CN105175522B - Crowtoe AP2/ERF transcription factors and its encoding gene and application - Google Patents

Abstract

The invention discloses japonicus japonicus AP2/ERF transcription factor, its coding gene and application, and belongs to the field of AP2/ERF transcription factor and application. The present invention first discloses the AP2/ERF transcription factor gene isolated from japonicus japonicus, its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. The invention also discloses an AP2/ERF transcription factor domain capable of combining with cis-acting elements to initiate the expression of anti-stress response genes, the amino acid sequence of which is shown in SEQ ID NO.3. Functional transformation experiments proved that overexpressing the AP2/ERF transcription factor gene in plants can significantly improve the salt tolerance of transgenic plants, indicating that the encoded protein has the function of AP2/ERF transcription factor. The AP2/ERF transcription factor gene of the invention has an important application prospect in improving the resistance of plants to adversity stress.

Description

Radix japonicus AP2/ERF transcription factor and its coding gene and application

technical field

The present invention relates to transcription factors, in particular to an AP2/ERF transcription factor related to stress resistance isolated from Lotus corniculatus L., and the present invention also relates to the role of said transcription factors in improving plant resistance to adversity stress The application belongs to the field of AP2/ERF transcription factor and its application.

Background technique

The problems of soil salinization and secondary salinization are widespread in the world, especially in arid and semi-arid areas. Soil drought and salinization seriously hinder the growth of crops and reduce the yield of crops, which have become important factors restricting the sustainable development of irrigated agriculture in the world and affecting the ecological environment.

Lotus corniculatus L. is an excellent perennial leguminous forage widely planted in the world. It has the advantages of rich nutrition, tolerance to barrenness, acid and alkali resistance, grazing resistance, safe feeding and good palatability. It is not only a high-quality grass species for the construction of ecological agriculture, but also an important crop for soil improvement. It plays a unique role in being used as ground cover plants, maintaining water and soil, improving artificial pastures, and preventing soil desertification.

Plant stress resistance is a complex quantitative trait controlled by multiple genes. The enhancement of the expression of a single gene cannot fundamentally improve the resistance of plants to various adverse environments. Transcription factors (Transcriptional Factors, TFs) can interact with eukaryotic A type of DNA-binding protein that specifically binds to cis-acting elements in the gene promoter region, thereby activating or inhibiting the transcription and expression of downstream genes at a specific time and space.

Plant stress resistance-related transcription factors can be divided into five categories: AP2/ERF, bZIP, WRKY, MYB, and NAC. Among them, AP2/ERF transcription factor is a large supergene family unique to plants, with a very conserved DNA binding domain, which specifically binds to cis-elements such as ERE and GCC-box, regulates the expression of stress-responsive genes, and is involved in plant growth. Important transcriptional regulator of development, biotic/abiotic stress-responsive gene expression, and signal transduction. The AP2/ERF superfamily is divided into five subfamilies: AP2, DREB, ERF, RAV, and Soloist. At present, a large number of literatures have reported the research on the function and molecular mechanism of the DREB and ERF subfamily genes, but the individual subfamily (Soloist) There are few literature reports. Therefore, it is of great significance to isolate and identify AP2/ERF transcription factors from fine leguminous forage lotus japonicus and analyze their stress resistance functions for agricultural production, ecological environment construction, and soil improvement.

Contents of the invention

The technical problem to be solved by the present invention is to provide the AP2/ERF transcription factor and its coding gene isolated from Lotus corniculatus L.;

Another technical problem to be solved by the present invention is to provide a recombinant plant expression vector containing the above coding gene and a host cell containing the expression vector;

The third technical problem to be solved by the present invention is to apply the AP2/ERF transcription factor and its coding gene to improve the resistance of plants to adversity stress.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:

The present invention first discloses the AP2/ERF transcription factor encoding gene (named: LcAP2/ERF107) isolated from Lotus corniculatus L., and its polynucleotides are (a), (b), (c) , (d) or (e):

(a), the polynucleotide shown in SEQ ID NO.1; or

(b), a polynucleotide encoding the amino acid shown in SEQ ID NO.2; or

(c), a polynucleotide that can hybridize to the complementary sequence of SEQ ID NO.1 under stringent hybridization conditions, and the protein encoded by the polynucleotide still has the function of AP2/ERF transcription factor; or

(d), a polynucleotide having at least 90% or more homology with the polynucleotide shown in SEQ ID NO.1; or

(e) A polynucleotide variant in which one or more bases are deleted, substituted or inserted on the basis of the polynucleotide shown in SEQ ID NO.1, and the protein encoded by the polynucleotide variant Still have the function or activity of AP2/ERF transcription factor.

The AP2/ERF transcription factor encoded by the above-mentioned LcAP2/ERF107 transcription factor encoding gene of the present invention, its amino acid is shown in (a) or (b):

(a), the amino acid shown in SEQ ID NO.2;

(b) A protein variant still having AP2/ERF transcription factor function or activity derived from the amino acid shown in SEQ ID NO.2 by substitution, deletion or/and insertion of one or more amino acid residues.

Wherein, the sequence of the 108th to 159th amino acid residues at the amino terminal of SEQ ID NO.2 is the coding sequence of the AP2/ERF binding domain (shown in SEQ ID NO.3), and the 14th (121st) amino acid of the binding domain It is alanine (A), and the 19th (126th) amino acid is aspartic acid (D). This type of domain can combine with GCC box cis-acting elements to activate disease resistance defense genes or stress response genes expression.

