecting different recombinant vectors and transformation methods in the prior art, the exogenous gene target plant can exist and be expressed in the form of free plasmid, and the target gene can be integrated on the chromosome of the target plant by methods such as homologous recombination and the like, so that the transgenic plant with stable inheritance of the exogenous gene can be obtained.
In a preferred embodiment, the transformation process is a transient transformation process comprising, for peanuts: culturing and growing peanut seeds in dark for 15 days to obtain Huang Huamiao; culturing agrobacterium, re-suspending in the infection liquid, and standing at 28 ℃ for 0.5-2 h to form agrobacterium liquid; cutting the upper and lower hypocotyls of the yellow flower seedlings into stem discs respectively, and placing the stem discs into agrobacterium tumefaciens bacteria solution for infection; placing the stem disc in a culture dish for culture to obtain the peanut subjected to gene transient transformation; preferably, the thickness of the stem disc is 0.5-2 mm; preferably, the time of the infection is 0.5-2 hours; preferably, the stem discs are placed in a petri dish containing moist filter paper for cultivation.
In a preferred embodiment, the infestation liquid comprises MS liquid culture liquid containing 20. Mu.g/L acetosyringone; preferably, the agrobacterium includes GV3101, LBA4404, EHA105 or AGL1.
By using the transient transformation method, the recombinant vector carrying the exogenous gene can be transformed into the peanut, so that the peanut subjected to transient transformation of the gene can be obtained. The promoter is an endogenous promoter derived from peanut or has higher homology with the endogenous promoter, so that the problems of low efficiency, unstable inheritance, easy loss or easy methylation of the exogenous promoter after long passage time can be solved. The peanut promoter has high starting strength and can efficiently express genes in multiple tissues.
In a preferred embodiment, the method of conversion is a transient method of conversion, which for Nicotiana benthamiana comprises: culturing agrobacterium, re-suspending in the infection liquid, and standing at 28 ℃ for 0.5-2 h to form agrobacterium liquid; injecting agrobacterium tumefaciens bacteria liquid into the leaf backs of the Nicotiana benthamiana to obtain Nicotiana benthamiana subjected to gene transient transformation; preferably, the infestation liquid comprises MS liquid culture liquid containing 20 μg/L acetosyringone; preferably, the agrobacterium includes GV3101, LBA4404, EHA105 or AGL1.
By using the promoter, a large amount of exogenous genes can be expressed in Nicotiana benthamiana, and the promoter sequence capable of being used in Nicotiana benthamiana is expanded. In practical application, the method can be used commonly with the constructed molecular biological element which is explored in peanuts, and the workload and the working cost are greatly reduced. The problem of difference in gene expression amount caused by using different promoters in different plants is also solved.
In a fifth exemplary embodiment of the present application, there is provided the use of a peanut promoter, recombinant vector, host cell, or transformation method as described above in genetic transformation and/or breeding of plants.
The advantageous effects of the present application will be explained in further detail below in connection with specific examples.
Example 1 acquisition of pAhUBQ4 promoter
1. Identification of peanut ubiquitin gene and analysis of expression level
Genomic data of the cultivated peanut Arachis hypogaea cv.GIFrunner (accession number CV-93, PI 644011) was downloaded from https:// www.peanutbase.org/home. The reported protein sequence of Arabidopsis AtUBQ10 (AT 4G 05320) is downloaded in TAIR (https:// www.arabidopsis.org /) as a reference sequence, a peanut genome protein sequence library is searched by blastp, candidate peanut ubiquitin gene sequences are obtained, and the obtained peanut ubiquitin genes are confirmed by using CDD conserved domain database (https:// www.ncbi.nlm.nih.gov/CDD) to obtain 18 peanut ubiquitin genes in total.
