CN102268433B - Plant aging specific promoter and application thereof - Google Patents

Abstract

The invention relates to a plant aging specific promoter and application thereof. In the invention, a promoter capable of responding to plant aging signals and regulating high expression of target gene specificity is separated for the first time. The promoter is very useful for regulating the expression of the target gene in a plant aging process, so as to change the plant characters, state and aging process.

Description

A kind of plant senescence-specific promoter and application thereof

technical field

The invention belongs to the field of biotechnology; more specifically, the invention relates to a plant senescence specific promoter and application thereof.

Background technique

Promoter refers to a DNA sequence in a gene that can accurately and effectively initiate gene transcription, usually located upstream of the gene. Promoter is an important element in the regulation of gene expression. It interacts with trans-acting factors (transcription factors) such as RNA polymerase and other protein cofactors to regulate the level of gene transcription and the specificity of its temporal and spatial expression.

The most widely used constitutive promoter is the tobacco mosaic virus (CaMV) 35S promoter. At the same time, people attach great importance to cloning constitutive promoters from plants themselves and have achieved initial success. The promoters of genes such as actin (Actin) and ubiquitin (Ubiquitin) have been cloned. Mariani et al. successfully used the tobacco anther tapetum-specific expression gene promoter TA29 to drive the specific expression of nuclease Barnase and RnaseT1 genes in anthers, destroy the tapetum, and obtain male sterile rape. education system. Expressing the ipt gene in wheat leaves and increasing the content of cytokinin can slow down the degradation of chlorophyll and slow down the senescence of leaves.

At present, plant transgenic technology usually uses constitutive expression promoters, such as cauliflower mosaic virus CaMV 35S promoter. However, genes regulated by constitutive promoters are continuously and efficiently expressed in various stages and tissues of plant development, without specificity in time and space, which is not only an unnecessary waste of resources for plants, but also unfavorable for the healthy development of plants. If some genes that may change the agronomic traits are expressed in unnecessary parts and periods, they may cause certain toxic effects on the growth and development of plants.

In summary, it is necessary to develop new promoters with specific functions to effectively and accurately change the agronomic traits of plants and achieve plant variety improvement.

Contents of the invention

The object of the present invention is to provide a plant senescence specific promoter and its application.

In a first aspect of the present invention, there is provided an isolated plant (such as a leaf of a plant) senescence-specific promoter, said promoter:

(a) located at the 5' end of the gene encoding pheophorbide oxidase and its upstream;

(b) The base length is 500-3000;

(c) have the necessary sites to initiate transcription and a transcription initiation point; and

(d) It has the function of responding to plant senescence signals and initiating the specific expression of target genes.

In a preferred example, the pheophorbide oxidase is derived from Arabidopsis thaliana.

In another preferred example, in (a), it is located at -2800 upstream of the pheophorbide oxidase gene to the 200th position in the coding region. Preferably, it is located at -2000 upstream of pheophorbide oxidase to the 200th position in the coding region; more preferably, it is located at -1907 upstream of pheophorbide oxidase to the 186th position in the coding region.

In another preferred example, the promoter contains a TATA-box structure.

In another preferred example, the plant is a cruciferous plant.

In another preferred example, the plant is Arabidopsis.

In another preferred example, the promoter is:

(1) the promoter of the nucleotide sequence shown in SEQ ID NO: 1;

(2) The nucleotide sequence has more than 95% identity with SEQ ID NO: 1 and has a promoter that responds to plant senescence signals and activates the specific expression function of the target gene; or

(3) A promoter whose nucleotide sequence is completely complementary to the nucleotide sequence shown in SEQ ID NO:1.

In another aspect of the present invention, a vector containing the promoter is provided.

In another preferred example, the vector further contains a target gene operably linked to the promoter.

In another preferred embodiment of the present invention, the target gene is a structural gene.

In another preferred embodiment of the present invention, the target gene can encode a protein with specific functions.

In another preferred embodiment of the present invention, the target gene is a foreign gene.

