Application of GmACA24 promoter in improving soybean stress resistance

Abstract

The present invention discloses the application of GmACA24 promoter in improving the stress tolerance of plants, the application of reagents for regulating the expression of GmACA24 promoter in hosts in regulating the stress resistance of plants, the application of reagents for identifying the expression of GmACA24 promoter in hosts in identifying the stress resistance of plants, a method for improving the stress tolerance of plants by GmACA24 promoter, an expression vector for expressing GmACA24 promoter, and a host fungus for expressing the expression vector. The GmACA24 promoter, especially haplotype 1 (Hap1, Chr19.39775478: A), can improve the tolerance of plants in high temperature, high salinity and drought environments, and has positive significance and important influence on the cultivation of crops resistant to high temperature, salinity and drought.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of a GmACA24 promoter in improving soybean stress resistance. Background Art

[0002] Plant genetic engineering is widely used in agriculture to increase the value of food and feed. The purpose of genetic engineering is to introduce gene fragments with ideal traits into target plants and express them in space and time. At the same time, the promoter is a "gene switch" located upstream of the gene coding region, which turns on or off the functional activity of the gene. The specific cis-acting elements it contains are important targets for the binding of transcription initiation and regulatory proteins, and are also molecular biological clocks that determine the expression of target genes. The promoter of a coding gene usually contains a core promoter and is divided into three regions: core, proximal, and distal. In addition, the proximal and distal ends of the promoter contain different regulatory sequences, such as enhancers, silencing factors, insulators, and cis-elements, all of which help to finely regulate gene expression at the transcriptional level.

[0003] Promoter activity and specificity are two main parameters for regulating transgenic expression. Gene promoters in higher plants can be mainly divided into three types: constitutive promoters, tissue-specific promoters and inducible promoters. Since the use of constitutive promoters, such as cauliflower mosaic virus (CaMV35S), may have adverse effects on non-target organisms or ecosystems, inducible promoters and tissue-specific promoters provide guarantees for gene-specific expression. Tissue-specific and stress-inducible promoters can be used to purposefully reduce the effects of adverse environmental conditions (such as high temperature, drought, and salinity). Among them, tissue-specific promoters are very powerful "regulatory tools" for transgenic plants. Because they have the function of manipulating the expression of genes linked to them, they can be applied to specific stages of growth and development of organisms or specific tissues, thereby helping to reduce the energy consumption of plants. In addition, stress-inducible promoters are activated and induce gene expression only when under stress, enabling plants to maintain yield or productivity under adverse conditions, thereby improving the stress tolerance of plants. Summary of the invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide an application of a GmACA24 promoter in improving the stress tolerance of a plant, an application of an agent for regulating the expression of a GmACA24 promoter in a host in regulating plant stress resistance, an application of an agent for identifying the expression of a GmACA24 promoter in a host in identifying plant stress resistance, a method for improving the stress tolerance of a plant by a GmACA24 promoter, an expression vector expressing a GmACA24 promoter, and a host fungus expressing the GmACA24 promoter or an expression vector.

[0005] In the present invention, "adversity tolerance" and "stress resistance" are used synonymously.

[0006] In a first aspect of the present invention, there is provided the use of the GmACA24 promoter in improving the stress tolerance of plants, wherein the stress comprises at least one of high temperature, salinity and drought.

[0007] In some embodiments of the present invention, the GmACA24 promoter comprises the nucleotide sequence shown in SEQ ID NO:1.

[0008] In some embodiments of the present invention, the GmACA24 promoter includes a complementary sequence or a reverse sequence of the nucleotide sequence shown in SEQ ID NO:1.

[0009] In some embodiments of the present invention, the GmACA24 promoter is a nucleotide sequence as shown in SEQ ID NO:1.

[0010] In some embodiments of the present invention, the GmACA24 promoter is a complementary sequence or a reverse sequence of the nucleotide sequence shown in SEQ ID NO:1.

[0011] In the present invention, the nucleotide sequence shown in SEQ ID NO: 1 corresponds to haplotype 1 (Hap1, Chr19.39775478: A).

[0012] In some embodiments of the present invention, the plants include monocots and dicots.

[0013] In the present invention, in some embodiments of the present invention, the plants include soybean and Arabidopsis thaliana.

[0014] In the present invention, Arabidopsis thaliana is used as a model plant, which has the characteristics of short growth cycle, small genome, and easy access to a large number of mutants and genome resources, so it is suitable for functional research of all plants (Xu Pingli, Shan Lei, & Li Xinguo. (2006). Progress in genome research of model plant Arabidopsis thaliana. Shandong Agricultural Science (6), 3.).

