CN112251462B - Application of soybeans GmHSFA2 and GmHSP20a in enhancing heat resistance of plants in flowering period - Google Patents

Abstract

The present invention discloses a soybean heat shock transcription factor geneGmHSFA2And heat shock protein gene regulated therebyGmHSP20aThe application of the plant growth regulator in enhancing the heat resistance of plants in the flowering period. The invention provides an application of any one of the following substances 1) to 3) in enhancing the heat resistance of plants in the flowering period: 1) proteinGmHSFA2And/orGmHSP20a(ii) a 2) Encoding proteinsGmHSFA2And/or proteinsGmHSP20aThe gene of (1); 3) containing the encoded proteinGmHSFA2And/or proteinsGmHSP20aThe recombinant vector, expression cassette, transgenic cell line or recombinant strain of (a); the proteinGmHSFA2Is protein composed of an amino acid sequence shown in SEQ ID NO.2 in a sequence table; the proteinGmHSP20aIs protein composed of an amino acid sequence shown in SEQ ID NO.4 in a sequence table.

Description

Application of soybeans GmHSFA2 and GmHSP20a in enhancing heat resistance of plants in flowering period

Technical Field

The invention belongs to the technical field of biological gene engineering, and particularly relates to a soybean heat shock transcription factor gene GmHSFA2 and application of a heat shock protein gene GmHSP20a regulated by the soybean heat shock transcription factor gene GmHSFA2 in improving heat resistance of plants in a flowering period.

Background

Soybean is one of the important economic oil crops, contains rich protein and fat and is widely planted worldwide. Temperature is an important ecological factor affecting the physiological and biochemical processes of plants, and physiological damage to plants caused by high-temperature environment is called high-temperature stress. In recent years, with the rapid development of industrial and agricultural industries, greenhouse gases are emitted in large quantities, resulting in an increase in atmospheric temperature. Global warming is getting worse so that high temperature thermal damage becomes more frequent. Extreme high temperatures sometimes occur in Huang-Huai-Hai regions and southern regions of China in summer for nearly 30 years. The development process of plant male reproductive organs is extremely sensitive to temperature, and the continuous high temperature in summer can cause abnormal soybean male reproductive development, and finally, the yield of soybeans is reduced in different degrees. It is predicted that the global temperature in the 21 st century will continue to rise steadily with frequent thermal injury events, and high temperature will become one of the important features of global climate in the future. Therefore, the heat resistance of the soybean in the flowering period is improved, and the creation and cultivation of new heat-resistant soybean varieties are the key points for reducing the high-temperature stress hazard. With the increasing maturity of high-throughput gene identification methods and gene function verification techniques, a basis is provided for disclosing the molecular mechanism of the response of crops to high-temperature stress in the flowering period, and a foundation is laid for cultivating high-temperature resistant crops by means of genetic engineering. Wherein, the discovery and the analysis of the high temperature resistant related genes of the flowering period of the crops are key.

After sensing a high-temperature signal in the environment, the plant can adapt to the high-temperature environment by inducing the expression of genes related to high-temperature stress, including Heat-shock transcription factors (HSF) and Heat-shock proteins (HSP), and the HSF can recognize Heat-shock elements (HSE) of the HSP promoter region to promote the transcription expression of the Heat-shock elements. The plant HSFA2 is one of HSFA subfamily members, and can activate a series of HSP and high-temperature protection related genes under high-temperature stress, thereby improving the heat resistance of the plant. In recent years, the function of HSFA2 in participating in the regulation of plants against high temperature stress has been increasingly studied, which is considered as one of the cores of gene regulatory network systems for coping with high temperature stress. The current research on HSFA2 and HSP genes and high temperature stress during flowering stage is mainly focused on model plants Arabidopsis and tomato. At least 38 HSF genes and 51 HSP20 genes are known in soybeans, but reports that soybean HSF and HSP20 gene regulated by the soybean HSF participate in regulation of response to high temperature stress in a plant flowering period are not found. The GmHSFA2 gene and the downstream regulatory gene GmHSP20a thereof are cloned by a biological gene engineering technology and are subjected to functional verification, so that an important basis can be provided for improving the heat resistance of soybeans in the flowering phase, an important gene resource is provided for soybean heat-resistant molecule breeding, and the method has important value.

Disclosure of Invention

The invention aims to provide the functions of a downstream regulatory gene GmHSP20a of a soybean heat shock transcription factor gene GmHSFA2 and a coding protein GmHSFA2 thereof and GmHSFA2 and a coding protein GmHSP20a thereof in the process of responding to high-temperature stress of plants in the flowering period and the function of the soybean heat shock transcription factor gene GmHSFA2 in cultivating high-temperature resistant plants.

The purpose of the invention can be realized by the following technical scheme:

the invention protects the application of any substance of 1) to 3) in enhancing the heat resistance of plants in the flowering period:

1) proteins GmHSFA2 and/or GmHSP20 a;

2) a gene encoding protein GmHSFA2 and/or protein GmHSP20 a;

3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a gene encoding protein GmHSFA2 and/or protein GmHSP20 a;

the protein GmHSFA2 is (1) or (2) as follows:

(1) a protein consisting of an amino acid sequence shown in SEQ ID NO.2 in a sequence table;

(2) and (2) the protein which is derived from the protein (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 in the sequence table.

The protein GmHSP20a is (1) or (2) as follows:

(1) protein composed of an amino acid sequence shown in SEQ ID NO.4 in a sequence table;

(2) and (2) the protein which is derived from the protein (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.4 in the sequence table.

The GmHSFA2 can respond to high-temperature stress expression of soybean in the flowering phase, activate the expression of related heat-resistant genes such as downstream heat shock protein GmHSP20a and the like, and improve the heat-resistant capability of plants in the flowering phase.

The invention protects the application of any substance of 1) to 3) in cultivating plants with improved heat resistance in the flowering period:

1) proteins GmHSFA2 and/or GmHSP20 a;

2) a gene encoding protein GmHSFA2 and/or protein GmHSP20 a;

3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing a gene encoding a protein GmHSFA2 and/or a protein GmHSP20 a;

the protein GmHSFA2 is (1) or (2) as follows:

(1) a protein consisting of an amino acid sequence shown in SEQ ID NO.2 in a sequence table;

(2) and (2) the protein which is derived from the protein (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 in the sequence table.

The protein GmHSP20a is (1) or (2) as follows:

(1) protein composed of an amino acid sequence shown in SEQ ID NO.4 in a sequence table;

(2) and (2) the protein which is derived from the protein (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.4 in the sequence table.