In another preferred example of the present invention, the target genes include (but not limited to): β-glucosidase (GUS) gene, genes related to cell division and differentiation (including cytokinin, etc.), auxin metabolic pathway related Genes (including genes related to auxin synthesis and degradation, auxin transport genes, etc.), genes related to nutrient transport (such as prenyl transferase genes), genes related to improving plant quality or traits or phenotypes (such as human lactoferrin gene, lysine synthase gene, beta-carotene synthesis gene, linear and amylopectin synthase gene, etc.), and hormone synthesis-related genes in seeds, etc.

In another preferred embodiment of the present invention, the target gene is located downstream of the promoter and is directly adjacent to the sequence of the gene encoding the promoter region. Usually, the distance between the promoter and the target gene is less than 1000bp (preferably, less than 500bp; more preferably, less than 100bp; most preferably, less than 50bp).

In another aspect of the present invention, a genetically engineered host cell is provided, said cell:

contain said carrier; or

The exogenous promoter is integrated in its genome.

In another aspect of the present invention, there is provided a method for expressing a gene of interest during plant senescence, said method comprising:

Transforming plant cells with the construct, the construct containing the promoter and the gene of interest operably linked to the promoter;

selecting for plant cells transformed with the construct or chromosomally integrated with the construct; and

The plant cells are regenerated into plants in which the gene of interest is expressed during plant senescence.

In another preferred example, the method includes:

(a) providing an Agrobacterium carrying an expression vector, the expression vector contains a construct containing the promoter and a gene of interest operably linked to the promoter;

(b) contacting plant cells, tissues or organs (such as flower organs) with the Agrobacterium in step (a), so that the construct is transferred into plant cells, tissues or organs;

(c) selecting plant cells, tissues or organs into which the construct has been transferred; and

(d) regenerating the plant cells, tissues or organs in step (c) into plants in which the gene of interest is expressed during plant senescence.

In another aspect of the present invention, the use of the promoter is provided, and the promoter is used for responding to plant senescence signals and promoting the expression of a target gene in the process of plant senescence.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1 shows the change of the expression level of AtPaO gene with the number of days of dark induction under the condition of dark induction.

Figure 2 shows the results of GUS staining under natural senescence regulation of transgenic plants. in,

A, Top view of transgenic plants, cotyledons and true leaves can be observed.

B. Senescence of each leaf of Arabidopsis thaliana. From left to right are all the rosette leaves of Arabidopsis thaliana, including: 2 cotyledons, the 1st true leaf, the 2nd true leaf, the 3rd true leaf...the 10th true leaf. It can be observed that the leftmost leaves are the yellowest, and the leaves are getting greener along the direction from left to right, that is, the leftmost leaves have the highest aging degree, and the aging degree of leaves decreases along the direction from left to right.

C and D, GUS staining results show the expression of GUS in each rosette leaf of Arabidopsis. It can be observed that the leftmost leaf has the darkest blue, and along the direction from left to right, the blue color changes from dark to light.

Figure 3 shows the results of GUS staining under natural senescence regulation of transgenic plants.

A, The senescence site of each leaf of Arabidopsis thaliana. From left to right are some rosette leaves of Arabidopsis thaliana, including: the 1st, 3rd, 5th, 7th, 9th, 11th, and 13th rosette leaves. It can be observed that the first senescent parts are the leaf tip and leaf edge, as indicated by the arrow.

B. The results of GUS staining show the expression of GUS in each rosette leaf of Arabidopsis thaliana. It can be observed that the tip and edge of the leaf are first observed to be stained blue by the GUS staining solution.

Figure 4 shows the results of GUS staining of leaves under dark induction. It can be observed that the staining of leaves on the 0th day of dark treatment is not obvious, but GUS staining can be observed on the edge of the leaves after 2 days of dark induction, and GUS gene expression can be observed in most parts of the leaves on day 4.