[0015] The second aspect of the present invention provides the use of an agent for regulating the expression of the GmACA24 promoter in a host in regulating plant stress resistance, wherein the stress resistance includes at least one of high temperature resistance, saline-alkali resistance and drought resistance.

[0016] The third aspect of the present invention provides the use of a reagent for identifying the expression of the GmACA24 promoter in a host in identifying plant stress resistance, wherein the stress resistance includes at least one of high temperature resistance, saline-alkali resistance and drought resistance.

[0017] The fourth aspect of the present invention provides a method for improving the stress tolerance of plants, the method comprising upregulating the expression of the GmACA24 promoter gene in the plant and / or promoting the production of its encoded product, wherein the stress comprises at least one of high temperature, salinity and drought.

[0018] In some embodiments of the present invention, upregulating the expression of the GmACA24 promoter gene and / or promoting the production of its encoded product includes upregulating the DNA molecule expression of the GmACA24 gene, upregulating the mRNA molecule expression of the GmACA24 gene, and upregulating the protein molecule expression of the translation product of the GmACA24 gene.

[0019] In some embodiments of the present invention, the GmACA24 promoter gene comprises a nucleotide sequence as shown in SEQ ID NO:1.

[0020] In some embodiments of the present invention, the method for improving plant stress tolerance specifically comprises the following steps: transfecting a host bacterium with an expression vector comprising a GmACA24 promoter gene; and infecting a wild-type plant with the host bacterium comprising the expression vector.

[0021] A fifth aspect of the present invention provides a biomaterial containing a GmACA24 promoter, wherein the biomaterial comprises at least one of the following (1) to (2):

[0022] (1) an expression vector containing a GmACA24 promoter comprising the nucleotide sequence shown in SEQ ID NO: 1;

[0023] (2) A host bacterium containing a GmACA24 promoter comprising the nucleotide sequence shown in SEQ ID NO: 1 or the expression vector described in (1).

[0024] In some embodiments of the present invention, the expression vector includes a plasmid, a viral vector, an artificial chromosome and a transposon.

[0025] In some embodiments of the present invention, the viral vector includes an adenoviral vector and a lentiviral vector.

[0026] In some embodiments of the present invention, the artificial chromosome includes a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a P1-derived artificial chromosome (PAC), a mammalian artificial chromosome (MAC) and a human episomal artificial chromosome (HAEC).

[0027] In some embodiments of the present invention, the expression vector is a plasmid.

[0028] In some embodiments of the present invention, the plasmid uses pCAMBIA3301-GUS as a vector backbone.

[0029] In some embodiments of the present invention, the plasmid further comprises a pMD19-T vector.

[0030] In some embodiments of the present invention, the host bacteria include Escherichia coli Trans1-T1 and Agrobacterium tumefaciens EHA105.

[0031] The beneficial effects of the present invention are:

[0032] The present invention proposes for the first time that the GmACA24 promoter, especially haplotype 1 (Hap1, Chr19.39775478: A), can significantly improve the tolerance of plants in high temperature, high salinity and drought environments. The GmACA24 promoter, as a stress-inducible promoter, is activated and induces gene expression only when under stress, so that the plant can maintain yield or productivity under adverse conditions, thereby improving the plant's tolerance to adversity. Therefore, the application of the GmACA24 promoter has positive significance and important impact on the development and cultivation of crops resistant to high temperature, salinity and drought. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the haplotype analysis result of soybean GmACA24 promoter, Figure 1 A is the p-value significance analysis of two haplotypes (haplotype 1 and haplotype 2). Figure 1 B is the haplotype analysis of the effective grain number trait of the GmACA24 gene;

[0034] Figure 2 The PCR detection results of soybean GmACA24 promoter, where M: DNA Marker DL2000; "+" is the positive control; "-" is the negative control; "1" is the PCR amplification product of GmACA24 promoter;

[0035] Figure 3 is the plasmid map of the expression vector pCAMBIA3301-proGmACA24-GUS (proGmACA24::GUS);

[0036] Figure 4 To analyze the expression pattern of GmACA24 in soybean seedling roots under different stress conditions;

[0037] Figure 5 The results are genomic PCR identification and glufosinate screening of proGmACA24::GUS transgenic Arabidopsis; Figure 5A is the PCR identification result of the genome of transgenic Arabidopsis thaliana, M: DNA Marker DL2000, +: positive control, 1-4: PCR identification of the genome of transgenic Arabidopsis thaliana; Figure 5 B: Identification results of T2 generation seed segregation ratio;