In the application, the gene of the coding protein GmHSFA2 has a nucleotide sequence shown in SEQ ID NO.1 in a sequence table.

In the application, the gene of the coding protein GmHSP20a has a nucleotide sequence shown in SEQ ID NO.3 in a sequence table.

In the application, the promoter of the GmHSP20a has a nucleotide sequence shown as SEQ ID NO. 5.

Preferably, the promoter HSE element of the GmHSP20a has a nucleotide sequence shown as SEQ ID NO. 6.

The nucleotide sequence of the region of the HSE element with the upstream of 100-500bp of the initiation codon of the soybean heat shock protein gene GmHSP20a is shown as SEQ ID NO. 6. The HSE element of the GmHSP20a promoter is recognized by GmHSFA2, thereby activating transcriptional expression of GmHSP20 a.

In the above application, the plant is a monocotyledon or dicotyledon; wherein the dicotyledons are leguminous plants or cruciferae plants.

The invention also provides a method for cultivating the plant with improved heat resistance in the flowering phase, which is characterized in that DNA of the encoded protein GmHSFA2 and/or GmHSP20a is introduced into a target plant to obtain a transgenic plant, wherein the heat resistance of the transgenic plant in the flowering phase is higher than that of the target plant: wherein the DNA molecule coding for the protein GmHSFA2 and/or GmHSP20a is introduced into the target plant through a recombinant vector.

The invention also protects a recombinant vector, which is obtained by inserting the gene of the coding protein GmHSFA2 and/or GmHSP20a into an expression vector, wherein the gene of the coding protein GmHSFA2 and/or GmHSP20a is expressed by the recombinant vector of the expression protein GmHSFA2 and/or GmHSP20 a:

the protein GmHSFA2 is (1) or (2) as follows:

(1) a protein consisting of an amino acid sequence shown in SEQ ID NO.2 in a sequence table;

(2) and (2) the protein which is derived from the protein (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 in the sequence table.

The protein GmHSP20a is (1) or (2) as follows:

(1) protein composed of an amino acid sequence shown in SEQ ID NO.4 in a sequence table;

(2) and (2) the protein which is derived from the protein (1) and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.4 in the sequence table.

The recombinant plasmid is obtained by inserting the soybean heat shock transcription factor gene GmHSFA2 or the heat shock protein gene GmHSP20a into a pCAMBIA3301 plant overexpression vector.

The agrobacterium used in the invention is agrobacterium EHA 105.

The invention also protects the application of the soybean heat shock transcription factor gene GmHSFA2 and the heat shock protein gene GmHSP20a in high temperature resistance of soybean in the flowering period.

The invention also protects the application of the soybean heat shock transcription factor gene GmHSFA2 and the heat shock protein gene GmHSP20a in cultivating high-temperature-resistant soybean varieties in flowering periods.

The invention also protects the application of the recombinant plasmid containing the soybean heat shock transcription factor gene GmHSFA2 and the heat shock protein gene GmHSP20a in the high temperature resistance of soybean in the flowering period.

The invention also protects the application of the recombinant plasmid containing the soybean heat shock transcription factor gene GmHSFA2 and the heat shock protein gene GmHSP20a in cultivating high-temperature resistant plants in the flowering period.

Has the advantages that:

the soybean heat shock transcription factor gene GmHSFA2 provided by the invention is positioned on the 14 th chromosome, and the length of the reading frame is 1095 bp. The gene has A conserved domain specific to HSFA2 such as DNA Binding Domain (DBD), Oligomerization domain with two adjacent hydrophobic peptides including the OD of HR-A/B, Nuclear Localization Signal (NLS) and activation domain (AHA). DBD present in the sequence of GmHSFA2 can bind to HSE element of downstream heat-resistant function gene promoter region, such as soybean heat shock protein GmHSP20a provided by the invention. GmHSP20a is located on chromosome 12 with a reading frame length of 636 bp. The gene comprises 4 HSE elements in a promoter region 500bp upstream of the initiation codon. Under high temperature stress, the HSFA2 can be combined on an HSE element of an HSP gene to help the protein inactivated by high temperature stress to be reassembled into an active high-grade structure and maintain normal cell functions, so that the heat resistance of the plant is improved, and therefore, the GmHSFA2/GmHSP20a has an important high temperature stress response function.

By using any vector capable of guiding the expression of the exogenous gene in the plant and introducing the GmHSFA2 or GmHSP20a gene into a plant cell, a transgenic plant with improved high-temperature stress resistance can be obtained. When a plant expression vector is constructed using the gene of the present invention, any one of an enhancer promoter and an inducible promoter may be added before the transcription initiation nucleotide. In order to facilitate the identification and screening of transgenic plant cells or plants, vectors to be used may be processed, for example, by adding a plant selectable marker (GUS gene, luciferase gene, etc.) or an antibiotic marker having resistance (gentamicin, kanamycin, etc.). The expression vector carrying GmHSFA2 and GmHSP20a of the present invention can transform plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and culture the transformed plant tissues into plants. The host to be transformed may be either a monocotyledonous plant or a dicotyledonous plant. The gene of the invention has high temperature resistance to soybean in the flowering phase, and especially has important significance in cultivating high temperature resistant soybean varieties in the flowering phase.

Drawings

FIG. 1 shows expression patterns of GmHSFA2 and GmHSP20a in mixed flower buds of soybeans before and after high-temperature treatment. The abscissa represents the number of days treated, and the ordinate represents the relative expression levels of GmHSFA2 and GmHSP20 a.

FIG. 2 shows a structural model of GmHSFA2 protein and a structural model of GmHSP20a gene. Wherein A is a GmHSFA2 protein structure model, DBD is an N-terminal DNA binding domain, OD is an oligomerization domain, NLS is a nuclear localization signal, and AHA is an acidic C-terminal activation domain; and B is a GmHSP20a gene structure model.

FIG. 3 shows the interaction between GmHSFA2 and GmHSP20a promoter in yeast.

FIG. 4 is a schematic diagram of the construction of the overexpression vectors of GmHSFA2 and GmHSP20a genes. Wherein A is a schematic diagram of the construction of an overexpression vector of the GmHSFA2 gene; FIG. B is a schematic diagram of the construction of an overexpression vector of GmHSP20a gene.

