CN113151269B

CN113151269B - African agapanthus dehydrin protein ApSK 3 Promoter sequence of gene and application thereof

Info

Publication number: CN113151269B
Application number: CN202110356363.9A
Authority: CN
Inventors: 张荻; 杨天宸; 盛江源
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-04-01
Filing date: 2021-04-01
Publication date: 2022-08-26
Anticipated expiration: 2041-04-01
Also published as: CN113151269A

Abstract

The invention discloses a promoter sequence of a agapanthus dehydratum protein ApSK3 gene and application thereof. Which comprises one of the following: a nucleotide sequence (1) which is a polynucleotide shown as SEQ ID No. 1; or a nucleotide sequence (2) which has more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology with the polynucleotide shown in SEQ ID No.1 and has the function of the polynucleotide shown in SEQ ID No. 1. The detection of GUS enzyme activity proves that the dehydrin gene has the activity of a promoter, and is enhanced by drought, salt, osmosis, low temperature, ABA, gibberellin and ethylene induced activity, thereby showing the effect of the dehydrin gene in responding to adversity stress in agapanthus. Compared with other promoters, the promoter is suitable for promoting the expression of a reporter gene such as a beta-glucuronidase gene GUS in a plant.

Description

African agapanthus dehydrin protein ApSK 3 Promoter sequence of gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a promoter of a agapanthus dehydratum protein ApSK3 gene.

Background

The Agapanthus praecox (Agapanthus praecox) is an excellent garden ornamental plant, is a perennial rooted stem flower of the agapantaceae family, is called 'blue lily', has the characteristics of long flowering phase, luxuriant flowers, elegant color and stout and tall and straight scape, is a high-grade fresh cut flower in developed countries, and enjoys the reputation of 'love flower' in European and American countries; meanwhile, the flower has strong drought and barren resistance, is commonly used as a potting, a flower environment and a slope protection ground cover material in gardens, has rare blue, purple and white colors when flowers bloom in summer, and has wide application prospect, thereby being an important garden and road greening flower.

Dehydrin (dehydrin) belongs to the second group LEA-II family of late-stage abundant proteins in embryos, is a protein with the most abundant functions in LEA, can be expressed in a large number in the late embryonic development stage and in the adverse environment of plants, and is widely distributed in different tissues of the plants. The dehydrin gene silencing compound can play an important role in protecting plants in the abiotic stress tolerance process, researches show that the stress resistance of plants over expressing the dehydrin gene under the adverse circumstances is obviously enhanced, and the silencing of the dehydrin gene can cause the tolerance of the plants under various abiotic stresses to be obviously reduced. Therefore, the dehydrin plays an important role in improving the stress resistance of plants, but the specific action mechanism of the dehydrin is not clear.

A promoter is a non-coding nucleotide sequence located upstream of a gene core sequence and specifically recognized and bound by RNA polymerase to control transcription initiation and downstream gene expression. The cis-acting element in the promoter responds to the adversity stress signal and can regulate and control the expression of the dehydrin gene under the adversity. Dehydrin gene promoters of a plurality of plants such as arabidopsis thaliana, wheat, cynodon dactylon and the like are cloned, and regulation and control modes of the dehydrin gene promoters under adversity stress are researched. However, the cloning of a garden ornamental plant agapanthus dehydratin gene promoter and the regulation mode thereof under the adverse circumstances have not been reported in any relevant documents.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to fill the blank of the cloning and regulation pattern analysis of the agapanthus dehydratum gene ApSK3 promoter, and also provides a nucleic acid sequence of the dehydratum gene promoter; the invention discloses a change mode of the promoter activity of the agapanthus dehydrin gene ApSK3 after the arabidopsis is stressed, which lays a foundation for regulating and controlling the time-space characteristics of ApSK3 gene expression by using genetic engineering technology in the future, lays a theoretical foundation for improving the agapanthus anti-adversity capability and molecular breeding work, and has great application value.

To achieve the above and other related objects, the present invention is accomplished by the following aspects.