The protein variants described in the present invention can be produced by genetic polymorphism or human manipulation, and these manipulation methods are generally understood in the art. For example, amino acid sequence variants or fragments of AP2/ERF transcription factors can be prepared by DNA mutation, wherein methods for mutagenizing or altering polynucleotides are well known in the art. Among them, a conservative substitution is to replace one amino acid residue with another amino acid with similar properties.

The LcAP2/ERF107 transcription factor coding gene of the present invention includes two forms of naturally occurring sequences and variants. "Variants" means substantially similar sequences, and with respect to polynucleotides, variants comprise deletions, insertions, and/or substitutions of one or more nucleotides at one or more sites in the native polynucleotide. With respect to polynucleotides, conservative variants include those that do not alter the encoded amino acid sequence due to the degeneracy of the genetic code. Naturally occurring variants such as these can be identified by available molecular biology techniques. Variant polynucleotides also include polynucleotides of synthetic origin, such as polynucleotide variants obtained by site-directed mutagenesis that still encode amino acids shown in SEQ ID NO.2, or by recombinant methods (such as DNA shuffling ). Those skilled in the art can screen or evaluate the function or activity of the protein encoded by the variant polynucleotide through the following molecular biological techniques: DNA binding activity, interaction between proteins, activation of gene expression in transient studies or transgenic plants The effect expressed in etc.

In the present invention, the amino acid (LcAP2/ERF107 transcription factor) shown in SEQ ID NO.2 is compared with the homologous AP2/ERF amino acid sequence of Arabidopsis thaliana and rice, and a phylogenetic tree is constructed. The results show that LcAP2/ERF107 and The homology relationship of At4g13040 of the Arabidopsis separate subfamily (Soloist) is the closest. Thus, LcAP2/ERF107 is a novel gene of a separate subfamily.

The invention also discloses a recombinant expression vector containing the gene encoding the LcAP2/ERF107 transcription factor and a recombinant host cell containing the recombinant expression vector; wherein, the recombinant expression vector is a recombinant plant expression vector.

The LcAP2/ERF107 transcription factor coding gene is operably connected to the expression control element to obtain a recombinant plant expression vector that can express the coding gene in plants; the recombinant plant expression vector can be composed of a 5' non-coding region, SEQ The nucleotide and 3' non-coding region shown in ID NO.1; wherein, the 5' non-coding region may include a promoter sequence, an enhancer sequence or/and a translation enhancing sequence; the promoter It can be a constitutive promoter, an inducible promoter, a tissue or organ-specific promoter; the 3' non-coding region can include a terminator sequence, an mRNA cleavage sequence, and the like. Suitable terminator sequences can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase termination regions.

In addition, those skilled in the art can optimize the nucleotide shown in SEQ ID NO.1 to enhance the expression efficiency in plants. For example, polynucleotides can be synthesized using codon-biased codons of the target plant for optimization to enhance expression efficiency in the target plant.

The recombinant plant expression vector may also contain a selectable marker gene for selection of transformed cells for selection of transformed cells or tissues. Marker genes include: genes encoding antibiotic resistance, genes conferring resistance to herbicidal compounds, and the like. In addition, the marker genes also include phenotypic markers, such as β-galactosidase and fluorescent protein.

The present invention also relates to introducing the gene encoding the LcAP2/ERF107 transcription factor into plants to improve the resistance of plants to adversity stress.

The invention discloses a method for improving the resistance of plants to adversity stress, which comprises the following steps: (1) constructing a recombinant plant expression vector containing the gene encoding the LcAP2/ERF107 transcription factor isolated from Lotus japonicus; (2) converting the The constructed recombinant plant expression vector is transformed into plants or plant cells; (3) breeding and screening to obtain transgenic plants with improved resistance to adversity stress.

The present invention also discloses a method for cultivating a new variety of transgenic plant resistant to adversity stress, comprising the following steps: (1) constructing a recombinant plant expression vector containing a gene encoding the LcAP2/ERF107 transcription factor isolated from Lotus japonicus; (2) ) Transforming the constructed recombinant plant expression vector into plants or plant cells; (3) Cultivating and screening new transgenic plant varieties with improved resistance to stress.

Wherein, the adversity stress includes: high salinity, drought or low temperature.

Transformation protocols and protocols for introducing the polynucleotide or polypeptide into a plant may vary depending on the type of plant (monocot or dicot) or plant cell used for transformation. Suitable methods for introducing the polynucleotide or polypeptide into plant cells include: microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-speed ballistic bombardment. In specific embodiments, the gene encoding the LcAP2/ERF107 transcription factor of the invention can be provided to plants using various transient transformation methods. In other embodiments, the gene encoding the LcAP2/ERF107 transcription factor of the present invention can be introduced into the plant by contacting the plant with a virus or viral nucleic acid. Usually, such a method involves constructing the gene encoding the LcAP2/ERF107 transcription factor of the present invention. body into the viral DNA or RNA molecule.

Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986.5:81-84). The present invention can be used to transform any plant species, including but not limited to: monocots or dicots. More preferably, the target plants include crops, vegetables or ornamental plants, fruit trees, etc., for example, may be corn, rice, sorghum, wheat, soybean, potato, barley, tomato, bean, peanut or sugarcane.

