CN115786366A - Eggplant cold-resistant regulatory gene, eggplant cold-resistant regulatory protein and application of eggplant cold-resistant regulatory protein - Google Patents

Abstract

The invention discloses an eggplant cold-resistant regulation gene, protein and application thereof. The base sequence of the eggplant cold-resistant regulation gene is shown in SEQ ID NO.1; the amino acid sequence of the eggplant cold-resistant SmERF1 protein encoded by the SmERF1 gene is shown in SEQ ID NO.1. Shown in ID NO.2. The present invention clones the eggplant cold-resistance regulatory gene SmERF1 and its encoded protein for the first time. The expression of the SmERF1 gene is induced by low temperature, high temperature and drought, and the expression effect induced by low temperature is the most significant. The eggplant cold-resistance regulating SmERF1 gene provided by the present invention can improve the poor growth of plants in low temperature, endow plants with good growth characteristics in low temperature environment, and is also suitable for the improvement of genetic traits in eggplant cold resistance research, plant molecular marker assisted breeding and genetic engineering fields. Molecular breeding for cold resistance in eggplant lays the foundation.

Description

A kind of eggplant cold resistance regulation gene, protein and application thereof

technical field

The invention belongs to the field of biotechnology, and in particular relates to an eggplant cold resistance regulating gene, protein and application thereof.

Background technique

Under natural conditions, the growth and development of plants are inevitably affected by adversity stresses such as low temperature, high temperature, drought and salt stress. In severe cases, plants may even die, which seriously affects agricultural production and the ecological environment. Therefore, improving the stress resistance of crops has important research significance for the development of agricultural production. ERF transcription factors play an important role in plant response to low temperature, drought, high temperature and salt stress mainly by regulating the transcription of stress response genes. ERF genes have been cloned from many plants, such as soybean, potato, tomato and Arabidopsis. The overexpression of tomato TERF2/LeERF2 genes in tobacco and tomato regulates the induced expression of cold resistance-related genes and enhances the cold resistance of transgenic plants. The overexpression of tomato SlERF5 gene enhanced the drought resistance and salt resistance of tomato. Overexpression of Arabidopsis AtERF53 gene can enhance the heat resistance of transgenic plants by regulating ABA signal transduction pathway and proline synthesis. The overexpression of PtaERF194 gene in poplar improves the resistance of plants to drought stress by increasing water use efficiency and limiting water loss. From the above results, it can be seen that different ERF transcription factors have different functions in plants, and it is of great research significance to discover new transcription factors and clarify their biological functions. As an important vegetable crop, eggplant is seriously harmed by adversity stress compared with other Solanaceae vegetables. However, there are few research reports on eggplant ERF genes in response to adversity stress.

Contents of the invention

Purpose of the invention: Aiming at the deficiencies in the prior art, the present invention provides an eggplant cold-resistant regulation gene, which can improve plants such as eggplant that grow poorly in low temperature, and endow it with good growth characteristics in a low temperature environment.

The invention also provides the cold-resistant protein encoded by the cold-resistant gene and its application.

Technical solution: In order to achieve the above objectives, the present invention provides an eggplant cold-resistance regulatory gene SmERF1, the base sequence of which is shown in SEQ ID NO.1.

The eggplant cold resistance regulatory protein SmERF1 encoded by the eggplant cold resistance regulatory gene SmERF1 of the present invention has an amino acid sequence as shown in SEQ ID NO.2.

Wherein, the length of the coding region of the eggplant cold resistance regulation gene is 849bp, named as SmERF1. The gene does not contain an intron structure, and the gene encodes an eggplant cold resistance regulatory protein, and the amino acid sequence contains an AP2 structural domain in total.

The primer pair for amplifying the SmERF1 gene according to the present invention, the base sequences of the primer pair are shown in SEQ ID NO.3 and SEQ ID NO.4.

The application of the eggplant cold resistance regulation gene SmERF1 or the eggplant cold resistance regulation protein SmERF1 in improving plant quality.

Wherein, the improvement of plant quality refers to the application of improving poor growth of plants in low temperature and endowing plants with good growth in low temperature environment.

The eggplant cold-resistance regulation gene SmERF1 or the eggplant cold-resistance regulation protein SmERF1 of the present invention is used in cultivating plant stress-resistant varieties.

Wherein, the application is an application in cultivating cold-resistant plant varieties.

Wherein, the plant is eggplant or Arabidopsis.

