CN102732518A - FGI2 promoter expressed by regulatory gene under pathogen-induced condition and application of same - Google Patents

Abstract

The invention relates to an FGI2 promoter for regulating gene expression under the condition of pathogen induction and application thereof. The present invention isolates for the first time a promoter capable of responding to pathogen infection signals and regulating the specific expression of target genes. The said promoter is very useful for regulating the expression of the target gene to change the disease resistance of the plant under the state of pathogen infection.

Description

FGI2 promoter regulating gene expression under pathogen-induced conditions and its application

technical field

The invention belongs to the field of biotechnology; more specifically, the invention relates to an FGI2 promoter for regulating gene expression under the condition of pathogen induction and application thereof.

Background technique

Plant promoters play a key role in regulating plant gene expression, and can control plant expression in specific tissues, developmental stages, and different environmental conditions. Promoters can be divided into constitutive promoters, tissue-specific promoters and inducible specific promoters according to their functions. In terms of crop disease resistance, the current mainstream view in the world is that the expression of any disease-resistant gene by a constitutive promoter will cause the plant to be in a disease-resistant defense mode (mode), so that the plant defense system preferentially obtains energy and resource allocation, thereby affecting economics such as plant yield. traits. This is also considered to be the reason why the current application of genetically modified methods for disease resistance has not been successful. A reasonable way to solve this problem is to use inducible specific promoters to control the expression of disease resistance genes only when pathogens invade (Gurr, S.J., and Rushton, P.J. (2005a). Engineering plants with increased disease resistance: what are we going to express? Trends in Biotechnology 23, 275-282).

Pathogenesis-Related Proteins (PRs) refer to one or several proteins induced by plants in pathological or pathologically related environments. Plant pathogenic bacteria, fungi, viruses and viroids can all induce the production of pathogenic related proteins. Compounds with similar effects to pathogens and compounds that can induce similar stress conditions can also induce plants to synthesize disease process-related proteins, and related enzymes synthesized during natural aging also belong to disease process-related proteins (Liu Lihua et al., 1999). The earliest PR protein is Gianinazzi et al. (Gianinazzi, S., Pratt, H.M., Shewry, P.R., and Miflin, B.J. (1977). Partial-Purification and Preliminary Characterization of Soluble Leaf Proteins Specific to Virus-Infected Tobacco Plants.J Gen Virol 34 , 345-351) and Vanloon et al. found in tobacco respectively, and the researchers detected this type of protein in tobacco infected by tobacco mosaic virus TMV, and named it disease course-related protein. Later, it was found in various plants such as tomato and potato. Maize process-related protein is also called PRms (m stands for maize). Maize seedlings are infected with the pathogenic fungus (Fusarium moniliforme), and PRms mRNA can be detected in the corn radicle infected by the fungus. It has been found that corn seeds, radicles, and corn seedling leaves that germinate after absorbing water can produce PRms after being induced by compounds such as viruses, pathogenic fungi, mercuric chloride, or salicylic acid, and the PRms induced by different inducing factors are different. (Guo Honglian, Chen Jie, Gao Zenggui (1999). Advances in the research of corn process-related protein (PRm). Journal of Shenyang Agricultural University, 30(3), 372-374). Corn does not contain chitin and chitin, and only contains a small amount of glucan, but chitin and glucan are the main components of most fungi and bacteria except oomycetes. There is little or no chitinase and glucanase in healthy plants, but they can accumulate up to 40-100 times after infection induction, indicating that PRms play a role in disease resistance response. In vitro experiments proved that PRms can inhibit Fusarium moniliforme, Trichoderma recseian and Aspergillus and other common corn pathogens individually or synergistically. Histological observations also proved that PRms may play a role in maize disease resistance. When germinated maize seeds were infected by Aspergillus, incompatible PRms accumulated earlier than compatible PRms, indicating that incompatible reactions induced PRms to inhibit pathogens Invasion, while the affinity reaction can invade but may also prevent its expansion, and it has also been found in histology that the pathogen is mainly concentrated on the surface of the plant cell wall in contact with the fungus (Murillo, I., Cavallarin, L., and SanSegundo, B. (1997). The maizepathogenesis-related PRms protein localizes to plasmodesmata in maize radicles. Plant Cell 9, 145-156). However, the promoter construction and function of PRms in maize have not been reported in this field so far.

Contents of the invention

The object of the present invention is to provide a FGI2 promoter for regulating gene expression under the condition of pathogen induction and application thereof.

In a first aspect of the present invention, an isolated promoter is provided, said promoter:

(a) isolated from the upstream of the disease process-related protein coding gene;

(b) The base length is 800-2000;

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

(d) It has the function of responding to the signal of pathogen infection and initiating the specific expression of the target gene.

In another preferred example, the disease process-related protein is derived from corn.

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

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

In another preferred example, the length of the promoter is 1000-1800 bases.

In another preferred example, the length of the promoter is 1100-1500 bases.

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 pathogen infection signals and activates the specific expression function of the target gene;

or

(4) 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 example, the target gene encodes a protein with specific functions.

In another preferred example, the target gene is a foreign gene.

In another preferred example, the target gene is an antifungal protein gene and a disease process-related protein gene.

In another preferred example, the target gene is located downstream of the promoter and 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

There is an exogenous promoter integrated in its genome.

In another aspect of the present invention, there is provided a method for preparing a plant capable of responding to a pathogen infection signal, the method comprising: transforming a plant with a construct containing the promoter and the The promoter is operably linked to the gene of interest, and the gene of interest is expressed under the state of pathogen infection.

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 the plant cell, tissue or organ with the Agrobacterium in step (a), thereby making the described construct transfer to the plant cell, tissue or organ;

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

(d) regenerating the plant cell, tissue or organ in step (c) into a plant in which the gene of interest is expressed in a state of pathogen infection.

In another aspect of the present invention, the use of the promoter is provided, and the promoter is used for responding to pathogen infection signals and promoting the specific expression of the target gene.

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

In another preferred example, the pathogen is selected from but not limited to: bacteria and fungi.

