CN109232725B - Soybean C2H2 type single zinc finger protein transcription factor and encoding gene and application - Google Patents

Abstract

The invention relates to a soybean C2H2 type single zinc finger protein transcription factor, an encoding gene and application, and belongs to the field of plant genetic engineering. The amino acid sequence of soybean C2H2 type single zinc finger protein transcription factor is SEQ ID No. 2, and is used in breeding cold-tolerant soybean varieties. C2H2-type double zinc finger protein has been confirmed in domestic and foreign studies to play an important role in adversity stress, especially cold tolerance, while single zinc finger protein plays an irreplaceable role in plant growth and development. The present invention successfully excavated a single zinc finger protein. In addition to verifying that the zinc finger protein gene has the function of regulating growth and development, the focus is on whether it has the function of cold tolerance. Finally, through a series of experiments, a gene that is involved in the regulation of growth and development and has unique cold tolerance is found. The bifunctional gene also lays a solid foundation for further in-depth functional exploration in the later stage, and plays an important role in improving and cultivating new multifunctional germplasm resources.

Description

Soybean C2H2 type single zinc finger protein transcription factor, coding gene and application

Technical Field

The invention relates to the field of plant genetic engineering, in particular to a soybean C2H2 type single zinc finger protein transcription factor and application of a coding gene thereof in cold resistance of transgenic plants.

Background

Soybeans are important economic crops, are generally planted worldwide, are not only main sources of human proteins and lipids, but also important livestock feed and industrial raw materials, and have great medical value. Biotic and abiotic stresses, however, have a very adverse effect on the growth and development of soybeans, wherein low temperature severely affects the yield, quality and economic benefits of soybeans, causing severe economic losses. Therefore, it is important to elucidate the response mechanism of soybean to stress, identify important genes related to cold tolerance and use them to improve the salt tolerance of soybean.

Plant transcription factors can be continuously synthesized and transmit and amplify signals under the stimulation of stress, and the expression of a plurality of downstream genes is regulated, so that the physiological and biochemical changes of plants are caused to adapt to the external adverse environment, and the plant transcription factors gradually become the research hotspots of people in recent years. Zinc finger protein is a transcription factor with finger-like domains, originally discovered by Milier in 1983 in the transcription factor TF IIIA of Xenopus oocytes. Zinc finger proteins are classified into 9 major classes according to their different functions and sequences: c4, C6, C8, CCCH, C2HC, C2HC5, C2H2, C3HC4 and C4HC3, which are the most studied zinc finger proteins of C2H 2. The C2H2 zinc finger protein is composed of about 30 amino acids, and the conserved sequence of the zinc finger structure is as follows: C-X2-4-C-X3-P-X5-L-X2-H-X3-H (where C is cysteine, X is any amino acid, H is histidine, P is phenylalanine, and L is leucine). These sequences form a compact beta-alpha structure in which the alpha helix and two antiparallel beta chains sandwich the zinc ion, which forms a tetrahedral structure with two histidines at the C-terminal part of the alpha helix and two cysteines at the ends of the beta chains. The plant zinc finger protein not only participates in regulation and control in the processes of morphogenesis, pollen, embryo development and the like, but also plays an important role in abiotic stress.

So far, zinc finger proteins are divided into two types, one type is a protein with a single zinc finger structure, and mainly participates in the regulation of the characteristics of plant reproductive organs, seed development and growth and the like, while double zinc finger proteins mainly participate in various abiotic stresses, and a plurality of zinc finger protein genes of C2H2 type related to the abiotic stresses of plants are discovered at home and abroad. Wherein, low temperature can induce some zinc finger protein genes in the plant body to play a role so as to enable the zinc finger protein genes to resist low temperature stress.