Detailed ways

After extensive and in-depth research, the inventors isolated for the first time a promoter capable of responding to plant senescence signals and regulating the specific high expression of the target gene. The said promoter is very useful for regulating the expression of the target gene in the process of plant senescence so as to change the plant traits, state or senescence process. The present invention has been accomplished on this basis.

the term

As used herein, the "plant" is not particularly limited, including (but not limited to): fruit plants, vegetable plants, crops and the like. Fruit plants such as but not limited to: Citrus, Rosaceae, Cucurbitaceae, Musaceae plants and the like. Vegetable plants are for example but not limited to: plants of Asteraceae, Solanaceae, Lamiaceae, Umbelliferae, Brassicaceae (such as Arabidopsis). Crops such as but not limited to: Gramineae, Amaryllidaceae plants, etc.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, the "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed at a specific position relative to the nucleic acid sequence of the target gene, so that the transcription of the nucleic acid sequence is guided by the promoter region, thus, the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the "promoter" or "promoter region (domain)" refers to a nucleic acid sequence, which usually exists upstream (5' end) of the coding sequence of the gene of interest, and can guide the transcription of the nucleic acid sequence into mRNA . Generally, a promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription. Herein, the promoter or promoter region includes variants of the promoter, which are obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, and the like.

As used herein, the term "specific expression" refers to the expression of a gene of interest at a specific time and/or in a specific tissue. The "senescence-specific promoter" refers to the expression of the target gene in the process of plant senescence under the regulation of this type of promoter.

As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, a promoter is foreign to a gene of interest if the combination of the promoter and the sequence of the gene of interest does not normally occur in nature. A particular sequence is "foreign" to the cell or organism into which it has been inserted.

As used herein, "cis-regulatory element" refers to a conserved base sequence that regulates transcription initiation and transcription efficiency of a gene.

As used herein, "target gene" refers to a gene whose expression can be promoted or directed by the promoter of the present invention. Suitable target genes include, but are not limited to: genes related to improving plant quality, traits or metabolism. Suitable target genes include, but are not limited to: β-glucosidase (GUS) gene, genes related to cell division and differentiation (including cytokinin, etc.), genes related to auxin metabolic pathway (including genes related to auxin synthesis and degradation, auxin transport genes, etc.), nutrient transport-related genes (such as prenyltransferase genes), genes related to improving plant quality or traits or phenotypes (such as human lactoferrin genes, lysine synthase genes, beta carotene synthesis genes, linear and amylopectin synthase genes, etc.), and genes related to hormone synthesis in seeds, etc.

As used herein, the words "comprising", "having" or "comprising" include "comprising", "consisting essentially of", "consisting essentially of", and "Consisting of"; "Consisting essentially of", "Consisting essentially of" and "Consisting of" are "contains" , The subordinate concept of "has" or "includes".

As used herein, the "aging process" of plants includes "natural aging process" and "induced (or artificial) aging process".

The "natural aging process" refers to the aging process initiated by plants in the process of growth and development. For example, the rosette leaves of Arabidopsis do not age at the same time, but gradually age according to leaf age, starting from the cotyledon and then the first true leaf, the second true leaf... to the tenth leaf true leaf. The process of plant aging can be known by observing the appearance of plants. For example, the leaves of most plants (such as Arabidopsis, etc.) change from green to yellow, which means that the leaves are senescent, and the more obvious the yellow, the higher the degree of senescence.

The "induced (or artificial) senescence process" refers to external conditions or thought to interfere with the growth and development process of plants, creating an atmosphere or environment for plants to senescence. For example, changing the growth environment of plants so that the plants grow under dark conditions, the plants will start the senescence process under the induction of darkness, and the senescence process will also increase with the increase of the induction time.

Promoter

The present invention provides a plant senescence-specific promoter, which has the following characteristics: (a) located upstream of the pheophorbide oxidase gene (called AtPaO in Arabidopsis) (preferably located at Pheophorbide oxidase upstream -2800 to the 200th position in the coding region); (b) has the function of responding to plant senescence signals and initiating the specific expression of the target gene; (c) has the necessary sites for initiating transcription and the transcription initiation point; And (d) the base length is 500-3000. As an embodiment of the present invention, the promoter has the promoter of the nucleotide sequence shown in SEQ ID NO: 1.

Hybridization of polynucleotides is a technique well known to those skilled in the art, and the hybridization properties of a particular pair of nucleic acids indicate their similarity or identity. Therefore, the present invention also relates to hybridization with the aforementioned specified nucleotide sequence with at least 50%, preferably at least 70%, more preferably at least 80% (such as 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical polynucleotides. The invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention.

"Stringent conditions" (or "stringent conditions") refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization When adding denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only if the identity between the two sequences is at least 50%, Hybridization occurs preferably at 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more or 90% or more, more preferably 95% or more. Also, hybridizable polynucleotides also have the function of responding to plant senescence signals.