In order to analyze the function of the LcAP2/ERF107 transcription factor isolated from Lotus corniculatus L. in the present invention, the present invention first constructs a gene encoding the transcription factor containing LcAP2/ERF107 (the nucleotide is shown in SEQ ID NO.1) The high-efficiency expression vector of recombinant plants was transformed into Arabidopsis thaliana by Agrobacterium-mediated method, and positive plants were obtained through hygromycin resistance screening, PCR and RT-PCR identification. The germination rate and survival rate of transgenic Arabidopsis under salt stress and the physiological and biochemical indicators of transgenic Arabidopsis under salt stress showed that the germination rate of transgenic LcAP2/ERF107 Arabidopsis seeds was extremely significant under high salt stress Under continuous high-salt stress, the survival rate of transgenic Arabidopsis seedlings was significantly higher than that of wild-type, indicating that the transcription factor LcAP2/ERF107 significantly improved the salt tolerance of transgenic plants. The measurement of physiological and biochemical indicators showed that transgenic LcAP2/ERF107 Arabidopsis improved salt tolerance by increasing relative water content and reducing cell membrane damage. Therefore, the transcription factor LcAP2/ERF107 is an excellent regulator of salt tolerance in the AP2/ERF family of japonicus japonicus.

Functional transformation experiments have proved that overexpressing the LcAP2/ERF107 transcription factor encoding gene in plants can effectively increase or improve plant resistance to stresses including high salinity, drought, low temperature, etc., and play an important role in improving plant stress resistance. The protein encoded by LcAP2/ERF107 gene has the function of AP2/ERF transcription factor.

Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

The present invention isolates and identifies a single subfamily gene LcAP2/ERF107 from japonicus japonicus, and its stress resistance function analysis shows that this gene significantly improves the salt tolerance of transgenic Arabidopsis thaliana, and is an excellent gene in japonicus japonicus Anti-retroviral factors provide genetic resources and technical support for stress-resistant molecular improvement and breeding of japonicus or other leguminous forages, and are of great significance for agricultural and animal husbandry production, ecological environment construction, and soil improvement.

Definition of terms involved in the present invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "transcription factor" refers to a class of DNA-binding proteins that can specifically bind to cis-acting elements in the promoter region of eukaryotic genes, thereby activating or inhibiting the transcription and expression of downstream genes at a specific time and space.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the explicitly designated sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted at position 3 with mixed bases and/or deoxyinosine residues (Batzer et al. , Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J.Biol.Chem.260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol Cell.Probes 8: 91-98 (1994)).

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer of amino acid residues. That is, descriptions for polypeptides apply equally to descriptions of peptides and descriptions of proteins, and vice versa. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (ie, antigens), wherein the amino acid residues are linked via covalent peptide bonds.

The term "stringent hybridization conditions" means conditions of low ionic strength and high temperature known in the art. Generally, under stringent conditions, a probe will hybridize to its target sequence to a detectably greater extent (eg, at least 2-fold over background) than to other sequences. Stringent hybridization conditions are sequence-dependent and will be different under different environmental conditions, with longer sequences hybridizing specifically at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the stringency of hybridization or wash conditions. For the detailed guidance of nucleic acid hybridization, reference can be made to related literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes,"Overview of principles of hybridization and the strategy of nucleic acid assays.1993). More specifically, the stringent conditions are usually defined by It is selected to be about 5-10°C below the thermal melting point (Tm) of the specific sequence at a defined ionic strength pH. The Tm is the temperature at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium (at a given ionic strength, pH, and nucleic acid concentration) (50% of the probe is occupied at Tm at equilibrium because the target sequence is present in excess). Stringent conditions can be those in which the salt concentration is low at pH 7.0 to 8.3 At about 1.0M sodium ion concentration, usually about 0.01 to 1.0M sodium ion concentration (or other salt), and the temperature is at least about 30°C, and at least about 60°C for long probes (including, but not limited to, greater than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selectivity or For specific hybridization, a positive signal may be at least two times background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5×SSC and 1% SDS at 42° C. Incubate; or 5×SSC, 1% SDS, incubate at 65°C, wash in 0.2×SSC and wash in 0.1% SDS at 65°C. The washes can be performed for 5, 15, 30, 60, 120 minutes or longer.

The "multiple" in the present invention usually means 2-8, preferably 2-4, depending on the position of the amino acid residue or the type of amino acid in the three-dimensional structure of the AP2/ERF transcription factor; the " "Replacement" refers to the replacement of one or more amino acid residues with different amino acid residues; the "deletion" refers to the reduction of the number of amino acid residues, that is, the lack of one or more amino acid residues; The "insertion" refers to the change of amino acid residue sequence, relative to the natural molecule, the change results in the addition of one or more amino acid residues.

The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, pairing, or in the art. other known methods. Exogenous polynucleotides may remain as non-integrating vectors such as plasmids or may integrate into the host genome. The host cell can be a prokaryotic cell or a eukaryotic cell, and the host cell can also be a monocotyledonous or dicotyledonous plant cell.

The term "operably linked" refers to a functional linkage between two or more elements, which may be contiguous or non-contiguous.

The term "transformation" refers to a method of introducing a heterologous DNA sequence into a host cell or organism.

The term "expression" refers to the transcription and/or translation of an endogenous or transgene in a plant cell.

The term "coding sequence" refers to a nucleic acid sequence transcribed into RNA.

The term "recombinant plant expression vector" means one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors together with vectors with helper plasmids are the most commonly used for Agrobacterium-mediated transformation. Binary vectors usually include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc.