The method for analyzing the expression level of the SmERF1 gene of the present invention when eggplant responds to low temperature, high temperature and drought, the method comprises the following steps:

S1. Obtain the leaf tissues of the plants treated with low temperature, high temperature and drought stress for different periods of time, and extract their total RNA;

S2. Using the total RNA as a template, obtain cDNA by reverse transcription;

S3, using the first strand of cDNA as a template, respectively amplifying the SmERF1 gene and the specific primers of the internal reference gene APRT (JX448345) for fluorescence quantitative analysis to obtain the expression levels of the SmERF1 gene under different stresses in eggplant; The base sequence of the specific primer of the SmERF1 gene is shown in SEQ ID NO.5 and SEQ ID NO.6; the base sequence of the specific primer of the amplification internal reference gene APRT (JX448345) is as SEQ ID NO.7, Shown in SEQ ID NO.8.

In order to make full use of the advantages of molecular breeding, from the perspective of molecular biology methods, the present invention uses seamless cloning, sequencing and splicing to obtain the sequence of the coding region of Solanum solanum SmERF1 gene; and then conducts bioinformatics analysis on it , Detect the expression pattern of the SmERF1 gene in response to different abiotic stresses to further verify its function, reveal the role of the gene in cold resistance, and determine the genes related to cold resistance. The present invention clones the ERF transcription factor gene SmERF1 from eggplant and conducts bioinformatics and analysis of its response to low temperature, high temperature and drought stress, in order to further understand the function and mechanism of SmERF1 transcription factor in response to abiotic stress in eggplant Provide references.

The method for obtaining the SmERF1 gene of the present invention comprises the following steps: 1) extracting the total RNA of the leaf tissue of March Solanum, and reverse-transcribing it into cDNA; 2) using the reference genome sequence as a template to design primers (cP1F and cP1R); 3) using the steps 1) The reverse-transcribed cDNA is used as a template, and the primers designed in step 2) are used for cloning; 4) The PCR product obtained in step 3) is recovered, sequenced and sequenced.

The expression of SmERF1 gene was induced by low temperature, high temperature and drought, and the expression induced by low temperature was the most significant. The eggplant low temperature resistance gene SmERF1 provided by the invention is suitable for the improvement of genetic traits in the field of eggplant cold resistance research, plant molecular marker assisted breeding and genetic engineering, and lays the foundation for eggplant cold resistance molecular breeding.

The gene of the invention is of great significance for cultivating stress-resistant varieties of plants, especially for improving crop yield and quality of cold-resistant eggplant varieties.

Specifically, the eggplant cold resistance regulation gene SmERF1 of the present invention is obtained and expressed according to the following steps:

1) Extract the total RNA of March eggplant samples (the leaves of March eggplant plants that have grown for about 60 days);

2) Using the cDNA extracted from RNA reverse-transcribed in step 1) as a template, carry out PCR amplification to obtain the sequence of the coding region of the Solanum solanum SmERF1 gene, and the nucleotide sequences of the specific primers used in the PCR amplification are as follows :

cP1F:

GGACTCTAGAGGATCCATGGATTCATTCTCACTAGATTTGATAAGACAACA (SEQ ID NO. 3)

cP1: R:

GACCACCCGGGGATCCTTATGAAACCAAAAGTTGTGAGTATCCAAAACTTGG (SEQ ID NO. 4)

3) Recovering the PCR product obtained in step 2), ligation transformation, colony PCR screening, sequencing and sequence analysis.

4) SmERF1 gene expression level analysis during abiotic stress in eggplant, the method comprises the following steps:

S1. Obtain a plant sample and extract its total RNA;

S2. Using the total RNA as a template, obtain cDNA by reverse transcription;

S3. Using the first strand of cDNA as a template, amplify the SmERF1 gene and the specific primers of the internal reference gene APRT (JX448345) to perform fluorescence quantitative analysis to obtain the expression of the SmERF1 gene under different abiotic stresses; The base sequence of the specific primer for amplifying the SmERF1 gene is shown in SEQ ID NO.5 and SEQ ID NO.6; the base sequence of the amplified internal reference gene APRT (JX448345) is shown in SEQ ID NO.7 and SEQ ID Shown in NO.8.

Beneficial effects: compared with the prior art, the present invention has advantages and effects:

(1) The present invention has screened a brand-new eggplant cold-resistance regulatory gene SmERF1, which is the sequence of the eggplant ERF gene reported for the first time. Experiments have shown that the gene responds to the process of low temperature stress. The cloning and research of this gene are of great importance for understanding the cold-resistant mechanism of eggplant. Certain reference value;

(2) The eggplant cold-resistance regulating SmERF1 gene provided by the present invention can improve the poor growth of plants in low temperature and endow plants with good growth characteristics in low temperature environment. Gene selection for cold-resistant transgenic breeding of eggplant and other plants was established, and a preliminary foundation for cold-resistant molecular breeding of eggplant was laid.