[0038] Figure 6 The results of the analysis of GmACA24 promoter activity under different stress conditions are shown in Figure 2. Figure 6 A is the result of GUS staining. Figure 6 B is the GUS quantitative result. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0040] Example 1: Haplotype analysis of the GmACA24 promoter

[0041] The soybeans were divided into a control group and a high temperature stress group by artificial warming. The control group (439 soybean materials) adopted normal field management (the number of varieties and the sorting method were exactly the same as those of the high temperature group). The high temperature stress group adopted normal field management before high temperature stress. The flowering time was used as the grouping standard (97 in the early group, 103 in the middle group, 123 in the late group, and 116 in the very late group). The 439 soybean materials were divided into 4 groups for high temperature stress. The soybeans in each group were subjected to 7 days of high temperature stress treatment (48 (± 3) ℃) at the beginning of flowering (9:00-15:00 / day) to form an effective high temperature stress treatment effect. After the high temperature stress treatment, ventilation and heat dissipation were carried out to keep it consistent with the external environment temperature. Using soybean Glycine maxWm82.a2.v1 as the reference genome, TASSEL 5.0 software was used to conduct GWAS analysis on soybean heat resistance traits. The results revealed a significant site on chromosome 19, which was the promoter region of GmACA24. Further analysis revealed a significant SNP (A / C) in the promoter region of the gene. Haplotype analysis results showed that the number of effective grains of soybeans with haplotype 1 (Hap1, Chr19.39775478: A) under high temperature stress was significantly higher than that of haplotype 2 (Hap2, Chr19.39775478: C) ( Figure 1 A and Figure 1 B), in which the number of W-box elements in the promoter of haplotype 1 (Hap1, Chr19.39775478:A) was reduced, indicating that different haplotypes of the GmACA24 promoter have different stress resistance.

[0042] Example 2: Extraction of soybean genomic DNA

[0043] Soybean seeds of "Jiadou 20" containing haplotype 1 (Hap1, Chr19.39775478: A) were sown in liquid nutrient pots and cultured in an artificial climate chamber. Then 0.2 g of leaf tissue was taken to extract genomic DNA using the TPS method. The specific steps are as follows:

[0044] 1) Take 0.2 g of leaves and put them into a 1.5 mL centrifuge tube, add 200 μL of TPS extract (0.5 M Tris-HCl, 0.5 M EDTA, 2 M KCl), and crush them with a 1 mL pipette tip;

[0045] 2) Place the crushed leaves in a 75°C water bath for 20 min, then centrifuge at 12,000 rpm for 10 min;

[0046] 3) Take the supernatant and place it in a new centrifuge tube, add an equal volume of isopropanol, and centrifuge at 5000 rpm for 10 min;

[0047] 4) Discard the supernatant, add 200 μL 70% ethanol to the precipitate, and centrifuge at 5000 rpm for 5 min;

[0048] 5) Discard the supernatant, dry the precipitate, and add 40 μL of sterile water to dissolve the DNA.

[0049] Example 3: Cloning of GmACA24 promoter and construction of expression vector

[0050] The genomic DNA of soybean "Jiadou 20" containing haplotype 1 was used as a template, and the GmACA24 promoter was cloned using the forward primer proGmACA24-F and the reverse primer proGmACA24-R. The sequence of the GmACA24 promoter is SEQ ID NO: 1 (Hap1, Chr19.39775478: A), the sequence of the forward primer proGmACA24-F is: 5'-TTCAGTTCTTTGTTTGCTTAAG-3' (SEQ ID NO: 2), and the sequence of the reverse primer proGmACA24-R is: 5'-ATACCGGCTGCTACGTTTAC-3' (SEQ ID NO: 3). The PCR system is shown in Table 1. The PCR program is 95°C denaturation for 10 seconds, 58°C annealing for 5 seconds, and 68°C extension for 10 seconds, for a total of 35 cycles. The agarose gel electrophoresis results of the PCR products are shown in Table 1. Figure 2As shown. After the PCR product was detected by agarose gel electrophoresis, it was connected to the pMD19-T cloning vector. After successful sequencing, the homologous recombination technology was used to connect the plant expression vector pCAMBIA3301-GUS to generate the expression vector pCAMBIA3301-proGmACA24-GUS. The plasmid map of the expression vector pCAMBIA3301-proGmACA24-GUS is shown in Figure 3 Then the promoter of the GmACA24 gene in the expression vector pCAMBIA3301-proGmACA24-GUS was sequenced and its sequence was confirmed to be SEQ ID NO: 1 (Hap1, Chr19.39775478: A).