In another preferred example, the pathogen is selected from but not limited to: Fusarium graminearum, Magnaporthe oryzae, and Fusarium oxysporum.

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

Description of drawings

Figure 1. The vector map of pTG-19T-FGI2 promoter (the promoter in the vector map is marked as Fgi-2promoter).

Figure 2. The expression vector map of pCAM-FGI2 promoter (the promoter in the vector map is marked as Fgi-2promoter).

Figure 3. Detection of GUS activity in Fgi-2promoter::GUS gene-transferred rice leaves induced by Fusarium graminearum. A: T0 generation leaf infection; B: T0 generation leaf water control; C: T1 generation coleoptile infection; D: T1 generation coleoptile water control; E: wild-type leaf infection; F: wild-type leaf water control ; G: 35S promoter drives GUS transgenic plant leaf infection; H: 35S promoter drives GUS transgenic coleoptile infection.

Fig. 4A. Comparison of the relative GUS enzyme activity of rice transfected with Fgi-2promoter::GUS gene induced by different pathogenic bacteria and control.

Fig. 4B. Comparison of Fgi-2 promoter-induced expression folds in Fgi-2 promoter::GUS gene transfected rice under different pathogenic bacteria and control induction.

Detailed ways

After extensive and in-depth research, the inventors isolated for the first time a promoter capable of responding to pathogen infection signals and regulating the specific expression of a target gene. The said promoter is very useful for regulating the expression of the target gene to change the disease resistance of the plant under the state of pathogen infection. The present invention has been accomplished on this basis.

the term

As used herein, the "plant (or crop, crop)" includes any kind of plant, as long as the plant is suitable for gene transformation operation (transgenic operation), such as various crops, flower plants, or forestry plants, etc. The plant may be, for example, a dicotyledonous plant, a monocotyledonous plant, or a gymnosperm. For example but not limited to: rice, wheat, barley, sorghum, corn, and rye of Poaceae; Chinese cabbage and pakchoi of Brassica in Brassicaceae; Arabidopsis in Brassicaceae, in addition to tobacco, melon and fruit , vegetables, rapeseed and so on. More specifically, said plants include (but are not limited to): wheat, barley, rye, rice, corn, sorghum, sugar beet, apple, pear, plum, peach, apricot, cherry, strawberry, raspberry, blackberry, bean , lentils, peas, soybeans, canola, mustard, poppies, olean, sunflowers, coconuts, castor oil plants, cocoa beans, peanuts, gourds, cucumbers, watermelons, cotton, flax, hemp, jute, citrus, lemons, grapes Grapefruit, spinach, licorice, asparagus, cabbage, Chinese cabbage, bok choy, carrot, onion, potato, tomato, green pepper, avocado, cinnamon, camphor, tobacco, nuts, coffee, eggplant, sugar cane, tea, pepper, grapevine , oyster grass, bananas, natural rubber trees and ornamental plants. As a preferred mode of the present invention, the plants are gramineous plants, including but not limited to rice, wheat, barley, sorghum, corn, and rye.

As used herein, unless otherwise specified, the "pathogen" refers to a microorganism that infects a plant, usually a microorganism that is harmful to a plant, and causes plant diseases and insect pests. The pathogens are, for example, pathogenic fungi, pathogenic bacteria, plant viruses, Phytophthora, and nematodes. More specifically, the pathogen is selected from the group consisting of: pathogenic fungus Fusarium graminearum (Fusarium graminearum) bacteria (also referred to herein as Fusarium graminearum, also known as Gibberella zeae), rice blast fungus (Magnaporthe oryzae ), Fusarium oxysporum, 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" means that the target gene is expressed at a specific time and/or in a specific tissue and/or in a specific state. The "promoter expressed under pathogen-induced conditions" means that under the regulation of such a promoter, the target gene is specifically expressed in the process of pathogen infection (including when the pathogen initially infects), while in the absence of Not expressed in the state of pathogen infection.

Generally, if the mRNA level is at least 5 times, preferably at least 10 times, more preferably at least 10 times higher in a specific state (the specific state herein generally refers to a state of pathogen infection) than in other states (such as a state without pathogen infection). If the expression level is 50 times higher, the promoter is considered to specifically drive the expression of the target gene at a specific time and/or in a specific state.

Since mRNA is only an intermediate product of gene expression, protein is the final product of gene expression. Therefore, in a more preferred situation, the activity of the protein product (such as GUS) is detected. If the protein activity is increased by more than 3 times, preferably more than 5 times, more preferably when the protein activity is not infected by pathogenic fungi, it can Preferably more than 10 times, then the promoter is considered to be more valuable for specifically driving the expression of the target gene at a specific time and/or in a specific state.

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 genes of interest include, but are not limited to: genes related to improving plant disease resistance, traits or metabolism. Suitable target genes include, but are not limited to: antifungal protein coding genes, disease process-related protein genes (pathogenesis-related genes, PR1-PR17). The specific disease process-related proteins include PR-1 (cysteine-rich secretary protein), PR-2β-1,3-glucanase, PR-3 Chitinase, PR- 4 Chitinas chitinase, PR-5Thaumatin-like sweet protein similar, PR-6 Proteinase-inhibitor I protease inhibitor, PR-7Endoproteinase endoproteinase, PR-8 chitinase type III chitinase, PR-9lignin- forming peroxidase, PR-10Ribonuclease-like ribonuclease-like protein, PR-11 Chitinase, PR-12 Defensin, PR-13 Thionin, PR-14 Lipid-transfer protein Protein, PR-15 Oxalate oxidase oxalate oxidase, PR-16 OxOLP Oxalate-oxidase-like oxalate oxidase similar protein, etc., or see van Loon, L.C., M.Rep, et al.(2006). "Significance of inducible defense-related proteins ininfected plants. "Annu Rev Phytopathol 44:135-62. The target gene can also be H.Wongand T.B.Ng, Gymnin, a potent defensesin-like antifungal peptide from the Yunnan bean Gymnocladus chinensis Baill, Peptides 24 (2003), pp.963-968.2; Benhamou N, Broglie K, Broglie R , Chet I. Antifungal effect of bean endochitinaseon Rhizoctonia solani: ultrastructural changes and cytochemical aspect of chitinbreakdown. Can J Microbiol 1993; 39: 318-28.3; Broekaert WF, Van Parijs J, Leyns F, Jooschi W, J Binding lectin from stinging nettlerhizomes with antifungal properties. Reported in Science 1989;245:1100-2.