Description of drawings

Fig. 1 is eggplant SmERF1 gene clone electrophoresis figure of the present invention;

Fig. 2 is the multiple sequence alignment analysis of the eggplant SmERF1 transcription factor of the present invention;

Fig. 3 is the phylogenetic analysis of eggplant SmERF1 transcription factor of the present invention;

Fig. 4 is the comparative analysis of eggplant SmERF1 transcription factor of the present invention and other species ERF transcription factors conserved motifs and structural domains;

Fig. 5 is the prediction result of the eggplant SmERF1 protein secondary structure of the present invention;

Fig. 6 is the prediction result of the tertiary structure of eggplant SmERF1 protein of the present invention;

Fig. 7 is the interaction network analysis of CbERF5 protein (SmERF1);

Fig. 8 is the expression pattern of eggplant SmERF1 gene of the present invention under different abiotic stresses;

Fig. 9 is the process of inducing callus after eggplant leaf infection in March;

Fig. 10 is the root growth situation of eggplant seedlings under low temperature stress (4 DEG C);

Fig. 11 is the root length situation of eggplant seedlings under 4 ℃ stress;

Figure 12 is the result of the comparison of the CDS sequence of the SmERF1 gene by NCBI Blastn;

Figure 13 is the comparison result of SmERF1 transcription factors.

Detailed ways

The present invention will be further described below in conjunction with drawings and embodiments.

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental consumables used in the examples were all purchased from conventional biochemical reagent companies unless otherwise specified.

Example 1

Cloning of Eggplant SmERF1 Gene

1. Acquisition of plant material

The plant material used in this experiment is Sanyueqiu, an excellent germplasm resource of eggplant. The experimental materials were provided by Huaiyin Institute of Technology. The second and third true leaves from the top to the bottom of the March nightshade were collected for RNA extraction, using the three-month nightshade grown in a greenhouse pot for about 60 days and having the same size.

2. Extraction of RNA

According to the instructions of the Plant RNA Extraction Kit (HiPure HP Plant RNA Kit), RNA was extracted from the treated March nightshade leaves, and the integrity of the RNA was identified by electrophoresis, and then the purity and concentration of the RNA were measured on a spectrophotometer. cDNA was obtained by reverse transcription using PrimeScript ^TM RT reagent Kit with gDNA Eraser, which was used for subsequent gene cloning.

3. Cloning of SmERF1 gene

According to the eggplant reference genome sequence, Snapgene was used to design the primers for seamless cloning of SmERF1 gene.

cP1F:

GGACTCTAGAGGATCCATGGATTCATTCTCACTAGATTTGATAAGACAACA (SEQ ID NO. 3)

cP1R:

GACCACCCGGGGATCCTTATGAAACCAAAAGTTGTGAGTATCCAAAACTTGG (SEQ ID NO. 4)

Using the synthesized eggplant cDNA as a template, perform PCR, amplify to the expected length, recover and connect to the pBI121 vector, transform Escherichia coli DH5α, colony PCR screen positive clones, extract plasmids and send them to Sangon Bioengineering Co., Ltd. (Nanjing) Sequencing was performed, and the sequencing results showed that the open reading frame sequence of the SmERF1 gene was 849bp ( FIG. 1 ). See SEQ ID NO.1 for the detailed sequence. According to the CDS open reading frame sequence, the amino acid sequence of SmERF1 was deduced, with a total of 282 amino acid residues, a molecular weight of 31933.74Da, and a theoretical isoelectric point (pI) of 6.18. See the sequence shown in SEQ ID NO.2 for the detailed sequence.

Example 2

Sequence and Phylogenetic Analysis of Eggplant SmERF1 Transcription Factor

The present invention compares the CDS sequence of the SmERF1 gene by NCBI Blastn, and finds that the homology between the SmERF1 gene and the potato ERF gene is the highest (the highest identity can only reach 85.16%) (Figure 12); after the comparison by Blastp, the SmERF1 gene is found The encoded amino acid sequence has the highest homology (57.05% identity) with tobacco ERF transcription factor (Fig. 13). Using Jalview software, the amino acid sequence of eggplant SmERF1 was compared with 20 known AP2/ERF transcription factor sequences, and it was found that all AP2/ERF family transcription factors contained AP2 domains, and the domains had typical WLG element, indicating that the protein belongs to the AP2/ERF transcription factor family; further analysis found that the 14th and 19th amino acids in the domain are alanine and aspartic acid, so the SmERF1 protein belongs to the AP2/ERF family ERF subfamily (Figure 2). Using the large likelihood method of MEGA X software to construct a phylogenetic evolutionary tree, the results showed that the SmERF1 protein was most closely related to NsERF4 in Nicotiana lindenae, and farther related to the ERF transcription factors of tomato and Arabidopsis (Fig. 3). The reason for the analysis is that due to the large number of ERF transcription factor family members, the sequence differences between SmERF1 and tomato and Arabidopsis ERF transcription factors are relatively large, resulting in low homology among the three species.