The present invention also includes 50% or more (preferably more than 60%, more than 70%, more than 80%, more preferably more than 90%, most preferably more than 95%, such as 98%, 99%) of any promoter sequence of the present invention. %) identical nucleic acid, which also has the function of responding to plant senescence signals and initiating the specific expression of the target gene. "Identity"refers to the level of similarity (ie, sequence homology, similarity, or identity) between two or more nucleic acids, in terms of percentage positions that are identical.

It should be understood that although the promoter and its functions derived from Arabidopsis are provided in the examples of the present invention, the promoters derived from other similar plants that have certain identity (conservation) with the promoter also include Within the scope of the present invention, as long as those skilled in the art can easily obtain the promoter from other plants according to the information provided in the application after reading the application.

Initiate target gene expression

The promoter of the present invention can be operably linked to a gene of interest, which can be foreign (heterologous) with respect to the promoter. There is no particular limitation on the nucleic acid sequence of the target gene (such as a structural nucleic acid sequence), and the target gene preferably encodes a protein with specific functions, such as some proteins with important properties or functions in agriculture or plant improvement .

The promoter of the present invention can also be operably linked to an improved gene sequence of interest which is foreign (heterologous) to the promoter. The gene of interest can be modified to produce various desired properties. For example, the target gene can be improved to increase the content of essential amino acids, improve the translation of amino acid sequences, change post-translational modifications (such as phosphorylation sites), transport translation products outside the cell, improve protein stability, insertion or deletion cell signaling, etc.

Additionally, promoters and genes of interest can be engineered to downregulate specific genes. This is generally accomplished by linking the promoter to the gene sequence of interest directed in antisense reverse. Those of ordinary skill in the art are familiar with such antisense technology. Any nucleic acid sequence can be modulated in this manner.

Any of the aforementioned promoters and/or gene sequences of interest can be contained in the recombinant vector.

As a mode, the recombinant vector includes the promoter of the present invention, and includes a multiple cloning site or at least one restriction site downstream of the promoter. When the target gene needs to be expressed, the target gene is linked into a suitable multiple cloning site or restriction site, so that the target gene is operably linked to the promoter.

As another way, the recombinant vector includes (from 5' to 3' direction): a promoter, and a target gene. If necessary, the recombinant vector may also include a 3' transcription terminator, a 3' polynucleotide signal, other non-translated nucleic acid sequences, transport and targeting nucleic acid sequences, resistance selectable markers, enhancers or operators.

Methods for preparing recombinant vectors are well known in the art. The term "recombinant expression vector" refers to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vectors well known in the art. In conclusion, any plasmid and vector can be used as long as it can be replicated and stabilized in the host.

Methods well known to those skilled in the art can be used to construct an expression vector containing the promoter and/or target gene sequence of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein (GFP), etc.

In addition to the promoter of the present invention, the recombinant vector may also contain one or more other promoters. Said other promoters are, for example: tissue-specific, constitutive or inducible. For example, cauliflower mosaic virus 19S and 35S (CaMV19S CaMV35S), enhanced CaMV, tobacco RB7, etc.

The vector containing the above-mentioned appropriate promoter and the gene of interest can be used to transform appropriate host cells so that they can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells and the like. Those of ordinary skill in the art will know how to select appropriate vectors and host cells.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. Transformation of plants can also use methods such as Agrobacterium transformation or biolistic transformation, such as leaf disk method, immature embryo transformation method, flower bud soaking method and the like. Transformed plant cells, tissues or organs can be regenerated into plants by conventional methods, so as to obtain transgenic plants.

As a method, the method for preparing transgenic plants is: the vector carrying the promoter and the gene of interest (the two are operably linked) is transformed into Agrobacterium, and the vector fragment containing the promoter and the gene of interest is integrated into the plant by Agrobacterium on the chromosome. The transgenic recipient plants involved are, for example, Arabidopsis, tobacco, fruit trees and the like.