Description of drawings

Figure 1 is the amplification result of the full-length sequence of LcAP2/ERF107CDS;

Figure 2 is an analysis of the conserved domain of the transcription factor LcAP2/ERF107 structure;

Figure 3 is the phylogenetic analysis of the transcription factor LcAP2/ERF107;

Figure 4 is the tissue expression analysis of LcAP2/ERF107;

Figure 5 is the expression pattern analysis of LcAP2/ERF107 under different stress conditions;

Figure 6 is a flow chart of the construction of the plant expression vector pH7WG2D-LcAP2/ERF107; wherein, A: construction of the entry vector; B: construction of the plant overexpression vector;

Fig. 7 is the PCR identification result of recombinant plasmid pH7WG2D-LcAP2/ERF107;

Figure 8 is the colony PCR of Agrobacterium GV3101 containing the expression vector pH7WG2D-LcAP2/ERF107;

Fig. 9 is the PCR detection result of the DNA level of Arabidopsis transgenic LcAP2/ERF107;

Figure 10 is the PCR detection of the RNA level of the transgenic LcAP2/ERF107 Arabidopsis; wherein, L8, L13, and L15 are different strains of the transgenic LcAP2/ERF107 Arabidopsis;

Figure 11 shows the germination rate of transgenic LcAP2/ERF107 Arabidopsis seeds under salt stress; wherein, CK is wild-type Arabidopsis; L8, L13, and L15 are different lines of transgenic LcAP2/ERF107 Arabidopsis;

Figure 12 is the effect of salt stress on the survival rate of transgenic LcAP2/ERF107 Arabidopsis seedlings; wherein, WT is wild-type Arabidopsis; L8, L13, and L15 are different lines of transgenic LcAP2/ERF107 Arabidopsis;

Figure 13 shows the effect of salt stress on the physiological indicators of transgenic LcAP2/ERF107 Arabidopsis; where, A: relative water content; B: relative electrical conductivity; WT is wild type Arabidopsis; L8, L13, L15 are transgenic LcAP2/ERF107 Different strains of Arabidopsis.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, it should be understood that the described embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

Example 1 Isolation, Identification and Cloning of the Gene Encoding the LcAP2/ERF107 Transcription Factor in Lotus japonicus

1 Materials and methods

1.1 Materials

1.1.1 Plant material cultivation and stress treatment

Use japonicus 'Leo' as the material, select mature and plump seeds that are free from diseases and insect pests, soak them in 2% NaClO for 15-20 minutes, wash them with sterile water for 3 times, dry them, and put the sterilized seeds on the B5 medium , cultured in a light incubator for 30 days, treated with 150mM sodium chloride for 0h, 3h, 12h, and 24h, collected leaves were immediately frozen in liquid nitrogen, and stored in a -80°C refrigerator for later use.

Analysis of tissue expression specificity: The roots, stems, leaves, flowers, and seeds of J. japonicus were taken, quick-frozen in liquid nitrogen, and stored at -80°C until use.

Hormone-treated japonicus japonicus, the seedlings cultivated for 30 days were transplanted into MS medium containing 100 μM ABA, ACC, MeJA, SA, treated for 0 h, 3 h, 12 h, 24 h, young leaves were taken, liquid nitrogen quick-frozen, -80 °C Save for later use.

1.1.2 Strains and plasmids

Escherichia coli (Escherichia coli): DH5α preserved in the inventor's laboratory;

The pEASY-T-Simple cloning vector was purchased from Quanshijin Biological Company.

1.1.3 Solutions

(1) Preparation of TE (10mM Tris-HCl, pH 8.0; 1mM EDTA, pH 8.0):

1M Tris-HCl (pH 8.0) 1ml, 0.5M EDTA (pH 8.0) 0.2ml, ultrapure water to 100ml.

(2) 50×TAE buffer: 242g Tris base, 57.1ml glacial acetic acid, 100ml 0.05mol/L EDTA (pH8.0)

(3) 1.0M Tris-HCl buffer (1L): Dissolve 121.1g Tris base in 800ml of deionized water. After fully dissolved, add 40ml of concentrated HCl, and dilute to 1L with deionized water.

1.1.4 Medium

(1) LB liquid medium (1L, pH 7.4):

Peptone 10.0g, yeast extract 5.0g, NaCl 10.0g. If it is a solid medium, add 15.0-20.0g of agar powder.

(2) B5 medium (pH 5.8): B5 dry powder 4.43g, sucrose 20g, dissolved in 800mL deionized water, adjust the pH value to 5.8-6.0, dilute to 1L, add 15g agar powder or 2.5g Vegetable gel, sterilized under high temperature and high pressure at 121°C for 15 minutes. If preparing a medium containing antibiotics or hormones, cool the sterilized B5 medium to about 60°C, and then add the corresponding reagent to the required concentration.

1.2 Experimental method

1.2.1 Extraction of Radix japonicus total RNA

Refer to the instruction manual of the polysaccharide polyphenol plant total RNA extraction kit (catalogue number: DP441) of Tiangen Biochemical Company.

1.2.2 Synthesis of first-strand cDNA

Refer to the reverse transcription kit of Beijing Quanshijin Biotechnology Co., Ltd. ( gDNA Removal and cDNA Synthesis SuperMix) instructions.

1.2.3 Cloning of cDNA of LcAP2/ERF107 transcription factor

(1) Design and synthesis of primers

The primer sequences for cloning the full-length LcAP2/ERF107CDS are listed in Table 1.

Table 1 The sequence of primers for cloning the full length of LcAP2/ERF107CDS

(2) RT-PCR

Total RNA was extracted from leaves treated with 150mM NaCl for 12h and 24h, and RT-PCR was performed using the reverse-transcribed cDNA as a template. The PCR conditions were: 94°C for 5min; cycle; 72°C for 10 min; 4°C for storage.

(3) Recovery of PCR products

The target fragment was recovered using the Easypure PCR Purification kit of Beijing Quanshijin Biotechnology Co., Ltd., and the operation was performed according to the kit instructions.