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 "FGI2" and "FGI-2" can be used interchangeably.

Promoter

The present invention provides a promoter specifically expressed under the pathogen infection state, and the promoter has the following characteristics: (a) isolated from a gene encoding a possible disease course-related protein (abbreviated as FGI2 or PRms herein). Upstream; (b) 800-2000 bases in length; (c) has the necessary sites to initiate transcription and the transcription initiation point; and (d) responds to pathogens (such as fungi) infection signals and initiates specific expression of target genes Function. 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 (such as SEQ ID NO: 1) and there is at least 50%, preferably at least 70%, more preferably at least 80% (such as 85%) between the two sequences. %, 90%, 95%, 96%, 97%, 98%, or 99%) identical polynucleotides. The present invention particularly relates to a polynucleotide hybridizable under stringent conditions to the polynucleotide of the present invention (eg, SEQ ID NO: 1).

"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. Moreover, the hybridizable polynucleotide also has the function of responding to pathogen infection 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 pathogen infection 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 maize are provided in the examples of the present invention, the promoters derived from other similar plants having certain identity (conservation) with the promoter are also included in this invention. Within the scope of the 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.

In a specific embodiment of the present invention, the promoter sequence of the present invention was cloned by using the PCR method using corn genomic DNA as a template, and constructed on the plant expression vector pCAMBIA1300 with the fusion gene GUS, mediated by Agrobacterium Genetic transformation of rice, FGI2promoter::GUS transgenic plants were obtained, and the promoter activity and pathogen inducibility of the FGI2 promoter were analyzed. GUS activity can be detected in the transgenic rice callus, indicating that the promoter has activation activity. During the growth period of the seedlings, no GUS activity was detected in the roots and leaves, indicating that under normal growth conditions, the FGI2 promoter does not drive the expression of downstream genes; however, after the infection of Fusarium graminearum, the FGI2 promoter Driven GUS activity has upregulated expression. Therefore, it is considered that the FGI2 promoter has the initiating activity of driving the expression of the GUS gene in the early stage of infection by pathogenic fungi.

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 functional or structural nucleic acid sequence), and the target gene preferably encodes a protein with specific functions, such as certain proteins with anti-pathogen functions.

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. When used in plants, it can also be a eukaryotic expression vector. In conclusion, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. Several vector options are known to those skilled in the art.

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 gene gun 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, rice, wheat, corn, Arabidopsis, tobacco, fruit trees and the like.

In the example of the present invention, described recombinant vector is pCAMBIA vector, and it carries β-glucosidase (GUS) gene, constructs the promoter of the present invention to the upstream of GUS gene in this vector, transforms plant, promoter The expression of the GUS gene will be activated, and the activation is regulated by each cis-acting element in the promoter region, simulating the condition of the gene being activated and transcribed in vivo. 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. Generally, the following consensus has been reached in the field: if a promoter can drive the expression of GUS gene, it can also drive the expression of other target genes other than GUS gene.

application

According to an embodiment of the present invention, the early maize cells infected by Fusarium graminearum were obtained by laser microdissection technology, and a small amount of RNA was extracted therefrom through linear amplification to obtain tens of micrograms of cRNA for gene expression differential analysis (RT-PCR, real-time-PCR and chip multiple means). Comparing the gene expression of maize cells infected by Fusarium graminearum in the early stage and the same type of maize cells not infected by Fusarium graminearum, we found a gene whose expression was induced and up-regulated. The inventors named it FGI2, which encodes PRms .

According to an embodiment of the present invention, the FGI2 described in the present invention corresponds to the maize process-related protein gene (PRms), and the GenBank accession number of the maize process-related protein gene is X54325. The present inventor cloned the FGI2 promoter sequence by PCR method using corn genomic DNA as a template, constructed it on the plant expression vector pCAMBIA1300 with the fusion gene GUS, and obtained multiple plants through genetic transformation of rice mediated by Agrobacterium FGI2promoter::GUS transgenic plants were analyzed for the promoter activity and pathogen inducibility of the FGI2 promoter. GUS activity can be detected in the transgenic rice callus, indicating that the promoter has activation activity. During the growth period of the seedlings, no GUS activity was detected in the roots and leaves, indicating that under normal growth conditions, the FGI2 promoter does not drive the expression of downstream genes; after the Fusarium graminearum infection, the GUS activity driven by the FGI2 promoter was up-regulated . Therefore, it is considered that the FGI2 promoter has the initiating activity of driving the expression of the GUS gene in the early stage of infection by pathogenic fungi.

According to an embodiment of the present invention, the leaves of the transgenic rice in which the promoter of the present invention drives the GUS reporter gene has no expression of the GUS gene by GUS staining when it is not infected, and shows visible expression by GUS staining when it is infected. Transgenic rice with 35S promoter driving GUS reporter gene showed visible expression by GUS staining when uninfected. The 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. When plants are attacked (infected) by pathogens, the promoter will start the expression of downstream target genes (which can be any functional genes that can change agronomic traits of interest, especially genes that resist pathogens), so as to achieve The purpose of controlling the expression of a certain functional gene in the state of invasion.

The promoter obtained in the present invention can be used to drive specific other genes (such as anti-pathogen genes) in transgenic crops (such as rice, corn, etc.), so that the genes can be expressed in the early stage of transgenic plants being infected by pathogens, so as to achieve It plays the role of resisting pests and diseases and maintaining yield when there is no threat of disease, and keeps it closed when there is no threat of disease, which is conducive to the distribution of energy and nutrients and tilts towards high yield. By using the promoter of the present invention to drive the plant anti-pathogen gene, it can react quickly when being infected by pathogens, and defend against the infection of pathogens, so as to prevent the disease more effectively in the early stage of the disease, and at the same time it is different from the constitutive expression promoter. It can resist pathogens without affecting the yield of crops.