Sakamoto et al found that the expression of the tobacco STZ gene was enhanced under cold induction; mukhopadhyay et al, which transfers the rice zinc finger protein gene OSISAP1 into tobacco, shows the enhancement of low-temperature resistance; sugano et al found that the ZPT2-2 and ZPT2-3 genes transferred from petunia were susceptible to cold-induced expression under cold stress. The soybean zinc finger protein has few studies at home and abroad, and only SCOF-1 is a clear analysis, and is a nuclear-localized zinc finger protein gene induced by low temperature and ABA, and the constitutive over-expression of the zinc finger protein gene induces the expression of COR gene, so that the cold resistance of transgenic tobacco and Arabidopsis thaliana which are not subjected to cold resistance screening is enhanced.

Disclosure of Invention

The invention provides a soybean C2H2 type single zinc finger protein transcription factor, a coding gene and application.

The C2H2 type single zinc finger protein transcription factor provided by the invention is named GmC2H24-like, and is a protein with an amino acid residue sequence of SEQ ID N0.2 in a sequence table.

The amino acid residue sequence of SEQ ID N0.2 in the sequence table is a protein consisting of 251 amino acid residues, and is a C2H2 transcription factor of a typical single zinc finger protein structure in soybean.

The coding gene of GmC2H24-like is a DNA sequence of SEQ ID N0.1 in a sequence table, the DNA sequence of SEQ ID N0.1 in the sequence table is composed of 756 bases, and the reading frame of the gene is from 1 st to 756 th bases of a 5' end.

Any vector capable of guiding the expression of the exogenous gene in the plant is utilized to introduce the coding gene of GmC2H24-like provided by the invention into a plant cell, so that a transgenic cell line and a transgenic plant with improved cold resistance can be obtained.

The gene of the invention has important significance for cultivating soybean varieties with improved cold resistance of plants, and when the gene of the invention is used for constructing plant expression vectors, any enhanced promoter or inducible promoter can be added before transcription initiation nucleotide. In order to facilitate the identification and selection of transgenic plant cells or plants, vectors to be used may be processed, for example, by adding a plant selection marker (GUS gene, GFP gene, etc.) or an antibiotic marker having resistance (ampicillin, kanamycin, etc.). The expression vector carrying the GmC2H24-like 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 invention relates to application of a soybean C2H2 type single zinc finger protein transcription factor in cultivating a cold-resistant soybean variety.

The soybean C2H2 type single zinc finger protein transcription factor also participates in the regulation of growth and development.

The soybean C2H2 type single zinc finger protein transcription factor can be integrated into an arabidopsis genome and can be stably inherited in arabidopsis; the conductivity of the transgenic arabidopsis is obviously lower than that of wild arabidopsis, and the cell membrane of the leaf of the transgenic arabidopsis is slightly damaged by the adverse environment; the active oxygen scavenging capacity of the transgenic arabidopsis plant antioxidant system is higher than that of a non-transgenic plant; after the leaves of the transgenic arabidopsis are treated for 24 hours under the cold stress of 4 ℃, the leaves of the transgenic arabidopsis are less damaged, and the content of soluble sugar in the leaves of the transgenic arabidopsis is higher than that of proline in the leaves of wild arabidopsis and transgenic arabidopsis, so that the content of proline in the leaves of the transgenic arabidopsis is obviously increased.

The invention has the beneficial effects that: the C2H2 type double zinc finger protein is proved to play an important role in adversity stress, especially cold resistance in domestic and foreign research, while the single zinc finger protein plays an irreplaceable role in plant growth and development, the invention successfully excavates a single zinc finger protein gene, mainly researches whether the single zinc finger protein gene has the function of cold resistance in addition to verifying that the single zinc finger protein gene has the function of regulating and controlling growth and development, and finally discovers a dual-function gene which participates in the regulation and control of growth and development and has unique cold resistance through a series of experiments. In addition, the single zinc finger protein gene is cloned in Jilin 32 soybean, an expression map of the Jilin 32 soybean at each period is constructed in the laboratory, the expression map shows that the gene is expressed in each tissue, the expression level is higher in roots, stems and embryos, and a solid foundation is laid for the later-stage deep function research. Has important effect on improving and cultivating novel multifunctional germplasm resources.