In a first aspect, the invention provides a promoter sequence of a agapanthus dehydrin ApSK3 gene, which comprises one of the following:

a nucleotide sequence (1) which is a polynucleotide sequence shown as SEQ ID No. 1;

or a polynucleotide sequence having a nucleotide sequence (2) which has 80% or more homology (preferably 85% or more homology, more preferably 90% or more homology) with the polynucleotide sequence shown in SEQ ID No.1 and having the function of the polynucleotide sequence shown in SEQ ID No. 1.

The polynucleotide sequence in the nucleotide sequence (2) is specifically a polynucleotide which is obtained by substituting, deleting or adding one or more bases to the polynucleotide shown in SEQ ID No.1 and has the function of the polynucleotide shown in SEQ ID No. 1.

Specifically, the polynucleotide shown in SEQ ID No.1 is obtained by deletion, insertion and/or substitution of 1-100 bases or addition of 1-50 bases at the 5 '-end and/or the 3' -end, and has the function of the polynucleotide sequence shown in SEQ ID No. 1.

More specifically, the polynucleotide shown in SEQ ID No.1 is a polynucleotide formed by replacing 1-10 bases in the polynucleotide shown in SEQ ID No.1 with bases with similar or similar properties.

The invention provides a agapanthus dehydratus ApSK3 gene promoter with adversity stress response function and protection function, which consists of a nucleotide sequence shown as SEQ ID NO.1, and the activity of the promoter is induced by drought (PEG), high salt, osmosis, low temperature stress and phytohormones ABA, gibberellin and ethylene.

Further preferably, the promoter or the nucleotide sequence shown by SEQ ID NO.1 is subjected to substitution, deletion or addition of one or more bases, still has the promoter activity of the agapanthus dehydratum ApSK3 gene, and is induced by drought (PEG), high salt, osmosis, low temperature stress and phytohormones ABA, gibberellin and ethylene.

In the present invention, the term "agapanthus dehydratus ApSK3 gene promoter sequence" refers to a nucleotide sequence having an activity of promoting the expression of an agapanthus dehydratus ApSK3 gene located 5' -upstream of the Open Reading Frame (ORF) of the ApSK3 gene on the agapanthus genome.

In the present invention, "isolated DNA" or "purified DNA" means that the DNA or fragment has been isolated from the sequences which flank it in the natural state, and that the DNA or fragment has been separated from the components which accompany the nucleic acid in the natural state and from the proteins which accompany it in the cell.

The promoter is obtained by cloning from separated agapanthus DNA by a chromosome walking method, and replaces a CaMV35S gene expression frame on plasmid pBI 121. The modified recombinant plasmid containing the ApSK3 gene promoter is named as ApSK3-P, GUS.

In a second aspect, the invention provides the use of the polynucleotide as a promoter element for the expression of a plurality of proteins or polypeptides in a plant bioreactor.

In particular, the plant bioreactor may be a plant host cell.

More specifically, the promoter elements can be used to direct inducible expression of a variety of proteins or polypeptides of different origin in a plant host cell.

More specifically, the polynucleotide (i.e., promoter) does not promote expression of the downstream gene under normal growth conditions, but activity of the polynucleotide (promoter) is stimulated under stress and/or hormone induction to promote expression of the downstream gene.

Further, the proteins or polypeptides from different sources can be proteins with any molecular weight, such as various proteins from plant sources, various proteins from animal or bacterial sources, antibodies, and the like.

In a third aspect, the invention provides the use of the polynucleotide as an expression vector for a plant bioreactor.

The polynucleotide can be used as a promoter for guiding the expression of the foreign protein to construct or modify an expression vector of a plant bioreactor.

The plant bioreactor expression vector to be modified includes but is not limited to: pCAMBIA series vectors, pBI series vectors such as pBI121, pPZP series vectors, for example: pSN1301 (a transformable dicot), pUN1301 (a transformable monocot), pRTL2, pRTL2-GFP, pRTL2-CFP, pRTL2-RFP, pRTL2-YFP and the like.

In a fourth aspect, the present invention provides a plant bioreactor expression vector comprising at least one of said polynucleotides as a promoter element, wherein said promoter directs the expression of a foreign protein. Specifically, the beta-glucuronidase gene is GUS.

Preferably, the expression vector is obtained by replacing the CaMV35S promoter in the vector pBI121 with the polynucleotide (promoter).