FIG. 5 shows PCR identification of GUS genes of GmHSFA2 and GmHSP20a transgenic Arabidopsis thaliana. WT is wild type plant, 1, 2 and 3 are transgenic lines. Wherein A is GUS gene PCR identification of GmHSFA2 transgenic arabidopsis thaliana; b is PCR identification of GUS gene of GmHSP20a transgenic arabidopsis thaliana.

FIG. 6 shows the expression amount analysis of GmHSFA2 and GmHSP20a in WT and transgenic lines. WT is wild type plant, 1, 2 and 3 are transgenic lines. Wherein A is expression analysis of GmHSFA2 in WT and GmHSFA2 transgenic Arabidopsis; and B is the expression quantity analysis of GmHSP20a in WT and GmHSP20a transgenic Arabidopsis thaliana. ", and". x "represent 0.01. ltoreq. p < 0.05, respectively; p is more than or equal to 0.001 and less than 0.01; p < 0.001, and the like.

FIG. 7 shows the inflorescence heat tolerance identification of WT and transgenic lines. WT is wild type plant, 1, 2 and 3 are transgenic lines. Wherein A is inflorescence heat resistance identification of WT and GmHSFA2 transgenic arabidopsis; and B is the inflorescence heat resistance identification of WT and GmHSP20a transgenic Arabidopsis thaliana.

FIG. 8 shows the expression level analysis of GmHSFA2 downstream regulatory genes in WT and GmHSFA2 transgenic Arabidopsis thaliana before and after high temperature treatment.

FIG. 9 shows pollen fertility comparisons between WT and transgenic lines under normal and high temperature treatment. Wherein A is the pollen fertility phenotype of WT and transgenic line after 2 days of normal temperature and high temperature treatment; b is the pollen fertility phenotype of WT and transgenic line after 6 days of normal temperature and high temperature treatment. The pollen pointed by the black arrow is fertile pollen, and the pollen pointed by the white arrow is sterile pollen.

FIG. 10 is a statistical comparison of pollen fertility at normal and high temperature treatment of WT and transgenic lines.

FIG. 11 is a comparison of pollen properties of anthers under normal and high temperature treatment of WT and transgenic lines. Wherein A is the comparison of pollen property of the anther under the normal temperature of WT and transgenic strains; and B is the pollen dispersion comparison of the WT and the anther of the transgenic line after 2 days of high-temperature treatment. The black arrows point to loose pollen anthers, and the gray arrows point to loose pollen anthers.

The specific implementation mode is as follows:

the methods used in the following examples are conventional in the art unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The soybean conventional variety N4608, wild type Arabidopsis thaliana (Col 0) and the overexpression vector pCAMBIA3301 are provided by the national soybean improvement center of Nanjing agriculture university.

Example 1 Soybean GmHSFA2 and GmHSP20a respond to high temperature stress during the flowering phase of soybeans

Conventional variety N4608 of soybean was subjected to high temperature treatment using a plant incubator at the flowering stage (stage R1): treatment at 40 deg.C/34 deg.C (day/night) for 7 days (ambient temperature 30 deg.C/24 deg.C (day/night) as control). Mixing flower buds of N4608 plants with different sizes, which are treated under high temperature conditions for 0, 1, 3, 5 and 7 days and recovered to normal temperature for 1 day respectively, and immediately freezing in liquid nitrogen for storage at-80 ℃ for later use.

The total RNA of mixed flower buds with different sizes before and after high-temperature treatment is extracted by using a plant total RNA extraction kit (Tiangen), and real-time fluorescent Quantitative expression analysis (Quantitative real time PCR, qRT-PCR) of GmHSFA2 and GmHSP20a is carried out.

cDNA Synthesis for GmHSFA2 and GmHSP20a qRT-PCR analysis reverse transcription was performed according to the HiScript II Q RT Supermix for qPCR reverse transcription kit from Vazyme.

qPCR primers were designed using Primer5.0 software with GmHSFA2 and GmHSP20a gene CDS full length as target sequences and GmTubulin as reference gene.

GmHSFA2-QF：5'-TGAAGTGGGGGAGTTTGGAC-3'(SEQ ID NO.7)

GmHSFA2-QR：5'-AGTGGCTTGCAATCTGGTCT-3'(SEQ ID NO.8)

GmHSP20a-QF：5'-GCGACGATAT CTTCTCAGGT AATG-3'(SEQ ID NO.9)

GmHSP20a-QR：5'-GACCTGGTCC ATCATGTTCA GA-3'(SEQ ID NO.10)

GmTubulin-QF：5'-CTCTCTCTCT GTTTATCTCC GCAC-3'(SEQ ID NO.11)

GmTubulin-QR：5'-CTGAACGTGA AGGATCTCCC TC-3'(SEQ ID NO.12)

The cDNA was diluted 5-fold and used as a template for qPCR, and qPCR reaction was performed on a Bio-Rad CFX96 machine (CFX96 Touch, BIO-RAD, USA) using the AceQ qPCR SYBR Green Master Mix formulation system from Vazyme. The qPCR reaction procedure was: 30sec at 95 ℃; 95 ℃ 10sec, 60 ℃ 30sec, 40 cycles.

Results adopted 2^-ΔΔCqThe method can be used for relatively quantifying gene expression. Firstly, using Cq value of internal reference gene to normalize Cq value of target gene to obtain delta Cq value, then using Cq value of control sample to normalize Cq value of treated sample to obtain delta Cq value, using high-temperature treatment for 0 day as control, and finally using high-temperature treatment for 0 day as controlThe relative expression level ratio is calculated later, and the ratio of the expression amount is 2^-ΔΔCq。

As shown in fig. 1, GmHSFA2 and GmHSP20a respond to high temperature stress during soybean flowering, and GmHSP20a and GmHSFA2 have consistent expression trends.

Example 2 cloning of the Soybean GmHSFA2 and GmHSP20a genes and GmHSP20a promoters

The DNA and total RNA of the mixed flower bud of the conventional soybean variety N4608 are extracted by using a plant DNA and total RNA extraction kit (Tiangen), respectively. Total RNA was checked for RNA integrity by 1% agarose gel electrophoresis and RNA concentration was determined using NanoDrop 2000(Thermo Scientific). Synthesis of cDNA reverse transcription was performed according to the instructions of the TaKaRa PrimeScript 1st Strand cDNA Synthesis kit.