The main advantages of the present invention are:

(1) The promoter of the present invention belongs to a specific type of promoter. When the plant is infected by the pathogen, it responds to the infection signal to start the expression of the downstream gene, and the expression level is very low or not expressed when the plant is not infected by the pathogen, which can make the exogenous gene in the plant Effectively play a role in vivo, and at the same time reduce the adverse effects of the expression on plants.

(2) At present, some constitutively expressed promoters are mostly used in crop disease resistance, such as cauliflower mosaic virus (CaMV) 35S promoter, corn ubiquitin promoter, and the promoter obtained by the present invention is more than these constitutively expressed promoters Son is more specific. The promoter is only expressed when the crop is infected, but not during the normal period of the crop.

(2) The promoter of the present invention has the characteristic of rapid response.

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. For the experimental methods that do not indicate specific conditions in the following examples, usually according to the conditions described in J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, 2002, or according to the conditions suggested by the manufacturer . 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

Material

Maize variety B73 was purchased from The Maize Genetics Cooperation Stock Center (U.S. University of Illinois) or see Schnable, P.S., D.Ware, et al. (2009). "The B73 maize genome: complexity, diversity, and dynamics." Science 326 ( 5956): 1112-5, rice variety "Zhonghua 11" (Oryza sativa japonica Zhonghua 11) see Chinese patent 200510110629.9, Fusarium graminearum (Fusarium graminearum) strain PH-1 was purchased from FungalGenetics Stock Center (http://www. fgsc.net/) See Cuomo, C.A., U.Guldener, et al. (2007). "The Fusarium graminearum genome reveals a linkbetween localized polymorphism and pathogen specialization." Science 317(5843): 1400-2; Magnaporthe oryzae) bacterial strain ZB15 is provided by Institute of Genetics and Development, Chinese Academy of Sciences; Agrobacterium tumefaciens (Agrobacterium tumefaciens) bacterial strain EHA105 is referred to Hood, E.E. etc., 1993, New Agrobacterium helper plasmads for gene transfer to plants.Transgen.Res.2:208 -218; pCAMBIA1300 plasmid was purchased from CAMBIA Company; pTG-19T plasmid was purchased from TaRaKa Company. The concentrations of antibiotics used in the test were kanamycin (Kan) 50 μg/ml and ampicillin (Amp) 100 μg/ml. Restriction endonuclease, T4 DNA ligase, Taq DNA polymerase, etc. were products of TaRaKa Company; 5-bromo-4-chloro-3-indoleglucuronide (X-Gluc) was purchased from Qiancheng Biotechnology Company; ampicillin ( Amp) and Kanamycin (Kan) were purchased from Dingguo Biotechnology Company.

PRms gene expression detection

Design a pair of RT-PCR primers to detect gene expression:

FGI2RT-PCR primer F 1: GCAACATCGCCGGGATGAAG (SEQ ID NO: 4);

FGI2RT-PCR primerR1: ATGCGCTGCAACAAGCTCGT (SEQ ID NO: 5).

Cloning and sequence analysis of FGI2 promoter

According to the FGI2 gene sequence, the upstream and downstream primers of the FGI2 gene promoter containing HindIII and BamHI restriction sites were designed:

FGI2promoter primer F: 5'-GAGAAAGACCAGCGGGAAGA-3' (SEQ ID NO: 6);

FGI2 promoter primer R: 5'-TTGTAGAAGCTGCTACCTAGGGAG-3' (SEQ ID NO: 7).

Total DNA was extracted from maize leaves by referring to the CTAB method in Molecular Cloning: A Laboratory Guide. Using the total corn DNA as a template, the FGI2 promoter sequence was amplified by PCR with the above-mentioned upstream and downstream primers, connected to the pTG-19T plasmid, transformed into E.coli DH5-α competent cells, and obtained the recombinant plasmid by blue-white screening. Sent to Beauchamp Sequencing Company for sequence determination. The sequencing results were compared to http://www.ncbi.nlm.nih.gov/ and http://www.maizesequence.org/.

Construction and Transformation of FGI2(Fgi-2)promoter::GUS Expression Vector

The cloned FGI2 promoter fragment was digested with HindIII and BamHI, and after recovery, it was ligated with the HindIII and BamHI vector of pCAMBIA1300 to obtain the expression vector FGI2promoter::GUS fused with the FGI2 promoter and GUS gene; The vector was transformed into Agrobacterium tumefaciens EHA105 strain. Take immature rice seeds 12-15 days after pollination and disinfect them with 70% (v/v) alcohol for 1min, then disinfect them with 2% (v/v) sodium hypochlorite solution for more than 90min, and rinse them with sterile water for 4-5 times; Embryos were cultured in the dark at 26°C for about 7 days on the induction medium to induce callus tissue for later use. Referring to the method of Hiei et al. (Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Efficient Transformation of Rice (Oryza-Sativa L) Mediated by Agrobacterium and Sequence-Analysis of the Boundaries of the T-DNA. Plant Journal 6, 271-282), Agrobacterium-mediated genetic transformation, transformant screening and regeneration of transgenic plants.

Pathogen induction treatment

The FGI2promoter::GUS transgenic line without GUS expression in the roots and leaves of the T0 seedling stage was selected for pathogenic bacteria induction to verify the promoter function. The induction took two stages: seed germination stage and seedling stage.

The coleoptile infection method was adopted in the seed germination stage, and the promoter function was verified on the T1 generation of transgenic plants. Take the bud sheaths that have been sown for about 2 days for treatment, dip 1.0% (w/v) gelatin solution evenly around the bud sheaths with a cotton swab, cut off about 1-2 mm of the top leaf tips of the bud sheaths, and cause wounds. Take a cotton sliver and wrap it around ^the wound of the bud sheath, inoculate the sliver of Fusarium graminearum (Fusarium. Cultivate in an incubator at a temperature of 25° C. and a humidity of 75-100%. After 4-7 days, the GUS activity of the T1 generation of FGI2promoter::GUS transgenic plants was detected, and three repeated experiments were set up.