Drawings

FIG. 1 is a photograph showing the result of PCR amplification of GmC2H24-like of the present invention;

in the figure, M: DL2000 DNA Marker 1, 2: the PCR result of the target gene;

FIG. 2 is an electrophoretogram showing the expression result of GmC2H24-like of the present invention in Escherichia coli Rosetta (DE 3);

in the figure, M: low molecular weight protein Marker, 1: pET28a empty elution control, 2: pET28a-GmC2H24-like uninduced cell control, 3: pET28a-GmC2H24-like induced bacteria;

FIG. 3A is a graph of the expression of the gene of interest GmC2H24-like, a positive control pGAL4 and the empty plasmid pGBKT7 in Saccharomyces cerevisiae AH109 on a triple-deleted medium (SD/-Trp/-His/-Ade 10mM 3-AT);

FIG. 3B is a beta-galactosidase activity assay following expression of the gene of interest GmC2H24-like, a positive control pGAL4 and the empty plasmid pGBKT7 in Yeast AH 109;

FIG. 3C is a diagram showing the gene positions of three parts on a triple-deletion yeast medium and a beta-galactosidase activity assay plate;

FIG. 4 is a graph showing the tissue-specific expression level of the GmC2H24-like gene of the present invention.

FIG. 5A is a diagram showing the result of PCR identification of the GmC2H24-like of the present invention in a part of T2 generations of transgenic Arabidopsis thaliana;

in the figure, M: DL2000 Marker; +: a positive control; -: negative control; 1-5: part of transgenic Arabidopsis lines;

FIG. 5B: is a bar test strip identification result diagram of the GmC2H24-like in partial T2 generation transgenic arabidopsis thaliana;

in the figure, WT: wild type Arabidopsis thaliana; 1.2, 4: part of transgenic Arabidopsis lines;

FIG. 6A is a diagram of gene expression levels of wild type and transgenic T3 generation Arabidopsis thaliana analyzed by real-time fluorescent quantitative PCR at room temperature of 22 ℃;

wherein, each group of roots, stems and leaves is designed with two types of controls and transgenes, the left side bar graph is the expression condition of wild arabidopsis thaliana, and the right side bar graph is the expression condition of transgenes arabidopsis thaliana;

FIG. 6B is a graph of gene expression levels of wild type and transgenic T3 generation Arabidopsis thaliana analyzed by real-time fluorescent quantitative PCR after 4 ℃ low temperature treatment;

wherein, each group of roots, stems and leaves is designed with two types of controls and transgenes, the left side bar graph is the expression condition of wild arabidopsis thaliana, and the right side bar graph is the expression condition of transgenes arabidopsis thaliana.

Detailed Description

Example 1 cloning of the Soybean GmC2H24-like Gene

Total RNA from 32 roots, stems, leaves and seeds of the soybean variety Jilin was extracted separately using an RNAlso Reagent (from TaKaRa), and the integrity of the RNA was checked by 1% agarose electrophoresis. The cDNA was synthesized according to the instructions for Reverse Transcriptase M-MLV (RNase H-). Primers, 5'-TAGCTTGAAAACTTAGCACAG-3' and 5'-TAACAGCACATACAGAGCAAA-3', were designed according to GmC2H24-like (NM 001255238). PCR amplification was performed according to the following reaction system and conditions: a25. mu.l system containing 10 XPCR Buffer 2.5. mu.l, 2.5mM dNTP mix 2. mu.l, 1. mu.l each of 10. mu.M primer 1 and primer 2, cDNA 1. mu.l, Taq DNA Polymerase (purchased from TIAN GEN, 2.5 u/. mu.l) 0.5. mu.l, and deionized water 17. mu.l. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 8 min; 30 cycles of 94 ℃ for 30sec, 58 ℃ for 30sec, and 72 ℃ for 60 sec; post extension at 72 ℃ for 8 min. The GmC2H24-like obtained by PCR amplification consists of 756 base pairs, the reading frame is from 1 st to 756 th base of the 5' end, and the protein consisting of 251 amino acid residues is encoded, wherein the protein comprises a C2H2 single zinc finger protein conserved domain.