In a fifth aspect, the invention provides a host cell comprising said expression vector or having exogenous said polynucleotide integrated into its genome.

In a sixth aspect, the present invention provides a method for expressing a protein or polypeptide, comprising the steps of:

1) constructing recombinant expression vectors of the proteins or the polypeptides from the different sources by adopting the expression vectors;

2) transfecting the recombinant expression vector obtained in step 1 into a host, and culturing the host under conditions suitable for expression of the protein or polypeptide.

Preferably, the host may be a host cell, and in one embodiment of the invention, the host is tobacco lamina.

Preferably, the induction under the condition of properly expressing the protein or the polypeptide is specifically performed by PEG, mannitol, NaCl, low temperature (4 ℃), ABA, GA or ethylene.

Preferably, after step 2), the protein or polypeptide may be isolated from the culture, or the plurality of different sources of protein or polypeptide may be detected by a detection instrument. In one embodiment of the invention, the protein or polypeptide is GUS.

The recombinant expression vectors for said various proteins or polypeptides from different sources can be constructed by conventional recombinant DNA techniques, such as by inserting the gene segment of interest into the multiple cloning site of the plant bioreactor cell expression vector and placing it under the control of the promoter of the present invention. The gene segment of interest should at least comprise the entire coding region of the gene for a number of different sources of proteins or polypeptides.

The obtained host cell transferred with the recombinant expression vector can be cultured by a conventional method to express the foreign protein or polypeptide. Depending on the host cell used, the medium used in the culture may be selected from various known suitable media or culture solutions, and the culture may be carried out under conditions suitable for the growth of the host cell and the expression of the foreign protein or polypeptide. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties or adding a purification tag. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, treatment with a protein precipitant (salting-out method), centrifugation, wall breaking by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations thereof.

According to the cDNA sequence of the agapanthus dehydratum ApSK3 protein gene, a promoter sequence of the agapanthus dehydratum ApSK3 gene on the genome, which is closely related to agapanthus response stress, is cloned by a genome walking method. The promoter of ApSK3 gene is connected with GUS, Arabidopsis thaliana is transformed by agrobacterium-mediated method, and the response of the promoter to drought, osmosis, high salt, low temperature (4 ℃/8 ℃), high temperature (40 ℃/45 ℃) and ABA is detected. The GUS enzyme activity detection proves that the promoter has the activity of the promoter, and the activity is enhanced under four stresses of drought, osmosis, high salt and low temperature and three hormones of ABA, GA and ethylene. In order to further define the regulation function of different cis-acting elements on the promoter on an adversity signal, an expression vector formed by fusing a plurality of ApSK3 promoter fragments with different length deletions with a GUS gene is constructed and arabidopsis thaliana is transformed, and the detection result of the activity of the GUS enzyme shows that the response of the ApSK3 promoter on stress and hormone is mainly regulated by the cis-acting elements such as MBS, ABRE, ERE and the like. Thus, the effect of the dehydrin gene in agapanthus responding to drought, osmosis, high salt, low temperature stress, ABA, gibberellin and ethylene is shown. In addition, the promoter of the present invention is suitable for promoting the expression of a reporter gene such as a β -glucuronidase gene, a GUS gene, in plants, as compared with other promoters, and has an activity characteristic of being inducible and controllable.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a sequence of cis-regulatory elements deduced from bioinformatic analysis of the promoter sequence of ApSK3 gene, wherein the cis-regulatory elements are indicated by boxes and the names of the elements are noted;

FIG. 2 is the structure diagram of ApSK3-P, GUS, as the plant reactor expression vector where the ApSK3 gene promoter is located;

FIG. 3 is a schematic diagram of a segment of an ApSK3 promoter deleted expression vector;

FIG. 4 shows the change of GUS enzyme activity expression in different treatments after Arabidopsis thaliana was transformed with two plant reactor expression vectors (pBI121, ApSK3-P:: GUS);