(4) Ligation of target fragment and cloning vector

The recovered PCR product was ligated to the pEASY-T-Simple vector, the ligation reaction system was shown in Table 2, and ligated at 25°C for 10 min.

Table 2 Ligation reaction system

(5) Preparation of Escherichia coli competent

Refer to the specific method written in the document "One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution" (Chung et al., 1989).

(6) Transformation of Escherichia coli (heat shock method)

Carry out with reference to the specific method listed in the book "Molecular Cloning Experiment Guide" (Third Edition, J. Sambrook).

(7) Colony PCR identification

Use the white single colony picked on the LB plate containing Kan, carry out colony culture and PCR verification, use the universal primer M13, the amplification conditions are: 94°C for 3min, (94°C for 45sec, 55°C for 45sec, 72°C for 1min) 30 colonies Cycle, 72°C for 10min. After the reaction, the PCR products were identified by 1.5% agarose gel electrophoresis, and the positive clones obtained by screening were sent to the Sequencing Department of the Major Engineering Open Laboratory of the Institute of Crop Science, Chinese Academy of Agricultural Sciences for sequencing.

1.2.4 Analysis of the expression pattern of the gene LcAP2/ERF107 under different stress conditions

The tissue expression specificity of gene LcAP2/ERF107 in roots, stems, leaves, flowers and seeds was analyzed by RT-PCR.

Take the roots, stems, leaves, flowers, and seeds of japonicus japonicus, freeze them quickly in liquid nitrogen, and store them at -80°C for later use. For the specific steps of extracting total RNA, refer to the instructions of the polysaccharide and polyphenol plant total RNA extraction kit from Tiangen Biochemical Company ( Spin column type, catalog number: DP441), using agarose gel electrophoresis and ultraviolet spectrophotometer (Biodropsis BD-2000) to detect the quality and purity of the extracted RNA. cDNA was synthesized with reference to the reverse transcription kit of Beijing Quanshijin Biotechnology Co., Ltd. ( gDNA Removal and cDNA Synthesis SuperMix) instructions. Using cDNA as a template and primers as specific primers for gene LcERFs, RT-PCR was carried out, and the PCR product was electrophoresed on 2% agarose gel. The RT-PCR product was the ubiquitin protein gene polyubiquitin (AW720576) of 139 bp as a control.

Table 3 Specific primer sequences for RT-PCR or qPCR

The qRT-PCR method was used to analyze the expression changes of the gene LcAP2/ERF107 induced by hormones (ABA, ACC, MeJA, SA) and abiotic stress (NaCl) at different times.

The 30-day-old japonicus japonicus seedlings were treated with 100 μM ABA, ACC, JA, SA, and 150 mM NaCl for 0 h, 3 h, 12 h, and 24 h, respectively, and the young leaves were taken and frozen in liquid nitrogen. Total RNA was extracted according to the above method, and the first reverse transcription was performed. qRT-PCR was used to analyze the relative expression of salt stress response genes, and the ubiquitin gene polyubiquitin (AW720576) was used as an internal reference gene. For the reaction system of qRT-PCR, refer to the quantitative PCR kit ( Green qPCR SuperMix, Cat. No.: AQ131), see Table 4 and 5 for details. When performing qRT-PCR, the experiment was repeated three times for each sample, and three technical replicates were performed, and the relative expression of genes was calculated using the 2 ^-ΔΔCT method (Livak and Schmittgen, 2001).

Table 4 qRT-PCR reaction system

Table 5 qRT-PCR amplification program

2 Experimental results and analysis

2.1 Cloning of LcAP2/ERF107 gene and its bioinformatics analysis

The present invention uses 30-day-old japonicus seedlings treated with 150mM NaCl for 24 hours as material, extracts total RNA from leaves and reverse-transcribes it into cDNA, and uses the full-length primers in Table 1 to amplify LcAP2/ERF107 by RT-PCR The complete ORF sequence of the gene (shown in SEQ ID NO.1), the result is shown in Figure 1, and submitted to Genbank to obtain the accession number: KC357712. The open reading frame of the gene LcAP2/ERF107 is 699bp, encoding 232 amino acids (shown in SEQ ID NO.2). Using SMART software to analyze the conserved domain of transcription factor LcAP2/ERF107, the 108th to 159th amino acid residue sequence is the coding sequence of the AP2/ERF binding domain (shown in SEQ ID NO.3), the results are shown in Figure 2, the binding domain The 14th amino acid is alanine (A), and the 19th amino acid is aspartic acid (D). This type of domain can combine with the GCC box cis-acting element to activate the disease resistance defense gene or stress response gene. Express.

The isoelectric point of protein LcAP2/ERF107 is predicted to be 9.62 and the molecular weight is 24.8kDa by ExPASy Compute pI/Mw tool software; the signal peptide is analyzed by SignalP 4.1 software, and the results show that: LcAP2/ERF107 protein has no signal peptide and is a non-secreted protein; ProtScale Software analysis of hydrophobicity, TMHMM software analysis of transmembrane structure, the results showed that: LcAP2/ERF107 protein is a hydrophilic protein, and no transmembrane structure. Using Motif scan software analysis, the results showed that 14-18 and 68-77 are serine (ser)-rich regions, and 162-167 is an acidic amino acid-rich region, which may play an important role in transcriptional activation.

ClustalX2 software was used to perform multiple sequence alignments of the amino acid sequences of LcAP2/ERF107 and the homologous AP2/ERF of Arabidopsis thaliana and rice, and a phylogenetic tree was constructed with MEGA5.0, as shown in Figure 3, LcAP2/ERF107 and the separate subfamily of Arabidopsis thaliana (Soloist) At4g13040 has the closest homology, so LcAP2/ERF107 is a novel gene of a separate subfamily.