In an example of the present invention, the recombinant vector is a PBI101 binary vector with its own β-glucosidase (GUS) gene, and the promoter of the present invention is constructed upstream of the GUS gene in the vector to transform the plant, The promoter will activate the expression of the GUS gene, and the activation is regulated by each cis-acting element in the promoter region, simulating the condition that the gene is activated and transcribed in vivo.

In the example of the present invention, the inventors found that under the initiation or guidance of the promoter, the GUS gene can be specifically expressed in the process of plant senescence. Therefore, it can be seen that the promoter of the present invention is a promoter that responds to the senescence signal and initiates the expression of the target gene after receiving the signal. Said promoters are particularly useful for improving plant quality during plant senescence. β-glucosidase (GUS) can catalyze the cleavage of a series of β-glucosides to produce substances with chromophores or fluorescence. Spectrophotometer, fluorometer or histochemical methods can be used for quantitative and spatial analysis of GUS activity . In the technical field, GUS gene has been widely used as a reporter gene of transgenic plants, bacteria and fungi, especially it can be used to study specific cells and tissue parts where foreign genes are expressed.

application

The senescence-specific promoter of the invention has important application value in theoretical research and agronomic improvement.

The invention can be widely used in plant genetic engineering: as an important tool in plant genetic engineering, the promoter can be fused with the target gene to transform the plant and obtain the transgenic plant through the transgenic carrier. As the plant grows older, the promoter will start the expression of the downstream target gene (which can be any functional gene that can change the agronomic traits of interest), and gradually increase the expression of the gene with the aging process, so as to achieve The purpose of artificially controlling the expression of a certain functional gene in the aging stage.

In crop production, some crop leaves are prone to premature senescence, which reduces the overall photosynthesis level of the plant and reduces photosynthetic products, resulting in low crop yield and poor quality. For example, the premature senescence of leaves and functions of some hybrid rice in the later stage of development leads to low seed setting rate and high empty seedling rate, which seriously affects the further development of hybrid rice yield potential. The promoter can be fused with related genes that inhibit senescence (such as cytokinin, genes on the auxin metabolic pathway, exogenous isopentenyl transferase (ipt), etc.), transforming plants, and providing genes that delay senescence along with the aging process positive regulation, thereby delaying the senescence process of plants. It can be widely used in the preservation of vegetables and the delay of foliage plants.

In addition, the present invention provides a vector construction method of the promoter, and the feasibility of regulating the expression of downstream GUS gene by the promoter under the induction of senescence signal is confirmed in plants. It provides a basis for further research on the promoter and aging phenomenon.

The main advantages of the present invention are:

(1) The promoter of the present invention belongs to a specific type of promoter. In the natural aging process, it responds to the aging signal to start the expression of downstream genes, and the expression level is very low in the stages and organs that have not started aging, which can not only make the exogenous genes play an effective role in the plant, but also reduce the damage to the plant. Adverse effects, the use of senescence-specific promoters to regulate gene expression is a good way.

(2) Due to the specificity of senescence, the promoter of the present invention can be used in senescence-related research, and provides a wide range of applications in delaying plant senescence.

(3) The promoter of the present invention has the characteristics of rapid response and enhanced function with aging process. That is, the older the plant, the higher the expression level of the downstream gene regulated by the promoter.

(4) This gene can also drive downstream gene expression in dark-induced detached leaves. With the prolongation of dark induction time, the expression level increased obviously. This feature can be used for in vitro storage of vegetables.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

I. Materials and methods

1 Promoter cloning and vector construction

Using the Arabidopsis genomic DNA as a template, the forward primer AtPaO_GUS_FP2': 5'CTCGAGTTGTTGCGGTGGTC 3' and the reverse primer AtPaO_GUS_RP2: 5'CGTCGAATCCGAAGTGGGTA 3' were designed to amplify about 2kb upstream of the start codon ATG of At3g44880 by PCR. DNA fragments.

The amplified promoter sequence was connected to the vector pMD19-T Vector (purchased from TAKARA), and then the promoter fragment connected to the pMD19-T Vector and the vector pBI101.3 containing the GUS reporter gene were respectively treated with the restriction endonuclease XhoI+ XbaI and SalI+XbaI were digested. The digested products were recovered and ligated with T4 DNA ligase.