Transcriptome data of 15 tissues (leaves, roots, flowers, non-soil-penetrating needles, primary expanding pods, aerial stems of primary expanding pods, stage 3 pods, stage 5 husks, stage 5 seeds, stage 6 husks, stage 6 seeds, stage 7 seeds, stage 8 seeds, stage 10 seeds) of the peanut Arachis hypogaea by cultivation were downloaded from https:// www.ncbi.nlm.nih.gov/biopjet/PRJNA 291488. The timing of the pods was established by Pattern 1974. Expression amount data of 18 ubiquitin genes of peanut in 15 tissues are searched in transcriptome data, and the expression amount data is positioned to an AhUBQ4 gene which is high in expression in 15 tissues (shown in figure 1).
2. Cloning of the promoter of the peanut AhUBQ4 Gene
Extracting DNA of cultivated peanut Arachis hypogaea cv.GIFrenner by adopting a CTAB method, and designing a primer pAhUBQ4-F based on the genome sequence of cultivated peanut Arachis hypogaea cv.GIFrenner by taking the DNA of cultivated peanut Arachis hypogaea cv.GIFrenner as a template: tcagctgccgattaatggaca (SEQ ID NO: 3) and primer pAhUBQ4-R: gggtcttcacaaagatttgcat (SEQ ID NO: 4) to amplify the target promoter sequence and surrounding sequences together 991bp:
SEQ ID NO：2：tcagctgccgattaatggacagtggatcccaccgtgataataaaatctataaaagcccacttgtctatttttttatt ttgatttttgatttggtataccgtatacccctttctcagcccacttgccaattactacaacttgtgtgggcccaaatattatgttaaggatccacttataataagataacatctattaccacaaaaaaagagataataacaccgtggcggtcaatacgagaagagcgtttgcgtggtttgttgtttgtttgtgatggggaggtctgaggtgccttaactccaaccaacgccttccaatttccaacccttcacgtctggccaaacattgccgtgtctatagtgtcaattgacacctcaaccgtacacgtgtcgcaaactgataaggtaaatggtcaccctctttctgttgcgtaaataaggcggacacctctttatgctataaaggaacctccagaccccattgtttcttcacaattctcattttcattctctctttgttgtccgaaatccttcaaggtaccttcctcctctttctctttgcttttgattctggttccctttagttacatctgtagggttttggcatttcttcttaatcaccaatattatcggttttagattcgttatattatgtcttttgttgcctcgttccttggaaattttctctagatctgtttatactgcaatgatttatgatatttaattgttaaatctgatcggttttgatcacgcttttaaactgcaatttttagttgctttctgaaagatcgaaggcctttgtttcggatcgtaaattttctgaaaaaatctgagatcaattgtaccaattttgattataggttttttttttttttccgttgttgatcctgattcatctgtttatgacgatgcgaattttaatgtacgtttccttaactagtttcttatttaattgttgttaacgatgatgcagatgcaaatctttgtgaagaccc。
PCR amplification was performed using high fidelity enzyme (Takara R045A), 50. Mu.L system: primeSTAR Max Premix 25. Mu.L, pAhUBQ4-F (10. Mu.M) and pAhUBQ4-R (10. Mu.M) each 2. Mu.L, 200ng of DNA template, and the addition of ddH2O to 50. Mu.L. The PCR procedure was: denaturation at 98℃for 10s, annealing at 55℃for 5s, extension at 72℃for 10s,35 cycles. The PCR products were electrophoresed on a 1% agarose gel and the target bands were recovered from the gel (as shown in FIG. 2).
Example 2
Construction of pCAMBIA1381-pAhUBQ4-GUS vector
Constructing a target vector by adopting a homologous recombination method. The primer pAhUBQ4-42bp-inGUS-F was used: tgggcccggcgcgccgaattccctcagctgccgattaatgga (SEQ ID NO: 5) and the primer pAhUBQ4-42bp-inGUS-R: tcgacggatccccgggaattcatctgcatcatcgttaacaac (SEQ ID NO: 6), the target band recovered in the above step was amplified as a template, and the PCR system and procedure were identical to those described above. The PCR product was electrophoresed on a 1% agarose gel and the target band was recovered, at which time the band was a PCR product with homology arms.