2.2 Analysis of the expression pattern of gene LcAP2/ERF107 under different stress conditions

The tissue expression specificity of gene LcAP2/ERF107 in roots, stems, leaves, flowers and seeds was analyzed by RT-PCR method, as shown in Figure 4, the gene is expressed in roots, stems, leaves, flowers and seed tissues .

The qRT-PCR method was used to analyze the expression changes of the gene LcAP2/ERF107 induced by hormones (ABA, ACC, MeJA, SA) and abiotic stress (NaCl) at different times, and the results are shown in Figure 5. The gene LcAP2/ERF107 was treated with NaCl for 3h and 24h, and the expression level was up-regulated by about 2 times, indicating that the expression level was continuously induced by NaCl; ABA, ACC, JA and SA induced 3h, the expression level did not change significantly, and after induction for 24h, the expression level was significantly up-regulated, indicating that The expression of LcAP2/ERF107 is induced by the four major hormones ABA, ACC, JA and SA in the late stage. Therefore, the expression of gene LcAP2/ERF107 is induced by ABA, ACC, JA, SA and NaCl. It can be seen that LcAP2/ERF107 has different responses to salt abiotic stress and hormone treatments such as abscisic acid, ethylene, jasmonic acid, and salicylic acid, indicating that it may be involved in various abiotic stresses and signal transduction pathways .

Example 2 Construction of a plant expression vector containing the LcAP2/ERF107 gene and its salt-tolerance functional verification in Arabidopsis

1 Materials and methods

1.1 Materials

1.1.1 Plant material

Arabidopsis thaliana Columbia (Col-0) ecotype: preserved in the inventor's laboratory.

1.1.2 Strain and carrier

Agrobacterium tumefaciens strain GV3101 was preserved in the inventor's laboratory; gateway entry vector pDNOR201 and expression vector pH7WG2D were purchased from Invitrogen.

1.1.3 Medium

(1) YEB medium (1L)

Peptone 5g, yeast extract 1g, sucrose 5g, MgSO ₄ ·7H ₂ O 0.5g, deionized water to 1L, if it is a solid medium, add 15g agar powder, sterilize at 121℃ for 15min.

(2) LB medium (1L)

Tryptone 10g, sodium chloride 10g, yeast extract 5g, deionized water to 1L, if it is a solid medium, add 15g agar powder, sterilize under high temperature and high pressure at 121°C for 15min.

(3) MS medium

MS dry powder 4.43g, sucrose 20g, dissolved in 800mL deionized water, adjust the pH value to 5.8-6.0, dilute to 1L, add 15g agar powder or 2.5g plant gel to the solid medium, sterilize under high temperature and high pressure at 121℃ for 15min . If the medium containing antibiotics or hormones is prepared, cool the sterilized MS medium to about 60°C, and then add the corresponding reagent to the required concentration.

1.2 Experimental method

1.2.1 Primer design and synthesis

The primers containing the attb site were designed and synthesized, and the sequences are shown in Table 6.

Table 6 Primers used to construct plant expression vectors

1.2.2 Construction of plant expression vectors

(1) Construction of plant high-efficiency expression vector pH7WG2D-LcAP2/ERF107

Using primers LcAP2/ERF107-attb(F) and LcAP2/ERF107-attb(R) to amplify the complete open reading containing attb site from the full-length pEASY-T vector connected with the CDS of the gene LcAP2/ERF107 in Example 1 Box (shown in SEQ ID NO.1). The amplified product and the entry vector pDNOR201 were subjected to BP reaction, and the CCDB gene on the pDNOR201 vector was replaced with the LcAP2/ERF107 gene, and the entry vector containing the target gene LcAP2/ERF107 was constructed, transformed into Escherichia coli DH5α, and the recombinant plasmid was screened by PCR amplification and sequenced Identification. The correctly sequenced entry vector pDNOR201-LcAP2/ERF107 was subjected to LR reaction with the expression vector pH7WG2D, and the CCDB gene on the expression vector was replaced with the target gene LcAP2/ERF107 to construct a plant expression vector pH7WG2D-LcAP2/ERF107, which was transformed into Escherichia coli DH5α, PCR amplification identified recombinant plasmids.

(2) Mini-extraction of recombinant plasmids

For the plasmid extraction method, refer to the specific method listed in the Biotech Plasmid Miniprep Kit.

1.2.3 Agrobacterium transformation and cultivation

(1) Agrobacterium GV3101 competent cells were prepared by TSS method, and the specific method was the same as that of Escherichia coli competent cells. The heat shock method was used for the transformation method, and for specific steps, refer to the finely edited Molecular Biology Experiment Guide (Osper).

(2) PCR identification of Agrobacterium containing recombinant plasmid

Pick a single colony on the transformation plate of YEB (containing 50mg/L Spe, 50mg/L Rif) cultured at 28°C for 1-2 days, use untransformed Agrobacterium as a negative control, and use the plant expression vector plasmid pH7WG2D-LcAP2/ERF107 As a positive control, colony PCR identification was carried out.

1.2.4 Genetic transformation of Arabidopsis

Arabidopsis was transformed by inflorescence infection.

Cultivate the Agrobacterium containing the plant expression vector pH7WG2D-LcAP2/ERF107 to an _OD600 of 0.8-2.0, centrifuge at 5500rpm for 10-15min, discard the supernatant, resuspend the bacterial cell pellet with MS permeate, adjust the _OD600 to 0.8-1.2, for infecting Arabidopsis.