2 Obtaining of transgenic plants

The constructed vector was transformed into Agrobacterium strain C58 (obtained from University of California). Transgenic plants were obtained by conventional flower dipping. Transgenic plants were selected on MS medium containing 50ug/ml kanamycin.

3GUS histochemical staining

Remove the leaves of the transgenic plants with different treatments, put them in a centrifuge tube, add an appropriate amount of GUS staining solution and incubate overnight at 37°C, decolorize with 70% ethanol 2-3 times until the control material appears white. The GUS expression site was observed with the naked eye or under a microscope (blue is the GUS expression site), and the staining results were recorded by photography.

II. Example

Embodiment 1, amplification, cloning and sequence analysis of promoter

Using Arabidopsis thaliana genomic DNA as a template for PCR amplification, a DNA amplification product of about 2093bp was obtained, which was connected to pMD 19-T Vector, and the result was correct after sequencing. The full sequence of the promoter is shown in SEQ ID NO:1.

This sequence is the promoter of the key enzyme AtPaO in the chlorophyll degradation pathway in Arabidopsis. Therefore, the expression of AtPaO under dark induction conditions was detected by conventional Realtime PCR method.

It was found that under the dark induction condition, the expression level of AtPaO gene was up-regulated with the number of days of dark induction, as shown in Fig. 1 . Therefore, it can be seen that the promoter of the AtPaO gene is a senescence-specific promoter.

Embodiment 2, the construction of promoter plant expression vector

First use Xhol I and BamH I double enzyme digestion to recover the 2Kb promoter sequence, then use Sal I and BamHI double enzyme digestion pBI 101.3 (purchased from Takara), recover the large fragment, connect the recovered two fragments, transform Escherichia coli, extract The plasmid was digested and identified, and the promoter was correctly connected into the vector pBI101.3 to obtain pBI101.3-Promoter-GUS.

The recombinant vector pBI101.3-Promoter-GUS was transformed into Agrobacterium C58 by electric shock transformation method, amplified by PCR, and a band of about 2Kb was identified in 1% agarose gel electrophoresis, proving that pBI101.3-Promoter-GUS The vector has been transformed into Agrobacterium C58.

Embodiment 3, the acquisition of transgenic plants

Using Agrobacterium-mediated genetic transformation to obtain 10 transgenic plants, PCR identification, the results show that most of the lines can produce fragments with a size of about 2Kb, which proves that the promoter fragment fused with the GUS gene has been integrated into the Arabidopsis genome .

After GUS histochemical staining analysis, it was found that there were 6 lines carrying promoter and GUS chimeric gene, and these plants obtained under kanamycin resistance screening conditions could grow normally.

Example 4, GUS staining results under natural aging regulation of transgenic plants

The constructed fusion vector of the promoter and the reporter gene GUS was transformed into Arabidopsis thaliana, and 6 lines of transformed plants were obtained. The results of GUS staining under natural aging conditions showed that the staining was very light or even absent in the young leaves; And with the growth of leaf age, the staining becomes deeper and deeper. As shown in Figure 2, from left to right, all rosette leaves of Arabidopsis thaliana include: 2 cotyledons, 1st true leaf, 2nd true leaf, 3rd true leaf...10th true leaf The results of GUS staining also showed a changing trend from light to dark in leaves.

It is known in the prior art that the rosette leaves of Arabidopsis thaliana do not age at the same time, but gradually age according to leaf age, starting from the cotyledon, then the first true leaf, the second true leaf... to the 10th true leaf.

As shown in Figure 3: Among the leaves that start to senescence naturally, the yellow leaves are darker than the green leaves; while the same leaf, the first parts that start to senescence are the tip and the edge of the leaf. Blue staining by the GUS solution is the first to be observed, even though leaves have not yet shown obvious signs of yellowing visible to the naked eye. The above phenomena also further illustrate the sensitivity of the promoter to respond to aging signals.

Example 5, under dark induction, the results of GUS staining of leaves

Darkness is the promoter regulation of leaf senescence, and the expression of GUS gene downstream of this promoter can also be increased by inducing the detached leaves with darkness.