Designing PCR primers by using Primer5.0 software according to the CDS full length of soybean reference genome GmHSFA2 and GmHSP20a genes and the upstream 2000bp of the initiation codon of GmHSP20a as target sequences:

GmHSFA2-pMD19TF：5'-ATGTACATGG AGAGAATTAG AGTGA-3'(SEQ ID NO.13)

GmHSFA2-pMD19TR：5'-TTAGCCTAGA TAGTCCATAT GATCC-3'(SEQ ID NO.14)

GmHSP20a-pMD19TF：5'-ATGGCGTCCT CTCTCATTG-3'(SEQ ID NO.15)

GmHSP20a-pMD19TR：5'-CTACTCAACC TTAACACTGA TCAC-3'(SEQ ID NO.16)

pGmHSP20a-pMD19TF：5'-TTTGTCATTG ACATTGACGA A-3'(SEQ ID NO.17)

pGmHSP20a-pMD19TR：5'-TGAATTGAGA TTGCAAAAGG T-3'(SEQ ID NO.18)

PCR amplification was performed using the KOD Plus- (Toyobo) system of Toyobo using cDNA and DNA as templates, respectively, and the PCR reaction procedure was: 5min at 95 ℃; at 98 ℃ for 10sec, the denaturation temperature is set to 30sec according to the Tm value of the primer, at 68 ℃ for 2min, and 35 cycles; 10min at 68 ℃; keeping the temperature constant at 4 ℃.

And (3) after detecting, recovering and purifying the PCR product by 1% agarose gel electrophoresis, cloning the recovered fragment TA to a cloning Vector pMD19T Simple Vector (TaKaRa), transforming escherichia coli, selecting a single colony, culturing, detecting a positive strain by PCR, and sending the positive strain to a company for sequencing. The sequencing results were as follows:

the length of the gene sequence of GmHSFA2 is 1095bp, the nucleotide sequence is shown as SEQ ID NO.1, and the amino acid sequence of the gene coding protein is shown as SEQ ID NO. 2. As shown in FIG. 2A, the gene protein sequence has conserved domains such as N-terminal DNA binding domain DBD, oligomerization domain OD, nuclear localization signal NLS and AHA acidic C-terminal activation domain.

The length of the GmHSP20a gene sequence is 636bp, the nucleotide sequence is shown as SEQ ID NO.3, and the amino acid sequence of the gene coding protein is shown as SEQ ID NO. 4.

The length of the promoter sequence of the GmHSP20a gene is 2002bp, and the nucleotide sequence is shown as SEQ ID NO. 5. As shown in FIG. 2B, the promoter of the gene comprises four consecutive HSE elements (SEQ ID NO.6) within the region 500bp upstream.

Extracting plasmids by using a plasmid extraction kit (Axygen) to obtain positive recombinant clones pMD19T-GmHSFA2, pMD19T-GmHSP20a and pMD19T-pGmHSP20 a.

Example 3 interaction of the Soybean GmHSFA2 Gene with GmHSP20a

The pGADT7 plasmid was digested with the enzymes BamHI and XhoI, the pAbAi plasmid was digested with the enzymes KpnI and XhoI, the digested products were subjected to agarose gel electrophoresis (1% agarose), the linearized vector was recovered with a DNA gel recovery kit (Axygen), and the linearized vector was obtained, and the concentration of the objective linearized vector was determined using a ultramicro spectrophotometer NanoDrop 2000(Thermo Scientific).

Primers were designed based on the sequence of GmHSFA2 CDS and the sequence of the 100-and 500-bp HSE element upstream of the GmHSP20a start codon:

GmHSFA2-pGADT7F：

5’-CCCGGGTGGGCATCGATACGGGATCCATATGTACATGGAGAGAATTAG-3’(SEQ ID NO.19)

GmHSFA2-pGADT7R：

5’-TATCTACGATTCATCTGCAGCTCGAGTTAGCCTAGATAGTCCATATG-3’(SEQ ID NO.20)

pGmHSP20a-pAbAiF：

5’-AAAAGCTTGAATTCGAGCTCGGTACCTGAGAGAACAAATTGTAAAC-3’(SEQ ID NO.21)

pGmHSP20a-pAbAiR：

5’-TATACATACAGAGCACATGCCTCGAGAGGGGAATGGTTGGGACTTG-3’(SEQ ID NO.22)

PCR was carried out using the KOD Plus-system of Toyobo using pMD19T-GmHSFA2 and pMD19T-pGmHSP20a plasmids of example 2 as templates, respectively, and after PCR products were detected by 1% agarose gel electrophoresis and recovered and purified, the GmHSFA2 gene and pGmHSP20a (HSE element) were ligated to expression vectors pGADT7 and pAbAi, respectively, using a Vazyme Clonexpress one-step directed cloning seamless cloning kit using homologous recombination. DH5 alpha was transformed, positive strains were detected by PCR, and the plasmid was sequenced and extracted from the company to obtain the prey vectors pGADT7-GmHSFA2 of the GmHSFA2 gene and pGmHSP20a (HSE element), pAbAi-pGmHSP20 a.

The plasmid pAbAi-pGmHSP20a was self-activated and tested for toxicity: transforming the plasmid pAbAi-pGmHSP20a into Y1H Gold competent cells, coating SD/-Ura plates, selecting single spots from URA plates to be competent cells, transforming prey into bait competent cells in an unloaded mode, coating SD/-Leu which can grow to show that the recombinant bait plasmid is successfully transferred into host bacteria, coating plates SD/-Leu/Aba200 ng/mL, SD/-Leu/Aba 500ng/mL, SD/-Leu/Aba 800ng/mL and SD/-Leu/Aba 1000ng/mL to grow in sterile colonies, showing that the bait plasmid has no self-activation, and being used for the subsequent interaction verification of GmHSFA2 gene and GmHSP20 a.

FIG. 3 shows that prey plasmid PGADT7-GmHSFA2 was transformed into bait strain competent Y1H [ pAbAi-pGmHSP20a (HSE element) ], and after transformation, SD/-Leu/Aba plates were coated, and SD/-Leu, SD/-Leu/Aba plates 200ng/mL all had colony growth, indicating that prey proteins can bind to HSE element in the promoter region of GmHSP20a to activate reporter gene expression, indicating that there is interaction between GmHSFA2 and GmHSP20 a.

Example 4 construction of plant overexpression vectors for Soybean GmHSFA2 and GmHSP20a genes

The pCAMBIA3301 vector is digested by endonuclease BamHI, the digestion product is subjected to 1% agarose gel electrophoresis, a DNA gel recovery kit (Axygen) is used for recovering a linearized vector to obtain a linearized vector, and an ultramicro spectrophotometer NanoDrop 2000(Thermo Scientific) is used for detecting the concentration of the target linearized vector.