After about 4 weeks of Arabidopsis bolting, the main stem was cut off from the base to promote the growth of side stems for about a week. When the flower buds were full and in the dew-white stage, the Arabidopsis was transformed by dipping flowers (Clough and Bent, 1998). Soak in the above-mentioned permeate for 15sec-45sec, place it at an angle, culture in dark for 2-3 days, culture normally for one week, and infect Arabidopsis thaliana again once, and harvest mature seeds four weeks later.

1.2.5 Screening and identification of transgenic LcAP2/ERF107 Arabidopsis

(1) Resistance screening of LcAP2/ERF107 transgenic Arabidopsis

Spread the harvested T ₀ generation seeds aseptically on MS plates containing 25 mg/L hygromycin to grow T ₁ generation transgene-positive plants, cultivate them normally, and harvest individual seeds; select 50 seeds of T ₁ generation seeds Spread on a hygromycin-resistant plate, grow _T2 generation transgenic positive plants, select a line with a segregation ratio of 3:1 between positive plants and negative plants, cultivate normally, and harvest a single seed; select 50 _T2 generation The seeds were spread on the hygromycin-resistant plate, the homozygosity of the T ₃ generation was identified, and the seeds of the T ₃ generation of the homozygous line were harvested and preserved for subsequent functional analysis.

(2) PCR identified positive plants

A small amount of genomic DNA was extracted from transgenic Arabidopsis resistant seedlings by CTAB method, and positive plants were further screened by PCR detection. If the target fragment is amplified, but no fragment is amplified in the negative control, it is preliminarily proved that the target gene is integrated into the Arabidopsis genome.

(3) Further identification of positive plants by RT-PCR

For the Arabidopsis plants that were positive in the DNA level detection, the total RNA was extracted with TRIzol reagent, the cDNA obtained by reverse transcription was used as a template, and the positive transformed seedlings were screened by RT-PCR detection.

1.2.6 Salt tolerance function analysis of transgenic Arabidopsis

(1) Germination rate of transgenic Arabidopsis seeds under salt stress

Experiment of germination rate of transgenic Arabidopsis seeds under salt stress: After sterilizing wild-type and transgenic Arabidopsis seeds, they were planted on MS plates with 0mM, 100mM, 150mM, and 200mM NaCl respectively, and vernalized for 1-3 days , Cultivate in a light incubator for 7-15 days, observe and count the germination rate every day.

(2) Survival rate of transgenic Arabidopsis seedlings under salt stress

Salt tolerance test of seedlings: After sterilizing wild-type and transgenic Arabidopsis seeds, they were planted on MS plates, vernalized for 1-3 days, and cultured in a light incubator for 7 days. After further culturing for 2 weeks, water with 300 mM NaCl aqueous solution every 3-4 days, observe after 7-15 days, take samples, count the survival rate, and perform physiological index detection.

(3) Determination of physiological and biochemical indicators of transgenic Arabidopsis under salt stress conditions

Determination of relative moisture content:

The transgenic and wild-type Arabidopsis thaliana after NaCl stress treatment were rinsed with distilled water, and the surface moisture was blotted with absorbent paper. Take 3 individual plants for each treatment, measure their fresh weight quickly, put them into an oven at 120°C for 30 minutes, bake at 80°C for 24 hours until constant weight, and weigh their dry weight. For the dry and fresh weights measured above, the formula: relative water content (accounting for fresh weight %)=(fresh weight-dry weight)/fresh weight is used to calculate the water content.

Relative Conductivity Analysis:

Weigh 0.2g of the stress-treated Arabidopsis leaves into a 10ml EP tube, add 5ml of deionized water, soak naturally for 1h, then measure and record the conductivity J1, then put it in a boiling water bath and boil for 10min, cool to room temperature and measure the conductivity J2 and record.

Calculation formula of relative conductivity: relative conductivity=(J1-J0)/(J2-J0)×100%

(Note: J0 represents the conductivity value of deionized water).

2 Experimental results

2.1 Construction of plant expression vectors

2.1.1 Construction process of plant expression vector pH7WG2D-LcAP2/ERF107

The construction process of the plant expression vector pH7WG2D-LcAP2/ERF107 is shown in FIG. 6 .

2.1.2 PCR identification of recombinant plasmids

On the transformed LB (Spe+) plate of the recombinant plasmid, pick 10 white single colonies respectively, carry out colony PCR identification, the result has 1 positive clone, extract the plasmid of the positive clone, use the upstream and downstream primers of the gene LcAP2/ERF107 ( Table 1) and 35S upstream primer (35S-F: 5'-GACGCACAATCCCACTATCC-3') and the downstream primer of the target gene (LcAP2/ERF107-R) for PCR identification, the results are shown in Figure 7, and the target band was amplified , indicating that the plant expression vector was constructed correctly.

2.2 Identification of Agrobacterium containing expression vector

The recombinant plasmid pH7GW2D-LcAP2/ERF107 was transformed into Agrobacterium GV3101, and the transformed Agrobacterium was used as a template, and the untransformed Agrobacterium was used as a negative control, and the plasmid was used as a positive control, and PCR identification of the bacterial solution was carried out. The results can be seen in Figure 8, 10 single colonies are all positive clones, indicating that the recombinant plasmid has been successfully transferred into Agrobacterium.