FIG. 5 shows the change of GUS enzyme activity expression of five plant reactor expression vectors (ApSK3-P:: GUS, Sp-1167:: GUS, Sp-950:: GUS, Sp-646:: GUS and Sp-291:: GUS) after transforming Arabidopsis thaliana under different treatments.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the methods of testing, methods of preparation, and methods of preparation disclosed herein employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature and are described in particular in Sambrook et al, Molecular CLONING: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1 cloning of the African agapanthus dehydrin ApSK3 Gene promoter

1.1 obtaining of plant material:

taking agapanthus leaf tissues for extracting DNA;

1.2 extraction of DNA:

use of

Extracting agapanthus genome DNA by a Plant Genomic DNA Kit, taking 0.1g of agapanthus leaf tissue,after the liquid nitrogen was sufficiently ground, the powder was transferred to a 1.5ml EP tube; adding 250 μ L RB1 solution and 15 μ L RNase solution, mixing well, incubating in 55 deg.C water bath for 15min, centrifuging, and collecting supernatant. Adding 100 μ L PB1 solution, mixing, standing on ice for 5min, centrifuging, and collecting supernatant; adding 375 μ L BBI solution, adding the mixture into a centrifugal column, and centrifuging to remove eluate; adding 500 μ L CB1 solution, centrifuging and discarding the effluent; adding 500 μ L of WB1 solution, centrifuging to remove effluent, repeating for 1 time, centrifuging at 12000 Xg for 2min, and removing WB1 solution; placing the column on a new EP tube, adding 100 μ L of preheated EB solution (60-70 deg.C) into the center of the column, and centrifuging at 12000 × g for 1min to elute DNA; repeating the steps, and performing secondary DNA elution. The obtained DNA group was stored at-20 ℃ for further use.

1.3 cloning of the Gene promoter:

designing a primer according to the cDNA full-length sequence of the agapanthus dehydratum ApSK3 gene, and cloning the 5' -upstream promoter of the ApSK3 gene by adopting a chromosome Walking method (a gene Walking kit, which is purchased from Takara Clotech);

designing a downstream specific Primer ApSK3-SP1/2/3 to perform first chromosome Walking based on an ApSK3 encoding gene sequence, cloning a promoter sequence of ApSK3 by using an AP Primer in a Genome Walking kit as an upstream Primer, specifically using a PCR program shown in Table 1, obtaining an upstream promoter sequence at a 5' end after 3 rounds of thermal asymmetric nested PCR, and recovering and sequencing the fragment; and carrying out 3-round PCR by taking ApSK3-SP2-1/2/3 as a specific primer in the second chromosome walking, recovering and sequencing the PCR product, and splicing the two sequencing results by using Clustalx (1.81) software to obtain an ApSK3-P sequence SEQ ID NO:1 with the length of 2180 bp.

Primer ApSK3-SP1: 5'-TGTGGCCGGGGAGCTTCTGTTT-3' (SEQ ID NO:2)

Primer ApSK3-SP2: 5'-ACGTCCTGTTCGGTTACCACGG-3' (SEQ ID NO:3)

Primer ApSK3-SP3: 5'-CCACTTCCTCTTCGTCGCTCGA-3' (SEQ ID NO:4)

Primer ApSK3-SP2-1: 5'-AGCTAGAGAAACGAATTATCACATCCCTC-3' (SEQ ID NO:5)

Primer ApSK3-SP2-2: 5'-ACTTTCAAGCTCTCGACTCCGG-3' (SEQ ID NO:6)

Primer ApSK3-SP2-3: 5'-CCCTCTCTCACTCACCTCCACA-3' (SEQ ID NO:7)

TABLE 1 thermal asymmetric PCR reaction conditions

Bioinformatics prediction analysis was performed on ApSK3 promoter using plant cis-element database plantate. The results of the analysis are shown in table 2, the ApSK3 promoter contains structural elements common to various promoters and cis-acting elements related to adversity stress and hormone response.

TABLE 2 ApSK ₃ Cis-acting elements in gene promoters

Example 2 expression vector construction:

ApSK from example 1 was prepared using plasmid pBI121 (purchased from Youbao, Hunan, China) as template ₃ After the gene promoter sequence (SEQ ID NO.1) is subjected to enzyme digestion treatment by SphI and Smal I, the gene promoter sequence is connected between SphI and Smal I enzyme digestion sites of pBI121 to replace the original CaMV35S promoter, and ApSK is finished ₃ And (3) establishing an expression frame for expressing a GUS gene in a gene promoter sequence. The obtained recombinant plasmid was named ApSK ₃ GUS (see FIG. 2).