The leaf-cutting infection method was adopted at the seedling stage, and the promoter function was verified on the T0 generation and T1 generation of FGI2promoter::GUS transgenic plants. Take transgenic rice leaves that have grown for about 8 weeks, use pointed tweezers to scratch the back of the leaves, cut them from the veins, half of them are inoculated with PH-1 (bacteria suspension concentration is 1×10 ⁸ cfu/ml), and the other half are treated with water control. Wild-type "Zhonghua 11" rice was used as a negative control, and 35S promoter-driven GUS transgenic plants were used as a positive control. After 48 hours, the treated rice leaves were tested for GUS activity, and the experiments were repeated three times.

GUS activity detection

Qualitative method: referring to Jefferson's method (Jefferson, R.A. (1987). Assaying chimeric genes in plants: the GUS gene fusion system. Plant Molecular Biology Reporter 5, 387-405), the rice tissue was stained and GUS activity was detected. Stain at 37° C. for 8 hours, and decolorize with 95% (v/v) ethanol until the negative control material turns white. Observed under a microscope and photographed for preservation.

Quantitative measurement of GUS activity: Refer to Jefferson's method and optimize as follows:

1. Reagent preparation

1) GEB (GUS extraction buffer) 100ml

Store at 4°C.

2) stop buffer: 0.2M Na ₂ CO ₃ (21.2g dissolved in 1LddH ₂ O);

Dissolve 2.1g of anhydrous Na ₂ CO ₃ in 100ml of ddH ₂ O and store at room temperature.

3) 1mM 4-MU stock solution (dissolved by heating)

9.9 mg of 4-MU (4-methylumbelliferone) was dissolved in 50 ml of ddH ₂ O, and stored at 4°C in the dark.

4) 1μM 4-MU working solution

10.87μl 1mM 4-MU stock solution was dissolved in 10ml ddH ₂ O, and stored at 4°C in the dark.

5) 20mM 4-MUG

2.1 mg of 4-MUG (4-methylumbelliferyl β-D-glucuronide) was dissolved in 200 μl of ddH ₂ O, and stored at 4° C. in the dark (dissolved by heating).

2. Steps

1) Take 100 mg of rice leaf from the infected part, grind it with liquid nitrogen, dissolve it in 500 μl GEB, grind it completely in an ice bath, centrifuge at 14000 rpm at 4 °C for 10 min, and take the supernatant for the determination of GUS activity and protein concentration. Repeat the centrifugation once if necessary.

2) Add 50μl of 20mM substrate 4-MUG solution into 430μl GEB, add 20μl supernatant after preheating in 37℃ water bath, continue to react in 37℃ water bath and start timing, take 100μl reaction solution and add 1.9ml stop buffer after 60min The reaction was terminated, and stored in the dark at room temperature, ready to measure fluorescence.

3) Take 100 μl of the sample to be tested and put it into the ELISA plate, set 3 replicates for each sample, and use stop buffer as the blank control.

4) Take 1 μM 4-MU working solution, dilute it to 0, 5, 15, 30 μMol/L, take 100 μl of the reaction solution and add it to 1.9ml stop buffer to stop the reaction and make a standard curve.

5) Use BioTek Synergy2 multifunctional microplate reader to measure, the emission wave is 460nm, and the absorption wave is 360nm to detect the fluorescence value. GUS activity was calculated from the standard curve.

II. Example

Embodiment 1, plant endogenous FGI2 gene expression when pathogen invades or does not invade

The inventors used Real-Time PCR to detect the expression of the plant endogenous FGI2 gene under the condition of being invaded or not invaded by Fusarium graminearum.

Maize stalk cells infected by Fusarium graminearum for one day obtained by laser microdissection were detected by real-time reverse transcription PCR (infected tissue 1: adjacent cells adjacent to cells invaded by Fusarium graminearum, which had not been directly infected by F. graminearum graminearum invasion; infected tissue 2: cells directly invaded by Fusarium graminearum), compared with the same kind of corn stalk cells inoculated with water (Mock), the FGI2 gene expression level increased by 50-200 times (1st, 2nd, 3rd represent the data of three independent experiments).

The results are shown in Table 1 below.

Table 1. FGI2 gene expression detection results

Cloning and sequence analysis of embodiment 2, FGI2 promoter

The FGI2 promoter primer was designed according to the genome sequence, and the upstream 1293bp maize DNA fragment before the translation start site was amplified, which was named FGI2promoter.

The specific primer sequence used to clone the promoter is as follows:

FGI2promoter primer F: 5'-GAGAAAGACCAGCGGGAAGA-3' (SEQ ID NO: 8);

FGI2promoter primer R: 5'-TTGTAGAAGCTGCTACCTAGGGAG-3' (SEQ ID NO: 9).

The cloned promoter sequence (1305bp) was verified by sequencing as follows (SEQ ID NO: 1):