[0051] Table 1. PCR amplification system of GmACA24 promoter

[0052]

[0053] Example 4: Expression analysis of the GmACA24 gene in soybean seedling roots under different stress treatments

[0054] After the "Jiadou 20" soybean vermiculite was germinated, soybeans with consistent growth were selected for hydroponics. In the V1 period, drought (8% PEG8000), high temperature (48°C), and saline (70mM NaCl and 50mM NaHCO3) treatments were carried out, and sampling was carried out at 0h, 1h, 3h, 6h, 9h and 12h.

[0055] The samples were processed according to the following steps:

[0056] (1) Pre-cool the mortar with liquid nitrogen, add the sample into the mortar, and grind continuously until the liquid nitrogen evaporates completely. Repeat this process three times.

[0057] (2) After adding 1 mL of RNAiso plus (Takara, Code No. 9108) and homogenizing, the mixture was allowed to stand at room temperature for 5 min and then centrifuged at 12000 g for 5 min at 4°C.

[0058] (3) The supernatant was transferred to a new 1.5 mL centrifuge tube, 200 mL of chloroform was added, the tube was allowed to stand at room temperature for 5 min, and centrifuged at 12000 g for 15 min at 4 °C.

[0059] (4) The supernatant was transferred to a new centrifuge tube, an equal volume of isopropanol was added, the tube was inverted and mixed, and the tube was allowed to stand at room temperature for 10 min.

[0060] Centrifuge at 4°C for 10 min.

[0061] (5) Add 1 mL of 75% ethanol to the precipitate to wash it, centrifuge at 12000 g for 5 min at 4°C, and discard the supernatant;

[0062] (6) After opening and drying, dissolve in an appropriate amount of DEPC water and measure the concentration;

[0063] (7) Dilute the RNA to a final concentration of 400 ng / μL-800 ng / μL and perform electrophoresis. Store the RNA in a -80°C refrigerator;

[0064] (8) Take 500 ng of total RNA from each sample and use PrimeScript TM RT reagent Kit with gDNAEraser-PerfectReal Time (Takara, RR047A) kit. Reverse transcription is divided into two steps. The first step is genome removal reaction. The reagent system is shown in Table 2. The reaction program is 42°C, 2min. The second step is reverse transcription reaction. The reagent system is shown in Table 3. The reaction program is 37°C, 15min; 85°C, 5s.

[0065] (9) The cDNA obtained in step (8) was subjected to RT-qPCR using PremixEXTaqTM (Takara, RR820A) fluorescent quantitative reagent according to the reaction system and reaction conditions shown in Tables 4 and 5. The results are as follows: Figure 4 shown.

[0066] Table 2. Genome removal reaction system

[0067]

[0068] Table 3. Reverse transcription reaction system

[0069]

[0070] Table 4. RT-qPCR reaction system

[0071]

[0072] Table 5. RT-qPCR reaction conditions

[0073]

[0074] The results showed that GmACA24 was differentially expressed in the roots of soybean seedlings under different stresses ( Figure 4 ), the above results indicate that the GmACA24 promoter may play an important role in responding to high temperature, salinity and drought stresses.

[0075] Example 5: GmACA24 promoter-mediated genetic transformation of Arabidopsis

[0076] After Arabidopsis seeds are vernalized in a dark place at 4 degrees in a refrigerator for 3 days, they are planted in a culture environment with a ratio of vermiculite to soil of 8:2. When they grow to the 2-4 leaf stage, Arabidopsis of uniform size can be selected and transplanted into the infected pots. After transplanting, they are cultured in the dark for 1-2 days. When Arabidopsis grows to the flowering period, the blooming flowers and seeds are removed, leaving the buds for subsequent infection.

[0077] 1) Arabidopsis genetic transformation was carried out according to the following steps: pCAMBIA3301-proGmACA24-GUS recombinant plasmid was transferred into EHA105 tumefaciens competent Agrobacterium, gently mixed, and allowed to stand in an ice water bath for 10 minutes, then quickly frozen in liquid nitrogen for 5 minutes, and then 800 μL of antibiotic-free YEP liquid medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride) was added, and cultured at 28-30°C with shaking for 2-3 hours, and evenly spread on YEP solid medium plates containing kanamycin and rifampicin antibiotics and cultured for 48 hours, and finally a single clone was picked for bacterial liquid PCR identification for subsequent Arabidopsis infection;