Primer amplification is designed according to CDS sequences of GmHSFA2 and GmHSP20a genes respectively. The primer sequences are as follows:

GmHSFA2-pCAMBIA3301F：

5’-GAGCTCGGTACCCGGGGATCCATGTACATGGAGAGAATTAGAGTGA-3’(SEQ ID NO.23)

GmHSFA2-pCAMBIA3301R：

5’-CAGGTCGACTCTAGAGGATCCTTAGCCTAGATAGTCCATATGATCC-3’(SEQ ID NO.24)

GmHSP20a-pCAMBIA3301F：

5’-GAGCTCGGTA CCCGGGGATC CATGGCGTCC TCTCTCATTG-3’(SEQ ID NO.25)

GmHSP20a-pCAMBIA3301R：

5’-CAGGTCGACT CTAGAGGATC CCTACTCAAC CTTAACACTG ATCAC-3’(SEQ ID NO.26)

PCR reaction was carried out using KOD Plus-system of Toyobo with pMD19T-GmHSFA2 and pMD19T-GmHSP20a plasmids of example 2 as templates, respectively, PCR products were detected by 1% agarose gel electrophoresis, recovered and purified, and GmHSFA2 and GmHSP20a genes were ligated to plant over-expression vector pCAMBIA3301 by homologous recombination using a Vazyme Clonexpress one-step directed cloning seamless cloning kit, respectively. Transforming DH5 alpha, detecting positive strain by PCR, sending to company for sequencing, extracting plasmid, obtaining gene over-expression vector: pCAMBIA3301-GmHSFA2, pCAMBIA3301-GmHSP20a (FIG. 4).

Example 5 obtaining of transgenic Arabidopsis

1. Respectively transferring plasmids pCAMBIA3301-GmHSFA2 and pCAMBIA3301-GmHSP20a into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium tumefaciens;

2. inoculating the recombinant agrobacterium to YEB solid culture medium containing 50 mu g/mL Rif and 50 mu g/mL Ka, and culturing at the constant temperature of 28 ℃ for 36-48 h;

3. selecting a single clone in 1mL YEB culture solution containing 50 mu g/mL Rif and 50 mu g/mL Ka, carrying out PCR detection on a positive strain after shaking overnight culture at the temperature of 28 ℃ and the speed of 200 rpm;

4. after the bacteria detection is successful, the strains are respectively transferred into fresh YEB liquid culture medium containing 50 mu g/mL Rif and 50 mu g/mL Ka according to the proportion of 1:100 to OD₆₀₀About 1.0;

5. centrifuging at 4 deg.C for 10min at 4000g, and collecting thallus; resuspend with 5% sucrose solution (containing 0.02% Tween 20);

6. immersing the inflorescence of the arabidopsis into the bacterial liquid for 1min, covering the stained arabidopsis with a black plastic bag, and performing dark culture for 24 h; then transferred to normal growth conditions (16h light (23 ℃)/8h dark (18 ℃), humidity 75%) for culture. And carrying out PCR detection and qRT-PCR analysis on the obtained positive plants.

The full length of GUS gene is used as a target sequence, a specific Primer is designed by using Primer Premier5.0 software and is named as GUS-F/GUS-R, and the Primer sequence is as follows:

GUS-F：5'-CGATGCAGAT ATTCGTAATT ATGC-3'(SEQ ID NO.27)

GUS-R：5'-CATACCTGTT CACCGACGAC G-3'(SEQ ID NO.28)

the PCR procedure was as in example 2.

qRT-PCR analysis of GmHSFA2 and GmHSP20a transgenic Arabidopsis thaliana was performed according to the relevant procedure of example 1. And using AtActin2 as an internal reference gene, and using Primer5.0 software to design a specific Primer.

AtActin2-QF：5'-CTTGCACCAAGCAGCATGAA-3'(SEQ ID NO.29)

AtActin2-QR：5'-CCGATCCAGACACTGTACTTCCTT-3'(SEQ ID NO.30)

FIG. 5 shows the PCR identification result of GUS gene of transgenic Arabidopsis thaliana, and FIG. 6 shows the analysis result of GmHSFA2 and GmHSP20a qRT-PCR of transgenic Arabidopsis thaliana. The result shows that the GmHSFA2 gene is successfully integrated into the genome of Arabidopsis thaliana, can be stably inherited in Arabidopsis thaliana, and can be used for subsequent heat resistance function research.

Example 6, GmHSFA2 and GmHSP20a transgenic Arabidopsis inflorescence thermotolerance identification

Transgenic Arabidopsis thaliana and WT were treated with high temperature using a plant incubator, 45 ℃/40 ℃ (day/night) for 3 days, and 23 ℃/18 ℃ (day/night) for 1 day after recovery, and then the damage of the floral organs was observed using a stereomicroscope OLYMPUS SZ61 (Japan).

As shown in FIG. 7, the WT inflorescences were substantially full-bodied. The GmHSFA2 and GmHSP20a transgenic arabidopsis inflorescences were less damaged compared to WT. The inflorescences of the GmHSFA2 and GmHSP20a transgenic Arabidopsis plants show superior heat tolerance to WT.

Example 7 analysis of expression level of downstream regulatory gene of GmHSFA2

Total RNA of GmHSFA2 transgenic Arabidopsis thaliana and WT plants before and after high-temperature treatment in example 6 was extracted using a plant total RNA extraction kit (Tiangen), and qRT-PCR analysis was performed on potential downstream genes of GmHSFA 2. Specific primers were designed using Primer5.0 software with atacin 2 as an internal reference gene based on the reference gene sequence of each downstream regulatory gene.

Reverse transcription system, qRT-PCR analysis system and procedure and related data analysis reference example 1.

FIG. 8 shows that AtsHSP, AtHSP20, AtHSP40, AtHsp70 and AtHSP90 are all up-regulated in GmHSFA2 transgenic Arabidopsis thaliana compared to WT under high temperature treatment. The over-expression of GmHSFA2 is shown to induce the expression of HSP related genes, so that the heat resistance of the inflorescence of GmHSFA2 transgenic Arabidopsis at the flowering phase is improved.

Example 8 identification of Male fertility and thermotolerance of transgenic Arabidopsis thaliana

The transgenic arabidopsis thaliana and WT flowering plants of GmHSFA2 and GmHSP20a are treated for 4 hours at 42 ℃ by using a plant incubator, then are transferred to a normal growth environment (23 ℃/18 ℃, day/night) for culture, and male fertility (pollen fertility and anther pollen scattering property) identification is carried out within one week after high-temperature treatment.