AAGCTTGAGAAAGACCAGCGGGAAGAGAGAACGATGCGCGGGAGATATTTTTGTTCCGAGCTCGGTGCTAAGATCTACGACGCCGAGCTCGGGCCACGTCGACGCCACGTCGGCCCTCGCTAGCGAGCGCTGCAGCTAACTCGGTGCCAAGATCTATAGCGTGAGTTGTGTTAGCTCAGTGCCACGTCTCTTGGCGCCAAGCAAAGGGCCTAAAACTGCTATTAAGTATTCTAAGGGTCTAAACGTGAATATTTTTCATAAAATAGGCTAAAATACAAAAAAAATCAGGAGGTTCGCTGTGCTGCGAACAGAGGTGCGAGCGAATATAATAGGATGTTTGTGTAGTAATAAGCTAGTGTATTATCTTCTTAATCCTTACTTTTTTATTTGGTTTGTGAAATAGAATGAGGGCACGTTTGGTTTGATGGACTAAAGATTAGTCCCTCCATTTTAGTCTCATTTAATCCTTAA AAACGGTGGGACTAAAACAGAGACGACTAAACTGTTTTAGTCTCTAGTCCCTAAAGGGATGACTAAAAGGGACTAAACTATATAAATACCACTTTTTTACCCCTCTTTTATTTCAGTTGCACTAATGGTATGAGAATGCCAAAGGGTATTTCAGTCCTCTTATGATTCATTTAATATGTTTTGAATACTTTTAGTCCCTATAACCAAACAAGGTAGAGACTAAACTTTAGACTCTTAATTAAATTTTAGTTCATGGACTAAAGGAACCAAACAAGCCCTGAGTCGATCCATAACCATCTCATCTCATTCCTTATAGTTAGTTAGTTTGTTAGTTAACACTAATATAAGAAATGCGGTCACCTACTAAAGCTGAG AGACTCATGATGCACAACATCGCTGAATGTCGTCAGTTCAAACTTTACACGTTCGTAACAGAAATACAGATAAGAATGCATCTTATCCGTTTGACTTCAACTGCATTTTAGACTAGACAGCATGCACGAATTGACCATTCCATGAGGTCCTTTGATCTGTACAA TATATAAA TGCACCTAACAGAATCAAAGTTGCCTCAACGCTATGCATGTCCTGTTCGTGGAATTCTATTGAACTCATACGTAGAAAAGATTGGCATGCCGGCCATATTTTCGTGCAACGGGACTATTTTTCCAAGCATACTGGAGAAAATTCACATGCTCTCCACTAT TAATA GCCCCCACATATTTGCAG TATA GCATCCACAAATTAAACTCACGCACACACACAATTAGGTCACATACACACATCCCGGCTGCCTCCCTAGGTAACAGCTTCTACAAGGATCC

盒，以及W-box(Eulgem T，Rushton PJ，Schmelzer E，Hahlbrock K，Somssich IE(1999)Early nuclear events inplant defence signalling：rapid gene activation by WRKY transcription factors.EMBO J 18：4689-4699)。In the above sequence, the predicted TATA box,

box, and W-box (Eulgem T, Rushton PJ, Schmelzer E, Hahlbrock K, Somssich IE (1999) Early nuclear events inplant defense signaling: rapid gene activation by WRKY transcription factors. EMBO J 18: 4689-4699).

The FGI2promoter was connected to the T/A linker of the pTG-19T vector, and the recombinant vector pTG-19T-FGI2promoter was obtained through screening (Fig. 1). Sequence analysis showed that the cloned FGI2promoter sequence was consistent with the genome reported by Schnable, P.S., D.Ware, et al. (2009). "The B73 maize genome: complexity, diversity, and dynamics." Science 326 (5956): 1112-5 The sequence in the corresponding region is consistent.

A truncated FGI2 promoter (FGI2S) with a length of 878 bp was obtained in the same way and a vector was constructed for the analysis of the transgenic rice promoter. (The FGI2S promoter is reduced by 427 bases at the 5' end than the FGI2 promoter (SEQ ID NO: 1). The FGI2S sequence is as follows (SEQ ID NO: 2):

AAGCTTAGATTAGTCCCTCCATTTTAGTCTCATTTAATCCTTAAATTGCCAAACGGTGGGACTAAAACAGAGACGACTAAACTGTTTTAGTCTCTAGTCCCTAAAGGGATGACTAAAAGGGACTAAACTATATAAATACCACTTTTTTACCCCTCTTTTATTTCAGTTGCACTAATGGTATGAGAATGCCAAAGGGTATTTCAGTCCTCTTATGATTCATTTAATATGTTTTGAATACTTTTAGTCCCTATAACCAAACAAGGTAGAGACTAAACTTTAGACTCTTAATTAAATTTTAGTTCATGGACTAAAGGAACCAAACAAGCCCTGAGTCGATCCATAACCATCTCATCTCATTCCTTATAGTTAGTTAGTTTGTTAGTTAACACTAATATAAGAAATGCGGTCACCTACTAAAGCTGAGCAATAGACTCATGATGCACAACATCGCTGAATGTCGTCAGTTCAAACTTTACACGTTCGTAACAGAAATACAGATAAGAATGCATCTTATCCGTTTGACTTCAACTGCATTTTAGACTAGACAGCATGCACGAATTGACCATTCCATGAGGTCCTTTGATCTGTACAATATATAAATGCACCTAACAGAATCAAAGTTGCCTCAACGCTATGCATGTCCTGTTCGTGGAATTCTATTGAACTCATACGTAGAAAAGATTGGCATGCCGGCCATATTTTCGTGCAACGGGACTATTTTTCCAAGCATACTGGAGAAAATTCACATGCTCTCCACTATTAATAGCCCCCACATATTTGCAGTATAGCATCCACAAATTAAACTCACGCACACACACAATTAGGTCACATACACACATCCCGGCTGCCTCCCTAGGTAACAGCTTCTACAAGGATCC

The FGI2S promoter is connected to the T/A linker of the pTG-19T vector, and the recombinant vector pTG-19T-FGI2S promoter is obtained through screening.

Embodiment 3, construction and transformation of fusion gene expression vector

Recombinant plasmid pTG-19T-FGI2promoter was digested with HindIII and BamHI, the cloned fragment was recovered, ligated with the HindIII and BamHI double digestion plasmid vector of pCAMBIA1300, and the FGI2promoter::GUS fusion gene plasmid Fgi-2promoter was obtained after transformation and digestion verification ::p1300GN (Figure 2). The obtained recombinant expression vector was transformed into Agrobacterium EHA105 strain by freeze-thaw method, and confirmed by plasmid digestion analysis. The obtained clones were verified to be correct by DNA sequencing. The obtained Agrobacterium was called EHA105/Fgi-2promoter::p1300GN.

pTG-19T-FGI2S promoter was also operated to obtain Fgi-2Spromoter::p1300GN, and Agrobacterium EHA105/Fgi-2Spromoter::p1300GN.