Example 3 analysis of promoter expression Activity under different stress and exogenous hormone treatment

In order to research the response mechanism of the dehydrin gene ApSK3 under adversity stress and hormone treatment, a pBI121 empty vector (CaMV35S promoter) and ApSK3-P are respectively used for transforming agrobacterium GV3101 by GUS through a freeze-thaw method, and the obtained positive monoclonal antibody is used for transforming arabidopsis thaliana by using a flower dipping method. Transgenic Arabidopsis thaliana was treated with low temperature (8 ℃ C./4 ℃ C.), high temperature (40 ℃ C./45 ℃ C.), ABA (100. mu.M), gibberellin (0.2mM), and ethylene (2mM), respectively, with no treatment as a blank control, and a 35S strong promoter as a positive control. The specific treatment method comprises the following steps: disinfecting T3 generation Arabidopsis seeds, sowing the seeds on an MS culture medium, and respectively transferring the seeds into an MS solid culture medium containing 20 percent PEG, 400mmol/L mannitol and 200mmol/L NaCl after 14 days to respectively carry out drought, osmotic stress and salt stress; and transferring the sample to an environment with the temperature of 8 ℃/4 ℃ for low-temperature stress; transferring the mixture into an environment of 40 ℃/45 ℃ for high-temperature stress; spraying 100 mu mol/L abscisic acid (ABA), 2mmol/L ethylene and 0.2mmol/L gibberellin solution for hormone treatment, and carrying out quantitative detection on GUS enzyme activity on the obtained sample.

The results show that: under normal growth conditions, the ApSK3 full-length promoter (ApSK3-P) has certain expression activity (242 nM MU/min. mg); under different stresses and hormone signal treatment, the GUS enzyme activity of the positive control has no obvious change (about 742 nM MU/min. mg), and the activity of GUS protein driven by ApSK3-P has a significant difference (figure 3). ApSK3-P responds most remarkably to salt stress and ABA (GUS enzyme activity is 3.7 and 3.8 times of that of a control respectively); under drought, osmotic and low-temperature stress and gibberellin and ethylene treatment, the activity of GUS enzyme is improved by 2-2.8 times of that of a control group, and the promoter expression vector successfully constructed has expression activity and can be greatly up-regulated under the induction of various adversities and hormones. (FIG. 3, wherein CK represents blank control, i.e.: No treatment was applied to Arabidopsis thaliana).

Example 4 analysis of promoter deletion response to stress and hormone treatment

According to the distribution of the stress signal, the hormone signal response element and the endosperm development element, 5' end deletion segmentation and cloning are carried out on the ApSK3 promoter, and a binary expression vector of 5 promoter sequences of 2175bp, 1167bp, 950bp, 646bp and 291bp upstream of an initiation codon and a GUS reporter gene is constructed (figure 4, only action elements related to the hormone and the stress signal are listed). Promoter-deleted expression vectors were constructed with reference to example 2, and the obtained recombinant plasmids were designated Sp-2175 (ApSK3-P), Sp-1167, Sp-950, Sp-646 and Sp-291 according to the promoter length; reference example 3 Arabidopsis thaliana was transformed with the deletion expression vector, and GUS enzyme activity was measured after stress and hormone treatment.