Taking the whole small arabidopsis flowers which bloom the next day at about 10 o' clock every day, immersing the flowers in the prepared alexander staining solution, and incubating for 12-24 h at 65 ℃; the whole anthers were peeled off with tweezers, placed on a glass slide for observation under an OLYMPUS CX31(Japan) microscope and photographed using a photographing apparatus (OLYMPUS DP27, Japan).

The whole Arabidopsis flowers that were released every day were taken around 8-10 am, observed under a stereoscopic microscope OLYMPUS SZ61(Japan) and photographed using a photographing apparatus (OLYMPUS DP27, Japan).

FIGS. 9 and 10 show that pollen fertility of GmHSFA2 and GmHSP20a transgenic Arabidopsis and WT plants is normal at normal temperature, WT pollen shrinks at day 2 after high temperature treatment, and transgenic Arabidopsis is substantially normal; on day 6 after high temperature treatment, WT was nearly fully sterile, whereas transgenic Arabidopsis was only semi-sterile. FIG. 11 shows that at normal temperature, anthers of transgenic Arabidopsis and WT plants were normally pollen-dispersed, and on day 2 after high-temperature treatment, WT anthers were not pollen-dispersed, while transgenic Arabidopsis anthers were normally pollen-dispersed. It was shown that the male fertility of the transgenic arabidopsis thaliana, GmHSFA2 and GmHSP20a, exhibited a heat tolerance superior to WT. The combination of the consistency of the expression trends of GmHSFA2 and GmHSP20a under the high-temperature stress of soybean in the flowering phase (figure 1) and the demonstration of a yeast single hybrid experiment (figure 3) shows that GmHSFA2 can regulate GmHSP20a to improve the heat resistance of plants in the flowering phase. In addition, after high-temperature treatment, the male fertility of the GmHSFA2 transgenic Arabidopsis is higher than that of the GmHSP20a transgenic Arabidopsis, which indicates that the GmHSP20a is only one of the downstream regulated genes of the GmHSFA 2.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be protected.

Sequence listing

<110> Nanjing university of agriculture

Application of soybeans GmHSFA2 and GmHSP20a in enhancing heat resistance of plants in flowering period

<160> 30

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1095

<212> DNA

<213> Soybean (Glycine max)

<400> 1

atgtacatgg agagaattag agtgaaggag gaagaagcag tgacatgtgg tggtggttca 60

tcatcatcat catcttcgtc ttcaagcttt tctccacagc ctatggaagg gttgcacgag 120

gtgggtcccc ctccgtttct gagcaagatc tttgacatgg tagaagattc ttccacggac 180

tccattgtgt catggagcat ggctcgcaac agctttgtgg tgtgggattc tcacaagttt 240

tctgctgaca ttctgcctcg ttacttcaag cacggcaatt tctccagctt cattcgccag 300

ctcaacgcct atgggtttag gaaagttgat cctgatagat gggaatttgc aaacgaaggt 360

tttttggcag gacaaaggca cttattgaag accattaaga gaagaagaaa tgtgtcacag 420

agtttgcaac aaaaaggagg aagtggagct tgtgttgaag tgggggagtt tggactcgaa 480

ggtgagttgg agaggttgaa aagagacagg aacattttga tggcagaaat tgtgagattg 540

aggcaccaac aactgaactc aagggaacaa ttgaattcca tggagaccag attgcaagcc 600

actgagaaga aacagcagca aatgatgagt ttccttgcaa aagcactgag taacccatct 660

ttcacgaaac aattagtcca aaagaccccc caaagcagag aagtgttggg tgttgaaatc 720

aataggaaaa ggagactgac tgctagtcca agtgttgaga acttgcaaca agatgatcaa 780

gatttggcaa ctttggatta tccaagccat gatcgagatt tggcaactat ggagactgat 840

atggacactt ttttctcacc ggcttatgat aatgaactta gcagtgaaac caatgagcct 900

gcatcaattt cggttgagga tacaatcttg gaggactttc tcaacaaaga cttggttact 960

tggaatccgg aggatgaagt tataattggg gatagttccc aagtcgatgt gccggtggag 1020

gatttggttg caaaccctga tgattggagt gagcaattgc aggaccttgt ggatcatatg 1080

gactatctag gctaa 1095

<210> 2

<211> 364

<212> PRT

<213> Soybean (Glycine max)