By EcoRI-HF TM (NEB) cleavage of plasmid pCAMBIA1381-GUS, cleavage system: digestion buffer 3. Mu.L, ecoRI-HF TM Enzyme 1. Mu.L, plasmid 500ng, make up ddH 2 O to 30. Mu.L. And (3) electrophoresis is carried out at 37 ℃ for 2 hours by using 1% agarose gel, and the linear fragments after enzyme digestion are recovered by the gel. Constructing pCAMBIA1381-pAhUBQ4-GUS vector by adopting a noniprandial homologous recombinase (C112), wherein the reaction system is as follows: linearization vector 2. Mu.L, insert 2. Mu.L, 5 XCE II Buffer 2. Mu.L, exnase II 1. Mu.L, make up ddH 2 O to 10. Mu.L. The reaction was carried out at 37℃for 30min, and the mixture was cooled on ice. 2 mu L of recombinant product is taken, escherichia coli DH5 alpha (full-formula gold, CD 201-01) is transformed by a heat shock method, monoclonal propagation is selected, glycerol is added to positive clones which are correctly sequenced, and the positive clones are preserved at the temperature of minus 80 ℃ after the final concentration is 25%. In the homologous recombination process, the raw material of the insert fragment is 991bp (SEQ ID NO: 2) in total of the target promoter sequence and surrounding sequences, and in the process of constructing the plasmid by homologous recombination, part of CDS sequence at the 3' end is removed, and pAhUBQ4 (SEQ ID NO: 1) with 971bp is truly attached to the vector.
2. Agrobacterium-mediated transient transformation of Nicotiana benthamiana and peanut stem discs
Agrobacterium GV3101 competent cells were taken on ice, 2. Mu.L of pCAMBIA1381-pAhUBQ4-GUS vector was added after thawing, and mixed well, ice-bath for 5min, liquid nitrogen for 5min,37℃for 5min. mu.L of LB liquid medium was added thereto, and the mixture was cultured at 28℃and 200rpm for 2 hours. After centrifugation, the mixture was spread on LB medium containing rifampicin (25 mg/L), streptomycin (25 mg/L) and kanamycin (50 mg/L), and cultured at 28℃for 48 hours. Monoclonal propagation was picked up using primer pAhUBQ4-F: tcagctgccgattaatggaca (SEQ ID NO: 3) and GUS-SEQ-R: tggcacagcaattgcccg (SEQ ID NO: 7) colony PCR was verified, and positive clones were subjected to glycerol addition to a final concentration of 25% and stored at-80 ℃.
GV3101 Agrobacterium strain containing pCAMBIA1381-pAhUBQ4-GUS expression vector was used to transiently infect leaf of Nicotiana benthamiana by injection, with pCAMBIA 1381-2X 35s-GUS as control. GV3101 Agrobacterium 1:200 was inoculated into LB liquid medium (containing rifampicin and kanamycin), cultured at 28℃overnight at 200 rpm. The strain is collected by centrifugation at 5000rpm for 10min, the MS liquid culture solution is washed twice, the infection liquid (MS liquid culture solution containing 20 mug/L acetosyringone) is used for resuspending thalli, and the thalli are kept stand at 28 ℃ for 1 hour for later use.
Transient transformation of Nicotiana benthamiana: injecting the re-suspended agrobacterium liquid into the leaf back of tobacco leaves, shading for 24h, culturing for 24h under light, taking the leaves, and preserving one part of the leaves at-80 ℃ after GUS staining.