Embodiment 4, the acquisition of transgenic rice

Using Agrobacterium EHA105/Fgi-2promoter::p1300GN as the mediator, the constructed FGI2promoter::GUS chimeric gene was introduced into the primary callus derived from immature embryos in "Zhonghua 11" as described in "Materials and Methods" Among them, 21 independent transgenic rice lines (listed in Table 2) were obtained through screening and regeneration. Most of the transgenic plants grow normally and can bear fruit. In each independent transformed line, the total DNA of leaves was extracted from all individual plants, and analyzed with primers specifically paired with the GUS coding region, and specific products could be amplified, which preliminarily confirmed that the chimeric gene The T-DNA has been integrated into the rice chromosome. The following table shows that the roots and leaves of the callus and transgenic rice seedlings were tested for GUS activity, wherein 13 lines showed GUS activity when the callus was stained, and all of them did not show GUS activity in the detection of leaves at the seedling stage (Table 2 ).

Table 2

"Weak +" means that the color signal is weak.

Embodiment 5, Fusarium graminearum induces GUS expression result of transgenic rice

On the basis of the above analysis, 7 lines (11, 21, 26, 28, 36, 39, 43) that did not show GUS activity in the transgenic rice seedling stage were selected to induce the expression of Fusarium graminearum. As shown in Figure 3, the GUS activity was detected after the T0 generation leaves of FGI2promoter transgenic rice were infected by Fusarium graminearum for 48 hours, and there was GUS expression near the wound, but there was no GUS expression in the water treatment; GUS expression was expressed in the T1 generation plants when the coleoptiles were infected , water treatment without GUS expression. The transgenic rice with 35S promoter driving the GUS reporter gene showed visible expression by GUS staining when not infected (Table 3).

table 3

FGI2promoter-staining of each strain

(+: color development; -: no color development; space means no experiment)

EHA105/Fgi-2Spromoter::p 1300GN transgenic rice showed no GUS staining under F. graminearum treatment, indicating that the truncated Fgi-2Spromoter was not sufficient to drive F. graminearum-induced expression. The truncated promoter has no function of inducing expression, indicating that the first 427 base pairs in SEQ ID NO: 1 are necessary for inducing expression.

For comparison, the inventors also cloned the promoter (SEQID NO: 3) of another gene FGI1 (ANT-G2):

TCCGTTTCTCCATAGGTGTGTGCGTCGCAGTGGATTTAATGCTCCGTTTTTCCTGGAGCCTTGGAATCTTTAGATCTTTATGGAGTCTTTTTAGATCGTGAGATAATTGGTTGGTAATCGGGTAAGCGGGAAGAAAAAATTGTCGTATTTTGACCGCTGTTTTGTCATAGTGTCGGTGCCGCGTTAGATCTGTCTGTTATGAGCCGTGGATGGCGATCTGACGGCAGGCGACCGTCTCATTAGTACTTGTATAACTACCATTGTTTCAGTTTTTTTTTCAGTTCTTGAAAGTACTGAGTAATAGAGTGCACACCTATACTTTTGACATGCTATGATGTTCTTCGCTGGTTTTGTAATTAAATTTGTAAATCATGGTAATCTTTGAATGCTAAATTTTCTGAACTGTTTGAGCATGCTACAATGGGCATCTGTTCTATTTTGATGATTGTTGTCAAAACTAGAGTTATGGAACATGAAAAATATAAATGTGTATGTTTGTATGATTGCATTCATTGTTTATTTCTGTCTAATAGTTTTATCTGTTAGTGTTTGCACGGAGGCTCTCTGCTTTAGTTAGATGTGTCTAGCTTTGATTTGTTCTTACTCTATATTGAAACTGTAAAATATACTCATCCTTTATTGCACAGATGCGGAGGTCTTATTTTGTTTCTCTTTATGTAATGTCCTAAGTTTTGTGTGTTGACATTATGTTGCAGCCAACAGTTTTGAGG

TCCGTTTCTCCATAGGTGTGTGCGTCGCAGTGGATTTAATGCTCCGTTTTTCCTGGAGCCTTGGAATCTTTAGATCTTTATGGAGTCTTTTTAGATCGTGAGATAATTGGTTGGTAATCGGGTAAGCGGGAAGAAAAAATTGTCGTATTTTGACCGCTGTTTTGTCATAGTGTCGGTGCCGCGTTAGATCTGTCTGTTATGAGCCGTGGATGGCGATCTGACGGCAGGCGACCGTCTCATTAGTACTTGTATAACTACCATTGTTTCAGTTTTTTTTTCAGTTCTTGAAAGTACTGAGTAATAGAGTGCACACCTATACTTTTGACATGCTATGATGTTCTTCGCTGGTTTTGTAATTAAATTTGTAAATCATGGTAATCTTTGAATGCTAAATTTTCTGAACTGTTTGAGCATGCTA CAAT GGGCATCTGTTCTATTTTGATGATTGTTGTCAAAACTAGAGTTATGGAACATGAAAAATATAAATGTGTATGTTTGTATGATTGCATTCATTGTTTATTTCTGTCTAATAGTTTTATCTGTTAGTGTTTGCACGGAGGCTCTCTGCTTTAGTTAGATGTGTCTAGCTTTGATTTGTTCTTACTC TATA TTGAAACTGTAAAA TATA CTCATCCTTTATTGCACAGATGCGGAGGTCTTATTTTGTTTCTCTTTATGTAATGTCCTAAGTTTTGTGTGTTGACATTATGTTGCAGCCAACAGTTTTGAGG

The inventors constructed transgenic plants expressing GUS gene driven by FGI1promoter in the same way as described above to construct GUS gene expression driven by FGI2promoter, and detected the GUS staining status of each obtained line.

The GUS staining results of GUS gene expression driven by FGI1 promoter and FGI2 promoter showed that FGI2 was more strongly induced. In terms of the color development period, the T0 generation of the strain driven by the FGI1 promoter to express GUS was detected 48 hours after infection, and the detection was performed every 24 hours. It was found that GUS expression was detected in individual strains as early as 48 hours, and the color was very weak Most were detected after 7 days. Similarly, the T0 generation of the strains driven by the FGI2 promoter to express GUS was detected 48 hours after infection, and GUS expression was detected at 48 hours, and the color was very strong. After 4 days, most of the tested strains had GUS expression. It was also observed that the region of GUS expression was larger in FGI2 than in FGI1.