<400> 2

Met Thr Met Gly Ala Ile Ala Val Leu Gly Gly Gly Ala Val Thr Cys

1 5 10 15

Gly Gly Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Pro Ser Pro

20 25 30

Gly Pro Met Gly Gly Leu His Gly Val Gly Pro Pro Pro Pro Leu Ser

35 40 45

Leu Ile Pro Ala Met Val Gly Ala Ser Ser Thr Ala Ser Ile Val Ser

50 55 60

Thr Ser Met Ala Ala Ala Ser Pro Val Val Thr Ala Ser His Leu Pro

65 70 75 80

Ser Ala Ala Ile Leu Pro Ala Thr Pro Leu His Gly Ala Pro Ser Ser

85 90 95

Pro Ile Ala Gly Leu Ala Ala Thr Gly Pro Ala Leu Val Ala Pro Ala

100 105 110

Ala Thr Gly Pro Ala Ala Gly Gly Pro Leu Ala Gly Gly Ala His Leu

115 120 125

Leu Leu Thr Ile Leu Ala Ala Ala Ala Val Ser Gly Ser Leu Gly Gly

130 135 140

Leu Gly Gly Ser Gly Ala Cys Val Gly Val Gly Gly Pro Gly Leu Gly

145 150 155 160

Gly Gly Leu Gly Ala Leu Leu Ala Ala Ala Ala Ile Leu Met Ala Gly

165 170 175

Ile Val Ala Leu Ala His Gly Gly Leu Ala Ser Ala Gly Gly Leu Ala

180 185 190

Ser Met Gly Thr Ala Leu Gly Ala Thr Gly Leu Leu Gly Gly Gly Met

195 200 205

Met Ser Pro Leu Ala Leu Ala Leu Ser Ala Pro Ser Pro Thr Leu Gly

210 215 220

Leu Val Gly Leu Thr Pro Gly Ser Ala Gly Val Leu Gly Val Gly Ile

225 230 235 240

Ala Ala Leu Ala Ala Leu Thr Ala Ser Pro Ser Val Gly Ala Leu Gly

245 250 255

Gly Ala Ala Gly Ala Leu Ala Thr Leu Ala Thr Pro Ser His Ala Ala

260 265 270

Ala Leu Ala Thr Met Gly Thr Ala Met Ala Thr Pro Pro Ser Pro Ala

275 280 285

Thr Ala Ala Gly Leu Ser Ser Gly Thr Ala Gly Pro Ala Ser Ile Ser

290 295 300

Val Gly Ala Thr Ile Leu Gly Ala Pro Leu Ala Leu Ala Leu Val Thr

305 310 315 320

Thr Ala Pro Gly Ala Gly Val Ile Ile Gly Ala Ser Ser Gly Val Ala

325 330 335

Val Pro Val Gly Ala Leu Val Ala Ala Pro Ala Ala Thr Ser Gly Gly

340 345 350

Leu Gly Ala Leu Val Ala His Met Ala Thr Leu Gly

355 360

<210> 3

<211> 636

<212> DNA

<213> Soybean (Glycine max)

<400> 3

atggcgtcct ctctcattgc gaagcgcttc ctctcctctt ccctcctctc caggtccctc 60

cttcgccccg ccgcttccgc ttcccaccgc tctttcaaca ccaacgccat gcgccagtat 120

gacaaccgcg ccgacgacca cagcaccgac atcgatcgtc actccgaacg ctctttccct 180

agcactgcgc gccgcgacga tatcttctca gatgtgttgg atccattttt tccgactcgg 240

agtttgagcc aggttctgaa catgatggac caggtcatgg acaatccgtt cctctccgcg 300

tcgcgcggga tcggagctgg cgctggagtg cgtcgcggat gggacgcgag ggagacagag 360

gatgctctgc atctgcgcgt ggacatgcct gggctcggca aggaagacgt aaagatctcc 420

gtggagcaga acactctcat tatcaaaggt gaaggtgcta aagaaggcga tgaagaagag 480

agcgctcgtc gctacactag caggattgac ttgccggaca agctctacaa gattgaccag 540

atcagagctg agatgaagaa cggtgtgctc aaggtcgttg tgccgaaaat gaaggaggaa 600

gagaggaaag acgtgatcag tgttaaggtt gagtag 636

<210> 4

<211> 211

<212> PRT

<213> Soybean (Glycine max)

<400> 4

Met Ala Ser Ser Leu Ile Ala Leu Ala Pro Leu Ser Ser Ser Leu Leu

1 5 10 15

Ser Ala Ser Leu Leu Ala Pro Ala Ala Ser Ala Ser His Ala Ser Pro

20 25 30

Ala Thr Ala Ala Met Ala Gly Thr Ala Ala Ala Ala Ala Ala His Ser

35 40 45

Thr Ala Ile Ala Ala His Ser Gly Ala Ser Pro Pro Ser Thr Ala Ala

50 55 60

Ala Ala Ala Ile Pro Ser Ala Val Leu Ala Pro Pro Pro Pro Thr Ala

65 70 75 80

Ser Leu Ser Gly Val Leu Ala Met Met Ala Gly Val Met Ala Ala Pro

85 90 95

Pro Leu Ser Ala Ser Ala Gly Ile Gly Ala Gly Ala Gly Val Ala Ala

100 105 110

Gly Thr Ala Ala Ala Gly Thr Gly Ala Ala Leu His Leu Ala Val Ala

115 120 125

Met Pro Gly Leu Gly Leu Gly Ala Val Leu Ile Ser Val Gly Gly Ala

130 135 140

Thr Leu Ile Ile Leu Gly Gly Gly Ala Leu Gly Gly Ala Gly Gly Gly

145 150 155 160

Ser Ala Ala Ala Thr Thr Ser Ala Ile Ala Leu Pro Ala Leu Leu Thr

165 170 175

Leu Ile Ala Gly Ile Ala Ala Gly Met Leu Ala Gly Val Leu Leu Val

180 185 190

Val Val Pro Leu Met Leu Gly Gly Gly Ala Leu Ala Val Ile Ser Val

195 200 205

Leu Val Gly

210

<210> 5

<211> 2002

<212> DNA

<213> Soybean (Glycine max)