Transient transformation of peanut stem discs: cutting the upper and lower hypocotyls of yellow flower seedlings grown in the dark for 15 days into stem discs with the thickness of about 1mm, placing the stem discs in the resuspended GV3101 agrobacterium tumefaciens bacterial liquid containing pCAMBIA1381-pAhUBQ4-GUS expression vectors, infecting for 1h, placing the stem discs in a culture dish containing moist filter paper for dark culture for 48h, obtaining materials, dyeing one part to be GUS, and preserving the other part at the temperature of minus 80 ℃.
GUS histochemical staining
The infected tobacco leaves were cut (about 5mm width), peanut stem discs and negative control material were added to GUS staining solution (GUS staining kit, COOLABER, SL 7160) and incubated at 25-37℃for 5-8 hours. Subsequently, 100% ethanol decolorization was transferred to 70% ethanol for 2-3 times until the negative control material was white. The staining results are shown in fig. 3.
Calculating the expression level of exogenous Gene by qRT-PCR
Extraction of infected tobacco leaves, peanut stem discs and RNA, primeScript of negative control material using TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa, 9769) TM RT Master Mix (TaKaRa, RR 036) was reverse transcribed into cDNA. Exogenous GUS gene-specific primers were designed using Primer3 (https:// bioinfo. Ut. Ee/Primer3-0.4.0 /): GUS-QF: ctgatagcgcgtgacaaaaa (SEQ ID NO: 8); GUS-QR: ggcacagcacatcaaagaga (SEQ ID NO: 9). The tobacco internal reference gene primer is as follows: ntL25-QF: cccctcaccacagagtctgc (SEQ ID NO: 10); ntL25-QR: aagggtgttgttgtcctcaatctt (SEQ ID NO: 11). The peanut internal reference gene primer is as follows: ahEGF 1B-QF: aagcttccctggcaaagctcaa (SEQ ID NO: 12); ahEGF 1B-QR: ttcctcagctgccttcttatcc (SEQ ID NO: 13). With TBFast qPCR Mix (TaKaRa, RR 430) was reacted in an ABI 7500 real-time PCR system. The reaction system was 20. Mu.L, and the reaction conditions were: initial denaturation was set at 95℃for 5min, thermal cycling was set at 95℃for 30s and 60℃for 10s for 40 cycles. The output original result is obtained by calculating the relative expression quantity of the exogenous GUS gene by using a 2-delta delta Ct method through Excel 2010, and SPSS 2019 analyzes the data, see FIGS. 4 and 5.
Comparative example 1
Constructing pCAMBIA 1381-2X 35s-GUS recombinant vector, transiently transforming into Nicotiana benthamiana and peanut stem discs by using agrobacterium, performing GUS histochemical staining and qRT-PCR to calculate the expression level of exogenous genes, and adopting the experimental method as in example 2.
In FIG. 4, nt is a tobacco leaf not containing 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 pCAMBIA 1381-pAhUBQ4-GUS.
In FIG. 5, ah is a plasmid-free peanut stem disc (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 and 2X35S is a stronger promoter. Therefore, it is not normal that the expression level of pAhUBQ4 promoter in peanut is slightly lower than 2X35 s. Furthermore, the statistical analysis showed that the expression levels of GUS gene mediated by pAhUBQ4 promoter and 2X35s promoter were not significantly different in both peanut and tobacco (p < 0.05), so that the promoter efficiency of pAhUBQ4 promoter was considered to be equivalent to 2X35 s. The pAhUBQ4 promoter is used as a promoter for the first discovery of the endogenous broad-spectrum expression of the peanut, 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 embodiments of the present application achieve the following technical effects: the application discovers a strong promoter of peanut endogenous from peanut, and solves the problem that the endogenous promoter with broad-spectrum expression is lacking in the transgenic application of peanut in the prior art. The peanut endogenous promoter pAhUBQ4 or homologous genes thereof, or recombinant vectors and host cells containing the promoter can be used for expressing a large amount of exogenous genes in peanuts.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Sequence listing
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Claims (14)
1. Peanut promoterpAhUBQ4The saidpAhUBQ4Has the sequence of SEQ ID NO:1, and a nucleotide sequence shown in the specification.