Example 2 expression of Soybean GmC2H24-like Gene in prokaryote

Primers containing BamHI and SalI restriction sites are designed at the 5 'end and the 3' end of the gene respectively, and PCR amplification is carried out by taking a plasmid of pMD18-T-GmC2H24-like which is identified to be correct through sequencing as a template, wherein pMD18-T is purchased from TaKaRa company. The primers are as follows:

GmC2H24-like-YH-F：5’-CGCGGATCCATGAAACAACACTTTG-3’

GmC2H24-like-YH-R：5’-ACGCGTCGACTCAAAGTTTCAATGC-3’

the amplified product was purified by agarose gel DNA purification recovery kit (purchased from VitreeL), digested simultaneously with BamHI and SalI, recovered and purified, and ligated with pET28a (+) (purchased from Novagen) vector, digested simultaneously with BamHI and SalI. The ligation product was transformed into Escherichia coli (E.coli) DH 5. alpha. and, after validation, transformed into the host bacterium Rosetta (DE3) (from Novagen) for expression.

Prokaryotic expression of recombinant protein: the positive and empty vector strains were inoculated into 5mL (50. mu.g/mL kanamycin and 15. mu.g/mL chloramphenicol) of LB medium, respectively, and cultured overnight with shaking at 37 ℃. Inoculating 1% of the seed solution into 10mL LB medium, and performing shaking culture at 37 deg.C to OD₆₀₀About 0.6, IPTG (100mM) was added to the final concentration of 0.6mM, 0.8mM, 1.0mM, 1.2mM, respectively, and the mixture was subjected to shaking culture at 37 ℃ for 1 hour, 2 hours, 4 hours, and 6 hours, respectively, 1.5mL of the induced bacteria having different IPTG concentrations were taken, and the cells were collected by centrifugation at 5000rpm for 5min at 4 ℃.

SDS-PAGE analysis of expression products: the procedure was carried out according to the conventional method of SDS-PAGE electrophoresis.

Optimization of induction time and IPTG induction concentration shows that the optimal induction time of pET28a (+) -GmC2H24-like in Rosetta (DE3) is 4 hours, the optimal induction concentration of IPTG is 1.0mM, and GmC2H24-like protein (about 27.64KDa) has high expression under the condition.

Example 3 expression of the Soybean GmC2H24-like Gene in Yeast

The yeast expression vector was constructed in the same manner as in example 2. GmC2H24-like was subcloned into pGBKT7 (purchased from clontech) vector, and when correctly identified, transformed into yeast AH109 (purchased from clontech) for expression.

Yeast competence preparation and transformation method refers to Matchmaker^TMOne-Hybrid Library Construction&Screening Kit (clontech) instructions.

After the recombinant plasmid pGBKT7-GmC2H24-like is transferred into yeast AH109, the recombinant plasmid can grow on a defective culture medium SD/-Trp/-His/-Ade containing 10mM 3-AT, which indicates that GmMYB12B2 has transcription activation activity and is a transcription activator.

Example 4 tissue-specific expression of the Soybean GmC2H24-like Gene

Total RNAs of roots, stems, leaves, flowers and mature seeds of soybean variety Jilin 32 were extracted and reverse-transcribed into cDNAs in the same manner as in example 1. The expression of the GmC2H24-like gene in different tissues of soybean was detected by Real Time PCR according to SYBR O R Premix Ex Taq^TMII (from TaKaRa) instructions, using the Agilent Technologies Stratagene Mx305P amplification instrument. Wherein, soybean beta-tubulin is taken as an internal reference gene, and the primer sequence is tubulin-F: 5'-GGAAGGCTTTCTTGCATTGGTA-3'; tubulin-R: 5'-AGTGGCATCCTGGTACTGCA-3'. The primer of the GmC2H24-like gene is GmC2H 24-like-qpcr-F: 5'-AAAGAACAATAGCGAAGAG-3', respectively; GmC2H 24-like-qpcr-R: 5'-GAGGGAACCTGATGGTAG-3' are provided.