The results show (FIG. 5) that the expression activity tended to decrease with decreasing promoter length under any treatment. After PEG treatment, GUS enzyme activity of each deletion fragment of the promoter is obviously up-regulated by 1.4-2.3 times, and both the deletion fragments can respond to drought signals; sp-2175, Sp-1167 and Sp-950 can obviously respond to osmotic signals; whereas under NaCl treatment (Salt treated group in FIG. 5), only Sp-2175 and Sp-646 upregulated GUS enzyme activity; for low-temperature treatment, only Sp-2175 responds, so that the GUS expression level is obviously improved; after ABA treatment, Sp-2175 and Sp-1167 regulate GUS enzyme activity to be increased by 3.9 times and 1.22 times, and the-2175-950 fragment is an important region for responding to an ABA signal, and an ApSK3 promoter needs two ABA response elements ABRE to respond to the ABA signal; the-2175-291 region plays an important role for promoters in responding to ethylene signals, and is regulated by the ethylene response element ERE; the activity of the promoter-1167-646 region is obviously reduced under the treatment of gibberellin, and the phenomenon of crosstalk among hormone signals is also supposed to exist besides the direct regulation of gibberellin. By combining the results, the ApSK3 promoter has response activity to various stress signals, and-2175-1167 is a main regulation and control region responding to low-temperature stress and gibberellin signals; -2175-950 fragment is an important regulatory region in response to salt stress, osmotic stress, ABA signaling, where there are a number of stress response related cis-acting elements like TC rich repeats, MBS, ABRE; region-2175-291 is responsive to ethylene signaling, and the ethylene response element ERE plays a major regulatory role; all areas were able to respond to drought stress signals. These results indicate that the ApSK3 promoter response to stress and hormones is mainly regulated by TC rich repeats, MBS, ABRE, ERE, etc. cis-acting elements.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention. It should be noted that the prior art in the protection scope of the present invention is not limited to the examples given in the present application, and all the prior art which is not inconsistent with the technical scheme of the present invention, including but not limited to the prior patent documents, the prior publications and the like, can be included in the protection scope of the present invention.

In addition, the combination of the features in the present application is not limited to the combination described in the claims of the present application or the combination described in the embodiments, and all the features described in the present application may be freely combined or combined in any manner unless contradictory to each other.

The foregoing description has described specific embodiments of the present invention. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes or modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The embodiments and features of the embodiments of the present application may be combined with each other arbitrarily without conflict.

Sequence listing

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<400> 6

actttcaagc tctcgactcc gg 22

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 7

ccctctctca ctcacctcca ca 22

Claims

1. African agapanthus dehydrin protein ApSK ₃ The application of a promoter sequence of a gene is characterized in that the agapanthus dehydratum protein ApSK ₃ The promoter sequence of the gene serves as a promoter element for the expression of various proteins or polypeptides in a plant bioreactor;

the agapanthus dehydratum protein ApSK ₃ The promoter sequence of the gene is a nucleotide sequence (1), which is a polynucleotide sequence shown as SEQ ID number 1.

2. The use of claim 1, wherein said promoter element is used to direct the inducible expression of a plurality of proteins or polypeptides of different origin in a plant bioreactor.

3. The use according to claim 1, wherein the agapanthus dehydrin protein ApSK ₃ The promoter sequence of the gene is used as a promoter for guiding the expression of the foreign protein to construct or modify a plant bioreactor expression vector, and the plant bioreactor expression vector to be modified comprises: pCAMBIA series vectors, pBI series vectors and pPZP series vectors.

4. The plant bioreactor expression vector is characterized by comprising agapanthus hygrophicus ApSK ₃ The promoter sequence of the gene is used as a promoter element, and the expression of the foreign protein is guided by the promoter element; the agapanthus dehydratum protein ApSK ₃ The promoter sequence of the gene is a nucleotide sequence (1), which is a polynucleotide sequence shown as SEQ ID number 1.

5. The plant bioreactor expression vector of claim 4, wherein said plant bioreactor expression vector is obtained by replacing the CaMV35S promoter in the vector pBI121 with said polynucleotide.

6. A method of expressing a protein or polypeptide comprising the steps of:

step 1: adopting a plant bioreactor expression vector to be modified, and replacing a promoter of the plant bioreactor expression vector with a promoter comprising agapanthus hygiensis dehydrin protein ApSK ₃ A promoter element of a promoter sequence of the gene, and a plurality of recombinant expression vectors of proteins or polypeptides from different sources are constructed; the agapanthus dehydrin protein ApSK ₃ The promoter sequence of the gene comprises a nucleotide sequence (1), which is a polynucleotide sequence shown as SEQ ID number 1;

step 2: transfecting the recombinant expression vector obtained in step 1 into a host, and culturing the host under conditions suitable for expression of the protein or polypeptide.