[0078] 2) Use 10 ml YEP liquid medium to shake culture Agrobacterium tumefaciens containing pCAMBIA3301-proGmACA24-GUS recombinant plasmid, then inoculate into a conical flask containing 1000 ml YEP liquid medium, and culture at 28°C, 230 rpm for 20-24 h. The final OD 600 The value reaches 1.0-1.2; centrifuge at 4000 rpm for 15 min; add Arabidopsis infection buffer (as shown in Table 6)

[0079] Dissolve back, OD 600 Values ​​of 0.8–1.0 were used for infection of wild-type Arabidopsis thaliana;

[0080] 3) Infect Arabidopsis thaliana with Agrobacterium tumefaciens containing the pCAMBIA3301-proGmACA24-GUS recombinant plasmid for 7 minutes, seal with plastic wrap to retain moisture, keep away from light overnight for 12 hours, tear off the plastic wrap, culture the Arabidopsis thaliana normally under weak light, and harvest after maturity;

[0081] 4) Place the harvested Arabidopsis in a dark refrigerator at 4 degrees for 3 days for vernalization, then plant them in eight-row culture pots. When they grow to the four-leaf stage, use 1% glufosinate (Basta) to screen positive seedlings on the first, third, and fifth days;

[0082] 5) Transplant the still green seedlings into new pots, extract the genome after they grow up, and use the forward primer proGmACA24-F and the reverse primer proGmACA24-R to identify the transgenic Arabidopsis thaliana, and select the transgenic positive strains (such as Figure 5A), after maturity, T2 generation seeds were collected and single copy (3:1 segregation ratio) lines (such as Figure 5 B) for subsequent experiments.

[0083] Table 6. Preparation method of Arabidopsis infection buffer

[0084]

[0085] Example 6: Analysis and identification of GmACA24 promoter activity under adverse stress

[0086] 1) The proGmACA24::GUS (pCAMBIA3301-proGmACA24-GUS) transgenic Arabidopsis thaliana in Example 5 was germinated in MS medium, and the transgenic Arabidopsis thaliana was subjected to stress culture at 38°C, or in a medium containing 300 mM mannitol, or 148 mM NaCl+2 mM NaHCO3 at the 2-pair leaf stage, and samples were taken at 0 h, 3 h, 6 h, 9 h and 12 h, with 3 replicates at each time point;

[0087] 2) Take the Arabidopsis root tissue to be stained and add GUS staining solution to make the GUS staining solution completely immerse the root tissue;

[0088] 3) Incubate at 37°C for 1-24 hours. As the incubation time increases, blue gradually appears. When the expression level is high, the site or locus of GUS activity appears blue or blue spots.

[0089] 4) Immerse the tissue in 70% ethanol for 7 hours to remove chlorophyll until the negative control turns white. If the decolorization is not complete, repeat the decolorization step to completely remove the chlorophyll. Store the decolorized tissue sample in ethanol and observe it with the naked eye or under an optical microscope. The blue on the white background is the GUS expression site.

[0090] Figure 6 The results showed that Arabidopsis thaliana with haplotype Hap1 had no obvious expression in the roots under normal culture conditions, but was significantly expressed in the root pistil cells under high temperature, saline-alkali and drought stresses, and the expression level first increased and then decreased with the stress time. The expression level was highest under 9h stress time and decreased between 9-12h.

[0091] The above results indicate that Arabidopsis thaliana with haplotype Hap1 responds to adverse stress and is specifically induced to express in the pistil cells of the roots under adverse stress, especially with better expression characteristics under saline-alkali stress, and is more suitable for breeding new stress-resistant crop varieties using plant genetic engineering methods.

[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. GmACA24 The promoter is used to induce plant gene expression under adverse stress, wherein the adverse stress is at least one of high temperature, salinity and drought; GmACA24 The nucleotide sequence of the promoter is SEQ ID NO: 1; and the plant is soybean or Arabidopsis thaliana.

2. Contains GmACA24 The expression vector of the promoter GmACA24 The nucleotide sequence of the promoter is SEQ ID NO:

1.

3. The expression vector according to claim 2, characterized in that The expression vector is selected from the group consisting of a plasmid, a viral vector, an artificial chromosome and a transposon.

4. A host bacterium, characterized in that The host bacteria contain GmACA24 Promoter or the expression vector according to claim 2, wherein GmACA24 The nucleotide sequence of the promoter is SEQ ID NO:

1.

5. The host bacteria according to claim 4, characterized in that The host bacteria include Escherichia coli Trans1-T1 and Agrobacterium tumefaciens EHA105.

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