The PCR reaction system is as follows:

the data analysis uses KEYPLOT software, and the result is shown in figure 4, and the GmC2H24-like gene has the highest expression quantity in the root of soybean, which is 2.7 times of the expression quantity of the leaf; as soybeans gradually mature, the reproductive growth stage is entered. The expression of the GmC2H24-like gene is also increased. The expression pattern of the GmC2H24-like gene in soybean tissues indicates that the GmC2H24-like gene may have a certain regulation effect in soybean seeds.

Example 5 expression of the Soybean GmC2H24-like Gene in Arabidopsis thaliana

Arabidopsis thaliana Col. mu. mbia-0 ecotype was transformed with pCB 35S:GFP-zinc finger protein 4-like by the floral dip method. The specific method comprises the following steps:

5.1 cultivation of Arabidopsis thaliana

l) placing the seeds of arabidopsis into a 1.5 centrifuge tube, adding 1mL of sodium hypochlorite to disinfect for 3min, washing with sterile water for 5-7 times, soaking with sterile water, keeping out of the sun, storing at 4 ℃, and vernalizing for 4-6 days;

2) the vernalized seeds are sown on an MS culture medium, sealed by a sealing film and cultured in an artificial climate box with humidity of 80 percent, light intensity of 150 mu mol s < -1 > m < -2 >, light period of 16h and darkness of 8 h;

3) culturing under illumination for about 2 weeks, transplanting 4 true leaves of plantlets, pouring PNS nutrient solution, covering with preservative film, culturing under the above conditions, taking off the film after 3-5 days, and normally watering;

4) cutting off the main axis (the height of the plant is about 10cm) above the rosette leaves during flowering to promote the bolting of the secondary axis, performing a transformation experiment after 7-9 days, wherein the height of the plant is about 15cm, the largest inflorescence generates siliques, and stopping pouring nutrient solution or water one day before transformation.

5.2 amplification culture and harvesting of Agrobacterium

L) Agrobacterium containing the pCB 35S-GFP-zinc finger protein 4-like binary expression vector was cultured at 28 ℃ and 200rpm for 24 hours in 5mL of YEB liquid medium containing 50mg/L rifampicin and 100mg/L kanamycin;

2) carrying out amplification culture according to the volume ratio of 5mL to 500mL, and carrying out overnight culture under the same conditions until OD is approximately equal to 1.0-1.2;

3) centrifuging at 5000rpm for 15min, discarding supernatant, resuspending thallus in transformation medium (1/2 macroelement, 1 × microelement, 1 × ferric salt, 1 × organic, 5% sucrose, pH 5.7-5.8), and OD after resuspension is about 0.8-0.85; before flower invasion, adding a few drops of surface adsorbent (Tween) into the transformation medium to improve the infection rate;

5.3 vacuum infiltration method of transgenosis

1) Cutting off siliques of arabidopsis;

2) covering the flower pot of Arabidopsis thaliana with gauze, and fastening with rubber band. Reversely buckling the flowerpot on an infiltration tank filled with 400mL of bacterial suspension, immersing the floral foam of the plant in bacterial liquid, carrying out flower invasion for 10min, absorbing excess bacterial liquid by using absorbent paper after conversion, covering the bacterial liquid with a preservative film, and carrying out dark culture for 16-24 h;

3) after membrane uncovering, pouring enough water, and normally culturing; after one month, the water is gradually reduced, and when most siliques are mature, the watering is stopped;

4) individuals received seeds, which were considered as T0 generations.