2. A recombinant vector comprising the peanut promoter of claim 1.
3. A host cell transformed with the recombinant vector of claim 2, wherein the host cell is e.
4. A transformation method comprising transforming a foreign gene into a plant of interest, said foreign gene being located on said recombinant vector, using the host cell of claim 3, said plant of interest being peanut or nicotiana benthamiana.
5. The transformation method according to claim 4, wherein the transformation method is a transient transformation method comprising, for the peanut:
culturing and growing the peanut seeds in the dark for 15 days to obtain Huang Huamiao;
culturing agrobacterium, re-suspending in the infection liquid, and standing at 28 ℃ for 0.5-2 h to form agrobacterium liquid;
cutting the upper and lower hypocotyls of the yellow flower seedlings into stem discs respectively, and placing the stem discs into the agrobacterium tumefaciens bacteria liquid for infection;
and placing the stem disc into a culture dish for culture to obtain the peanut subjected to gene transient transformation.
6. The transformation method according to claim 5, wherein the thickness of the stem disc is 0.5-2 mm.
7. The transformation method according to claim 5, wherein the time of the infection is 0.5 to 2 hours.
8. The transformation method according to claim 5, wherein the stem disc is placed in a petri dish containing wet filter paper for cultivation.
9. The transformation method according to claim 5, wherein the invading fluid comprises MS liquid culture fluid containing 20. Mu.g/L acetosyringone.
10. The transformation method of claim 5, wherein the agrobacterium comprises GV3101, LBA4404, EHA105 or AGL1.
11. The transformation method of claim 4, wherein the transformation method is a transient transformation method comprising, for the tobacco of the benthamia:
culturing the agrobacterium, then re-suspending in an infection liquid, and standing at 28 ℃ for 0.5-2 h to form an agrobacterium liquid;
and injecting the agrobacterium tumefaciens bacteria solution into the back of the leaf of the Nicotiana benthamiana to obtain the Nicotiana benthamiana subjected to gene transient transformation.
12. The transformation method according to claim 11, wherein the invading fluid comprises MS liquid medium containing 20 μg/L acetosyringone.
13. The transformation method of claim 11, wherein the agrobacterium comprises GV3101, LBA4404, EHA105 or AGL1.
14. Use of the peanut promoter of claim 1, the recombinant vector of claim 2, the host cell of claim 3, or the transformation method of any one of claims 4 to 13 in genetic transformation and/or breeding of a plant, either peanut or nicotiana benthamiana.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001052620A2 (en) * | 2000-01-21 | 2001-07-26 | The Scripps Research Institute | Methods and compositions to modulate expression in plants |
| AU2008200749A1 (en) * | 2000-06-23 | 2008-03-13 | Syngenta Participations Ag | Promoters for regulation of plant gene expression |
| CN101506381A (en) * | 2006-06-21 | 2009-08-12 | 斯克里普斯研究学院 | DNA composition against tumor stromal antigen FAP and methods of use thereof |
| CN110628769A (en) * | 2019-09-23 | 2019-12-31 | 上海弥生生物科技有限公司 | Arahy7F1JQP gene promoter of peanut leaf-derived callus, recombinant expression vector and preparation method thereof |
| WO2023084416A1 (en) * | 2021-11-09 | 2023-05-19 | Benson Hill, Inc. | Promoter elements for improved polynucleotide expression in plants |
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| AU2008200749A1 (en) * | 2000-06-23 | 2008-03-13 | Syngenta Participations Ag | Promoters for regulation of plant gene expression |
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| CN110628769A (en) * | 2019-09-23 | 2019-12-31 | 上海弥生生物科技有限公司 | Arahy7F1JQP gene promoter of peanut leaf-derived callus, recombinant expression vector and preparation method thereof |
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