<400> 5

tttgtcattg acattgacga aacacgtgac gtggaggtgg gtctagccaa cagcattggc 60

gaaacaggta agacttctca gctaggttca ggtgttgcag agtagggcac agagggttca 120

cgtgggtttc gctaacatgg ctggcaaaac atgtgaagaa ggtatcccgc cattgcttct 180

ggcgagacaa gtgcttacgt ggcatgcatg catgcagggt cgtaggttga acgtagcagc 240

gtagcatagg aagcaggctt ttcaaaaacc ttttcagaaa gggtttcgca actgggagtg 300

gcaaaataag tttctctcct ataaaacgat cgtgcacgaa tgaatgggag catctgtgtg 360

aatttcttgg tgcttgttac gcttgtgctg gtggcgaaaa gcatgagttg tgtattcccc 420

tctttctcaa gcttggtaag tagttaatat ttgttgtatt tatttttttt ttctgttgct 480

tgataattaa attatttgtt ttaggtttag gtattataaa gtttgtatgc tatatttgtt 540

ttagatattc tatgccatta tttgtgttat atatatatat atatatatga taagttagtt 600

tgtaggttaa tatttttttt aaggtattct aagatattat ttaaatatat gatatgttag 660

tttgtattaa tttaatatat atgttgttta aatttttctt ttgttattaa aatatatatt 720

gtattattta tttaaattta tatctggtgc tgttacgtta tgctttgatg gtggcaaaaa 780

gcttgtgagt tccgtgttct ccgcccgtat ttggttatta atattttttg tgttctattt 840

gcagacagaa aatagtaatt aaaattactt aatgaattga ttatagatta ggaaaggaaa 900

aagaataaat aaattgtgtt cttaatacta attttaaact gttaaaatag gttaaattat 960

aattttgatt tattagtttt taaaatttat aatttaattg taatatttaa tttcctaatt 1020

ttataaacta acgattttgg tccctaatag attattaaca aataattata attaatagaa 1080

ttattaactt aattacaaat caattaaaaa gattaattat taaaacttta agaaaaaact 1140

attcacccta ctccctgcga caatgtgttt tttctttcat cagcaaacac gtattctact 1200

tctaccacca catgtgatga caccgtaaca acattgatca ataatctttt attaaatttt 1260

tgtaatgatt aataattttt atttaatata taattcactt aataatttta ttaatcacag 1320

ttattagtta ataaagaaga taaaaattgt taatttataa aattaatggg agtaaaatta 1380

caatttgatc tttaaaataa aatattaatc aaaaaatata tttagtagat atctgtccga 1440

aatattttaa ataaaaaatt gattacataa catgtgtcat aatcctaaat attaacagaa 1500

gttgagagaa caaattgtaa accgaggggt aggatctcat tttatctatc tcgacagaaa 1560

taatttattt aatgagaaaa tttatgattg attcttatta tagataaact tttctttaat 1620

taaaaacaat gacaaagtgg gaatgaaatt aatctttaag aaactatcgg aaaccatgtc 1680

tctgatccac gggaaaaaaa aaaaactgaa aagtttttaa cacagtgaat tatgaattaa 1740

gtgtggccac gatggtcgat ggctaataaa acggatatgc ttgctctcca aggcagaacc 1800

ttcgagaatc tcttagaagc gaataacact agtgactact ctggaacctt ctgacctttt 1860

tctagggtct tctagctcct ctcaagtccc aaccattccc cttatatcac ccactctcac 1920

tccccatttc cctgcacaaa gattttttcc cagcttgctc atcaagtcca agaaccaacc 1980

taccttttgc aatctcaatt ca 2002

<210> 6

<211> 295

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aggggtagga tctcatttta tctatctcga cagaaataat ttatttaatg agaaaattta 60

tgattgattc ttattataga taaacttttc tttaattaaa aacaatgaca aagtgggaat 120

gaaattaatc tttaagaaac tatcggaaac catgtctctg atccacggga aaaaaaaaaa 180

actgaaaagt ttttaacaca gtgaattatg aattaagtgt ggccacgatg gtcgatggct 240

aataaaacgg atatgcttgc tctccaaggc agaaccttcg agaatctctt agaag 295

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tgaagtgggg gagtttggac 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agtggcttgc aatctggtct 20

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gcgacgatat cttctcaggt aatg 24

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gacctggtcc atcatgttca ga 22

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctctctctct gtttatctcc gcac 24

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ctgaacgtga aggatctccc tc 22

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgtacatgg agagaattag agtga 25

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ttagcctaga tagtccatat gatcc 25

<210> 15

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atggcgtcct ctctcattg 19

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ctactcaacc ttaacactga tcac 24

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tttgtcattg acattgacga a 21

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tgaattgaga ttgcaaaagg t 21

<210> 19

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cccgggtggg catcgatacg ggatccatat gtacatggag agaattag 48

<210> 20

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tatctacgat tcatctgcag ctcgagttag cctagatagt ccatatg 47

<210> 21

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

aaaagcttga attcgagctc ggtacctgag agaacaaatt gtaaac 46

<210> 22

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tatacataca gagcacatgc ctcgagaggg gaatggttgg gacttg 46

<210> 23

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gagctcggta cccggggatc catgtacatg gagagaatta gagtga 46

<210> 24

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

caggtcgact ctagaggatc cttagcctag atagtccata tgatcc 46

<210> 25

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gagctcggta cccggggatc catggcgtcc tctctcattg 40

<210> 26

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

caggtcgact ctagaggatc cctactcaac cttaacactg atcac 45

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

cgatgcagat attcgtaatt atgc 24

<210> 28

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

catacctgtt caccgacgac g 21

<210> 29

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

cttgcaccaa gcagcatgaa 20

<210> 30

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ccgatccaga cactgtactt cctt 24

Claims (10)

1. The application of any one of the following substances 1) to 3) in enhancing the heat resistance of plants in the flowering period:

1) proteins GmHSFA2 and GmHSP20 a;

2) genes encoding protein GmHSFA2 and protein GmHSP20 a;

3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing genes encoding protein GmHSFA2 and protein GmHSP20 a;

the protein GmHSFA2 is a protein consisting of an amino acid sequence shown by SEQ ID NO.2 in a sequence table;

the protein GmHSP20a is a protein consisting of an amino acid sequence shown by SEQ ID NO.4 in a sequence table.

2. The application of any one of the following substances 1) to 3) in cultivating plants with improved heat resistance in flowering period:

1) proteins GmHSFA2 and GmHSP20 a;

2) genes encoding protein GmHSFA2 and protein GmHSP20 a;

3) a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing genes encoding protein GmHSFA2 and protein GmHSP20 a;

the protein GmHSFA2 is a protein consisting of an amino acid sequence shown by SEQ ID NO.2 in a sequence table;

the protein GmHSP20a is a protein consisting of an amino acid sequence shown by SEQ ID NO.4 in a sequence table.

3. The use of claim 1 or 2, wherein the gene encoding protein GmHSFA2 has the nucleotide sequence shown as SEQ ID No.1 in the sequence listing.

4. The use according to claim 1 or 2, characterized in that the gene encoding the protein GmHSP20a has a nucleotide sequence shown in SEQ ID No.3 of the sequence Listing.

5. The use as claimed in claim 4, wherein the promoter of GmHSP20a has a nucleotide sequence shown in SEQ ID NO. 5.

6. The use as claimed in claim 4, wherein the promoter HSE element of GmHSP20a has a nucleotide sequence shown in SEQ ID NO. 6.

7. The use according to any one of claims 1-2, wherein the plant is a monocotyledonous or dicotyledonous plant; wherein the dicotyledons are leguminous plants or cruciferae plants.

8. The use according to claim 3, wherein the plant is a monocotyledonous or dicotyledonous plant; wherein the dicotyledons are leguminous plants or cruciferae plants.

9. A method for breeding plants with improved flowering heat resistance, which comprises the step of introducing genes encoding proteins GmHSFA2 and GmHSP20a of claim 1 into target plants to obtain transgenic plants, wherein the flowering heat resistance of the transgenic plants is higher than that of the target plants: wherein the genes encoding proteins GmHSFA2 and GmHSP20a are introduced into the target plant through a recombinant vector.

10. A recombinant vector is characterized in that genes encoding proteins GmHSFA2 and GmHSP20a are inserted into an expression vector to obtain the recombinant vector expressing the proteins GmHSFA2 and GmHSP20 a:

the protein GmHSFA2 is a protein consisting of an amino acid sequence shown by SEQ ID NO.2 in a sequence table;

the protein GmHSP20a is a protein consisting of an amino acid sequence shown by SEQ ID NO.4 in a sequence table.

Images