5.4 selection of transgenic Arabidopsis seeds

1) Preparing an MS solid culture medium, sterilizing at high pressure, cooling, adding an appropriate amount of basta to a final concentration of 4mg/L, and subpackaging in a sterilized culture dish;

2) placing Arabidopsis thaliana T0 generation seeds into a 1.5mL centrifuge tube, adding 1mL sodium hypochlorite for disinfection for 5min, washing with sterile water for multiple times, keeping out of the sun at 4 ℃, vernalizing for 2-3 days;

3) transferring the seeds into an MS culture dish containing basta, adding a small amount of 0.3% agarose solution, gently shaking to uniformly distribute the seeds on a culture medium, and culturing in an artificial climate box;

4) transplanting when 4 true leaves grow, pouring PNS nutrient solution, harvesting seeds according to single plants after the seedlings are mature, and obtaining T1 generation seeds. And continuously screening the seeds of the T1 generation on an MS culture dish containing basta, and harvesting the seeds after maturation to obtain the seeds of the T2 generation until the seeds of the T3 generation are obtained for subsequent experiments.

5.5 molecular detection and bar test strip detection of transgenic arabidopsis of T2 generation

1) And (3) molecular detection: a part of transgenic Arabidopsis thaliana leaf total DNA of T2 generation was extracted according to the Universal Genomic DNA extraction-on Kit Ver.3.0 (purchased from TaKaRa) instruction. After 50-fold dilution, 1. mu.l of the solution was used as a template, and PCR amplification was carried out conventionally using primer 1 and primer 2, respectively, and a primer for the bar gene (bar-F: 5'-AAACCCACGTCATGCCAGCTC-3'; bar-R: 5'-CGACAAGCACGGTCAACTTC-3'). Wherein untransformed wild Arabidopsis thaliana is used as a negative control, and the plasmid pCB35S is used as a positive control, wherein GFP-zinc finger protein 4-like is used as a positive control.

2) bar test strip detection: taking fresh leaves of T2 generation transgenic arabidopsis plants, grinding into homogenate, slightly centrifuging, taking the homogenate of the leaves into a new sterile 1.5ml centrifuge tube, taking out a test strip (Libertylink strip), inserting the test strip into the homogenate for detection, observing after about 5 minutes, and displaying two strips as transgenic plants and only one indicator strip as wild non-transgenic plants. FIGS. 5A and 5B show the PCR identification result and the test strip detection result of GmC2H24-like in partial T2 generation transgenic Arabidopsis thaliana.

The results show that the GmC2H24-like gene has been successfully integrated into the Arabidopsis genome and can be stably inherited in Arabidopsis.

Example 6 determination of physiological and biochemical indices of wild type and transgenic GmC2H24-like Gene T3 generation Arabidopsis thaliana after Low-temperature treatment

The leaves of wild type and T3 generation Arabidopsis thaliana plants transformed with GmC2H24-like gene after 22 degrees and 4 degrees treatment are respectively taken, and each leaf is taken for three times of repetition. POD, soluble sugar, conductivity, proline content and malondialdehyde content were measured separately. Three transgenic lines and wild type arabidopsis thaliana were randomly selected as controls in the experiment, and the results were analyzed for the significance of the differences, and are shown in tables 1-5.

Table 1: determination result of relative conductivity of T3 generation arabidopsis thaliana transferred with GmC2H24-like gene part and wild type arabidopsis thaliana after low-temperature treatment

Relative conductivity

The results show that: under the condition of normal temperature, the relative conductivities of the leaves of the transgenic arabidopsis thaliana and the leaves of the wild arabidopsis thaliana are not obviously different, after the leaves are treated for 24 hours under the cold stress at 4 ℃, the conductivity of the transgenic arabidopsis thaliana is obviously lower than that of the wild arabidopsis thaliana, and the cell membranes of the leaves of the transgenic arabidopsis thaliana are slightly damaged by the.

Table 2: the Peroxidase (POD) activity of T3 generation Arabidopsis thaliana and wild type Arabidopsis thaliana transformed with GmC2H24-like gene part was measured.

Peroxidase POD Activity

The results show that: under the condition of normal temperature, the POD activity of the transgenic arabidopsis leaves and that of the wild arabidopsis leaves have no significant difference, and after the leaves are treated for 24 hours under cold stress at 4 ℃, the POD activity of the transgenic arabidopsis leaves is obviously higher than that of the wild arabidopsis leaves, so that the significance level is reached, and the active oxygen scavenging capacity of an antioxidant system of a transgenic arabidopsis plant is higher than that of a non-transgenic plant.