2.3 Identification of transgenic LcAP2/ERF107 Arabidopsis positive seedlings

2.3.1 PCR detection at the DNA level

Hygromycin-resistant seedlings were selected, their leaves were cut, and their DNA was extracted by CTAB method. Arabidopsis genomic DNA was used as a template, 35S-F and LcAP2/ERF107-R were used as primers, and PCR technology was used to detect hygromycin. The resistant seedlings were tested at the DNA level. The results are shown in Fig. 9. A total of 22 positive seedlings were detected, which indicated that the gene LcAP2/ERF107 had been integrated into the Arabidopsis genome.

2.3.2 PCR detection of RNA level

The leaves of 3 positive Arabidopsis plants identified at the DNA level were randomly selected as materials, the total RNA was extracted, and the cDNA obtained by reverse transcription was used as a template, and the positive seedlings were further detected by RT-PCR. The results are shown in Fig. 10, and all three strains were positive, which indicated that the gene LcAP2/ERF107 could perform normal transcription and expression in Arabidopsis thaliana to exert normal regulatory functions.

2.4 Germination rate experiment of transgenic Arabidopsis seeds under salt stress

Spread the wild-type and transgenic LcAP2/ERF107 Arabidopsis seeds on MS plates containing 0, 100, 150 and 200 mM NaCl respectively, and culture them for 10 days. In the medium, the germination rate was 80%, which was significantly higher than the germination rate of the wild type 45%, and in the MS medium of 200mM NaCl, the result was also very significant, the germination rate of the transgenic seeds was 50%, while the germination rate of the wild type The rate dropped sharply to 5%, indicating that the transgenic LcAP2/ERF107 Arabidopsis seeds significantly improved the resistance to high salt.

2.5 Survival rate experiment of transgenic Arabidopsis seedlings under salt stress

Three-week-old Arabidopsis seedlings were irrigated thoroughly with 300mM NaCl, and then watered with salt water every other week for a total of 3 times, with a period of 21 days. During this process, the phenotype was often observed and the survival rate was counted. It can be seen from Fig. 12 that the phenotypes of wild-type and transgenic LcAP2/ERF107 Arabidopsis were significantly different when high-salt stress was treated for 21 days. 95% of wild-type Arabidopsis had dry leaves and plants died, while 95% of transgenic Arabidopsis Mustard leaves were still green and the plant survival rate was 95%. It can be seen that under continuous high-salt stress, the transgenic Arabidopsis has a very significant salt-tolerant phenotype, indicating that the transcription factor LcAP2/ERF107 significantly improves the salt-tolerance of transgenic plants. Therefore, the transcription factor LcAP2/ERF107 is an Excellent regulator of salt tolerance in the /ERF family.

2.6 Determination of physiological and biochemical indicators of transgenic Arabidopsis under salt stress conditions

Under adversity stress, a series of complex physiological and biochemical changes will occur in plants to adapt to or resist the damage caused by the external environment. Therefore, this experiment measured the effects of salt-treated, untreated wild-type and transgenic LcAP2/ERF107 Arabidopsis. The physiological indicators of stress resistance, relative water content and electrical conductivity, can be seen from Figure 13A. Under normal conditions, the relative water content of the transgenic LcAP2/ERF107 Arabidopsis and the wild type has no significant difference. Under high-salt stress, the wild type The relative water content decreased to 70%, while the relative water content of the transgene remained at 85%, which was significantly higher than that of the wild type, which was also consistent with the phenotypic results; it can be seen from Figure 13B that under normal conditions, the transgenic The electrical conductivity of LcAP2/ERF107 and wild type is about 20%, under high salt stress, the electrical conductivity of wild type rises to 90%, and the electrical conductivity of transgenic is 45%, which is significantly lower than that of wild type. It shows that transgenic LcAP2/ERF107 Arabidopsis improves salt tolerance by increasing relative water content and reducing cell membrane damage.

Claims (10)

1. The AP2/ERF transcription factor encoding gene isolated from Lotus corniculatus L. is characterized in that its polynucleotide is shown in (a) or (b): (a), the polynucleotide shown in SEQ ID NO.1; or (b) A polynucleotide encoding the amino acid shown in SEQ ID NO.2. 2. The AP2/ERF transcription factor encoded by the coding gene of claim 1, characterized in that its amino acid sequence is shown in SEQ ID NO.2. 3. The structural domain of the AP2/ERF transcription factor that combines with the cis-acting element to initiate the expression of the disease resistance defense gene or the stress response gene, characterized in that its amino acid sequence is shown in SEQ ID NO.3. 4. The recombinant expression vector containing the coding gene of claim 1. 5. The recombinant expression vector according to claim 4, characterized in that: the recombinant expression vector is a recombinant plant expression vector. 6. The application of the coding gene according to claim 1 in improving the resistance of plants to adversity stress. 7. according to the described application of claim 6, it is characterized in that, comprise the following steps: (1) construct the recombinant plant expression vector that contains the described coding gene of claim 1; (2) transform the recombinant plant expression vector constructed into In plants or plant cells; (3) Breeding and screening to obtain transgenic plants with improved resistance to adversity stress. 8. A method for cultivating a new transgenic plant variety resistant to adversity stress, characterized in that it comprises the following steps: (1) constructing a recombinant plant expression vector containing the coding gene of claim 1; (2) constructing the recombinant plant expression vector The plant expression vector is transformed into plants or plant cells; (3) Breeding and screening to obtain new transgenic plant varieties with improved resistance to adversity stress. 9. The application of the AP2/ERF transcription factor according to claim 2 or the structural domain according to claim 3 in improving the resistance of plants to adversity stress. 10. The application according to claim 6, 7 or 9, characterized in that said adversity stress comprises: high-salt stress, drought stress or low temperature stress.