The Arabidopsis plants were placed in a dark environment, and the results are shown in Figure 4. The leaves of the transgenic plants treated with darkness for 21 days were analyzed by GUS staining. GUS staining could be observed at the edge of the leaves, and GUS gene expression was observed in most parts of the leaves at 4 days.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

<120> A plant senescence-specific promoter and its application

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ttcttggttc atgtcgtcca aatggagttc aaatgtttga aacggttgaa ggcatgatga 1080

ttgattttat tggagcttct cttaatgaaa ttgctaacca ggtacttcaa tctcataaaa 1140

cactagttac tcgtttgatc attacattat actaataaat ccataactaac atgttatgtt 1200

tttggctgtc tgttcaaaag ggtctaatag atcaacaaaa gcttgacact tttaagttgc 1260

ctatctatgc tccacaagcg gatgaattga agcaaatcat cgaggataac gggtgtttca 1320

cgattgaggt attcgaaaat attatacacg ccaagggaga gtatccgtta gaccccgagt 1380

ttttaacagt ctcctttaag gtcacggttg gaggatcagt agcttcacta tttgggcaag 1440

atggtatgga gaaaaccttt gagcttgtga aagagaagac acaagaaatg cttcctcaga 1500

tagccaaagc caaacccgga atgcaatacc tcattgtgct tcgaagaaac tgttttcatg 1560

atctatttag atctttgaaa cgagacttaa ataatgtaat tgcaatgatg tgtgtttgtg 1620

tgtgtatgtg ttgtattgtt tgttatttta aatagtctct ttcgtttatg tatatcacaa 1680

atacatatca caacgttcat atttgcaatg tgccaaaatc ctccacgtgt taaatccatt 1740

tgttcatagt ttctcgttac aagacaacaa atggtatcaa tgtccacgga aaaattgcat 1800

acttgcacgc aatctcttct tcttcttctt cgttcttctc gtagcttgaa ataagattca 1860

tcagaagagt aaataaacat caaaatccaa caaaccaaac tagaaaaatg tcagtagttt 1920

tactctcttc tacttctgca acaatcacca aatcccaatc caaaaagatt ccctttttat 1980

ctcccaccac aaaattccca ttaaaggtct caatttctcc atcaagatcg aaacttttcc 2040

acaacccttt acgcgtggcg gcgccgccgt ctgtacccac ttcggattcg acg 2093

Claims (9)

1. An isolated plant senescence-specific promoter, wherein said promoter:

(a) located at the 5' end and upstream of the encoding gene of the pheophorbide oxidase;

(b) has the necessary site for initiating transcription and transcription initiation point; and is

(c) Has the functions of responding to plant aging signals and starting the specific expression of target genes;

the promoter is SEQ ID NO: 1, or a promoter of the nucleotide sequence shown in 1.

2. The promoter of claim 1, wherein the pheophorbide oxidase is derived from arabidopsis thaliana.

3. The promoter of claim 1, wherein in (a) it is located from-1907 upstream of the pheophorbide oxidase to 186 th of the coding region.

4. A vector comprising the promoter of any one of claims 1 to 3.

5. The vector of claim 4, further comprising a gene of interest operably linked to said promoter.

6. A genetically engineered host cell, wherein said cell:

comprising the vector of claim 5; or

The promoter of claim 1 which is exogenous and integrated into its genome.

7. A method for expressing a gene of interest in the senescence process in a plant, comprising:

transforming a plant cell with a construct comprising the promoter of any one of claims 1-3 and a gene of interest operably linked to said promoter;

selecting plant cells into which said construct has been transferred or into which said construct has been integrated in the chromosome; and

regenerating said plant cell into a plant in which the gene of interest is expressed during the senescence process of the plant.

8. The method of claim 7, wherein the method comprises:

(a) providing agrobacterium carrying an expression vector, wherein the expression vector contains a construct, and the construct comprises the promoter of claim 1 and a target gene operably connected with the promoter;

(b) contacting a plant cell, tissue or organ with the agrobacterium of step (a) such that the construct is transferred to the plant cell, tissue or organ;

(c) selecting a plant cell, tissue or organ into which said construct has been transferred; and

(d) regenerating the plant cell, tissue or organ of step (c) into a plant in which the gene of interest is expressed during the senescence process of the plant.

9. Use of a promoter as claimed in any one of claims 1 to 3 for responding to a plant senescence signal and promoting expression of a gene of interest in the senescence process of a plant.

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