The method for quantitative detection can more accurately estimate the multiple of expression induced by pathogenic fungi. The specific experimental results are shown in Table 4 and Figure 4. The multiple of expression induced by Fusarium graminearum is in two independent transgenic lines (FGI2promoter transgenic plants) The average reached more than 20 times. The inventors believe that this induction factor is still underestimated, because only a small number of cells are infected by the fungus after infection and start to induce expression, while other peripheral cells in the sampling do not express, and the calculated value is the average value. The induction factor should be higher in infected cells.

The present inventor thinks that the FGI2 promoter may also be induced by the infection of some other pathogenic fungi (such as Magnaporthe grisea), and the specific experimental results are shown in Table 4 and Figure 4. The multiples of induced expression were equal in two independent transgenic lines reach more than 7 times.

Table 4. Quantitative detection of GUS enzyme activity in Fgi-2 promoter::GUS transgenic rice treated with Fusarium graminearum for 6 days or Magnaporthe grisea for 3 days

relative GUS unit 1L39 1L36 Pathogen-free water treatment control 0.06 0.03 Fusarium graminearum treatment experiment 1 2.07 0.55 Rice blast fungus treatment experiment 1 0.63 0.50 Rice blast fungus treatment experiment 2 0.86 0.18 standard deviation Pathogen-free water treatment control 0.09 0.10 Fusarium graminearum treatment experiment 1 0.06 0.02 Rice blast fungus treatment experiment 1 0.05 0.11 Rice blast fungus treatment experiment 2 0.02 0.04 Induced expression fold (compared with pathogen-free water treatment control) 1L39 1L36 Fusarium graminearum treatment experiment 1 32.27 21.84 Rice blast fungus treatment experiment 1 9.82 19.67 Rice blast fungus treatment experiment 2 13.41 7.24 standard deviation Fusarium graminearum treatment experiment 1 0.26 0.19 Rice blast fungus treatment experiment 1 0.25 1.03 Rice blast fungus treatment experiment 2 0.09 0.38

Note: Each data is the average of 3 measurements, 1L39 and 1L36 are two independent transgenic rice lines.

discuss

The present invention clones the corn FGI2 promoter, and uses the method mediated by Agrobacterium to transform rice. The results show that the leaves of the T0 generation of transgenic plants and the coleoptile stage of the T1 generation can be expressed when they are infected by Fusarium graminearum. No expression was found when Fusarium graminearum was infected, and the results showed that the promoter could be induced by Fusarium graminearum infection.

The promotor of the high-expression gene induced by Fusarium graminearum early stage of the present invention can be used in transgenic crops (rice, corn, etc.) It is expressed in the early stage of spore infection, so as to achieve the effect of antibacterial and yield protection when there is a threat of fungal disease, and keep it closed when there is no threat of disease, which is conducive to the distribution of energy and nutrients and tilts towards high yield. Maize process-related protein gene (PRms) is a gene that is highly expressed in the early stage of pathogenic fungi invading corn. The present invention obtains the promoter of the gene to drive the plant anti-fungal disease gene, and the obtained transgenic rice can be infected by Fusarium graminearum. , to respond quickly, induce the expression of disease-resistant genes, and defend against the infection of Fusarium graminearum, so as to prevent the disease more effectively in the early stage of the disease, and at the same time, it is different from the constitutive expression promoter, which can resist the disease and at the same time without affecting crop yields.

In addition, the present invention also proves that the FGI2 promoter is induced and expressed by various pathogenic fungi (such as Magnaporthe grisea). If it is induced by the infection of various pathogenic fungi, it may be used in the design of various disease resistances, so it may have a wider range of applications.

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.

Claims (10)

1. an isolating promotor is characterized in that, described promotor:

(a) separation is from the upper reaches of pathogenesis-related proteins encoding sox;

(b) base length is 800-2000;

(c) have necessary site and the transcripting start point that initiation is transcribed; And

(d) having the response pathogenic agent infects signal and starts the specific expressed function of goal gene.

2. promotor as claimed in claim 1 is characterized in that, the base length of described promotor is 1000-1800.

3. promotor as claimed in claim 1 is characterized in that, described promotor is:

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

(2) nucleotide sequence and SEQ ID NO:1 have 95% above homogeny and have the promotor that the response pathogenic agent infects signal and starts the specific expressed function of goal gene;

(3) nucleotides sequence is listed under the stringent condition and can hybridizes and have the polynucleotide that the response pathogenic agent infects signal and starts the specific expressed function of goal gene with the polynucleotide sequence of the arbitrary qualification in (1)-(2); Or

(4) the complete complementary promotor of nucleotide sequence shown in nucleotide sequence and the SEQ ID NO:1.

4. a carrier is characterized in that, described carrier contains the arbitrary described promotor of claim 1-3.

5. carrier as claimed in claim 4 is characterized in that described carrier also contains the goal gene that is operably connected with described promotor.

6. a genetically engineered host cell is characterized in that, described cell:

Contain the arbitrary described carrier of claim 4-5; Or

Be integrated with the arbitrary described promotor of claim 1-3 of external source in its genome.

7. one kind prepares and can respond the plant method that pathogenic agent infects signal; It is characterized in that; Described method comprises: construction is transformed plant; The goal gene that described construction contains the arbitrary described promotor of claim 1-3 and is operably connected with described promotor, said goal gene infect under the state in pathogenic agent expresses.

8. method as claimed in claim 7 is characterized in that, described method comprises:

(a) Agrobacterium of carrying expression vector is provided, contains construction in the said expression vector, the goal gene that described construction contains the described promotor of claim 1 and is operably connected with described promotor;

(b) vegetable cell, tissue or organ are contacted with Agrobacterium in the step (a), thereby make described construction change vegetable cell, tissue or organ over to;

(c) select vegetable cell, tissue or the organ that has changed said construction over to; And

(d) vegetable cell, tissue or neomorph in the step (c) are become plant, goal gene infects under the state in pathogenic agent and expresses in the said plant.

9. the purposes of the arbitrary described promotor of claim 1-3 is characterized in that, described promotor is used to respond pathogenic agent and infects signal and start goal gene specific expressed.

10. purposes as claimed in claim 9 is characterized in that described pathogenic agent is a phytopathogen.

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