Table 3: and (3) determining the content of Malondialdehyde (MDA) after the T3 generation arabidopsis thaliana and the wild arabidopsis thaliana are subjected to low-temperature treatment in the GmC2H24-like gene transferred part.

MDA malondialdehyde content

The results show that: under the condition of normal temperature, the malondialdehyde content of the transgenic arabidopsis leaves and the malondialdehyde content of the wild type arabidopsis leaves have no significant difference, after the leaves are treated for 24 hours under cold stress at 4 ℃, the malondialdehyde content of the transgenic arabidopsis leaves is obviously lower than that of the wild type arabidopsis leaves, the significance level is reached, and the transgenic arabidopsis plants are less damaged than the leaves of the non-transgenic plants.

Table 4: and (3) determining the content of soluble sugar after the T3 generation arabidopsis thaliana and wild arabidopsis thaliana are subjected to low-temperature treatment in the GmC2H24-like gene transferred part.

Soluble sugar content

The results show that: under the condition of normal temperature, the soluble sugar content of the leaves of the transgenic arabidopsis thaliana and the wild arabidopsis thaliana has no obvious difference, and after the leaves are treated for 24 hours under the cold stress of 4 ℃, the soluble sugar content of the leaves of the transgenic arabidopsis thaliana is higher than that of the wild arabidopsis thaliana.

Table 5: and (3) measuring the proline content of transgenic T3 generation arabidopsis thaliana and wild type arabidopsis thaliana of the GmC2H24-like gene part after low-temperature treatment.

Proline content

The results show that: under the condition of normal temperature, the proline content of the transgenic arabidopsis leaves and that of the wild arabidopsis leaves have no obvious difference, and the proline content of the transgenic arabidopsis leaves is obviously increased after the leaves are treated for 24 hours under the cold stress of 4 ℃.

Example 7, wild type and transgenic T3 generation arabidopsis thaliana gene expression before and after 4 degree treatment.

Total RNAs of wild type and T3 generation Arabidopsis thaliana plants transformed with GmC2H24-like genes before and after 4 degrees of treatment are respectively extracted and are reversely transcribed into cDNA, and the method is the same as the example 1. Primers were then designed for real-time fluorescent quantitation of pcr as in example 4. data analysis results show that transgenic plants were much improved over wild type both before and after 22 and 4 degrees treatment, and the results are shown in fig. 6A and 6B.

Sequence listing

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<120> soybean C2H2 type single zinc finger protein transcription factor, coding gene and application

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ctcaccaact cttccaatgt gacaaatcca atagagcttc atctccattc agatgctatc 180

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Tyr Ala Gly Pro Cys Ser Asp Asn Leu Thr Asn Ser Ser Asn Val Thr

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Leu Gly Gly His Gln Asn Ala His Lys Arg Glu Arg Thr Ile Ala Lys

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Arg Ala Met Arg Met Gly Ile Phe Ser Glu Arg Tyr Ala Ser Leu Ala

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Ser Ser Leu His His Gly Phe Ser Pro Thr Met Arg Pro Pro Glu Met

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Lys Ser Ser Ala Arg Phe Glu Gln Gly Tyr Val Gly Pro Pro Ile Phe

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Ser Ser Asn Leu Ser Phe Val Gly Ser Ser Leu Pro Pro Val Asp Leu

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Glu Asn Ser Thr Pro Glu Leu Thr Leu Lys Leu

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Claims (2)

1. The application of the soybean C2H2 type single zinc finger protein transcription factor in cultivating cold-resistant soybean variety is characterized in that: the amino acid sequence of the soybean C2H2 type single zinc finger protein transcription factor is SEQ ID NO. 2.

2. Use of nucleotides encoding the soybean C2H 2-type single zinc finger protein transcription factor of claim 1 for breeding cold tolerant soybean varieties, wherein: the sequence of the nucleotide is SEQ ID NO. 1.

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