Example 3

Comparative analysis of conserved motifs and structural domains of ERF transcription factors in eggplant and other species

The conserved motifs of ERF proteins in eggplant and other species were predicted and analyzed. Most ERF proteins contain 5 or 4 motifs, among which Motif 1 and Motif 2 are distributed in each ERF transcription factor, which are conserved regions of ERF proteins , and the eggplant SmERF1 protein contains five motifs (Motif 1, Motif 2, Motif 4, Motif 5 and Motif 6); then use the Batch-CD database in NCBI to predict and analyze the conserved domain of the transcription factor, eggplant SmERF protein and The ERF transcription factors of other species are the same, containing only one AP2 conserved domain; at the same time, the protein SmERF1 of the present invention is similar to the AP2 domain of NsERF4 transcription factor of Nicotiana linshii, and has only one AP2 structural domain, which further indicates SmERF1 belongs to the ERF subfamily of AP2/ERF family proteins (Fig. 4).

Example 4

Prediction of Secondary and Tertiary Structures of Eggplant SmERF1 Transcription Factor

The secondary structure of the new eggplant SmERF1 protein of the present invention is mainly composed of four structural elements: random coil, α-helix, extended chain and β-turn, and the proportions of the four structural elements are 53.90%, 27.30%, and 13.48%, respectively. % and 5.32% (Figure 5). Using the online analysis software SWISS-MODEL, the transcription factor AtERF1 (PDB: 2gcc.1.A) was used as a template to predict the tertiary structure of the transcription factor SmERF1. It was found that the SmERF1 transcription factor is mainly composed of random coils, α-helices and extended chains composition, and its random coils account for a large proportion (Figure 6). The prediction of the tertiary structure is consistent with the prediction of the secondary structure.

Example 5

The new eggplant SmERF1 protein of the present invention is compared with the CbERF5 transcription factor of Capsicum baccatum belonging to the same Solanaceae crop, and the sequence identity is 78.1%. The protein interaction relationship is used to construct an interaction network. CbERF5 transcription factor (Ethylene-responsive transcription factor 5) is mainly associated with two mitogen-activated protein kinases (Mitogen-activated protein kinase), one SNF1-related protein kinase regulatory subunit β-1 (SNF1-related protein kinase regulatory subunit beta- 1), 2 SNF1-related protein kinase regulatory subunit beta-2 (SNF1-related protein kinase regulatory subunit beta-2), 1 transcription factor KAN2 (Putative transcription factor KAN2), 2 guanine nucleotide binding protein subunits Base class β protein (Guanine nucleotide-binding protein subunit beta-like protein), a non-specific serine/threonine protein kinase (Non-specific serine/threonine protein kinase) and an uncharacterized protein (Uncharacterized protein) interacted as (Figure 7). It shows that eggplant SmERF1 transcription factor plays an important role in the process of plant growth and development and signal transduction.

Example 6

Prediction and analysis of gene structure and promoter cis-acting elements of eggplant SmERF1

The promoter sequence of SmERF1 gene contains 27 CAAT-boxes and 72 TATA-boxes which are the core elements of eukaryotes. Further analysis found that there are many functional elements in the promoter region, such as methyl jasmonate-induced cis-acting elements CGTCA-motif and TGACG-motif, anaerobic-induced cis-acting element ARE, abscisic acid-induced cis-acting element ABRE, growth The hormone response elements TGA-element and TGA-box and the circadian rhythm regulatory element circadian, etc. In addition, there are elements related to light response, such as CAG-motif, GA-motif, G-Box, GT1-motif, LAMP-element and TCCC-motif, etc. (Table 1). Surface SmERF1 transcription factor may be involved in plant growth and development and hormone signal transduction process.

Table 1 Some cis-acting elements in the promoter region of eggplant SmERF1 gene

The invention clones and obtains the eggplant ERF transcription factor gene SmERF1, the length of the coding region is 849bp, and it can encode 282 amino acids, and it is an unstable non-secreted hydrophilic protein located in the nucleus. SmERF1 contains a conserved AP2 domain, and the 14th and 19th amino acids in the domain are alanine and aspartic acid, and it has the highest homology with tobacco belonging to Solanaceae. The results of conserved motifs showed that Motif 1 and Motif 2 contained in ERF transcription factors are conserved regions of AP2 domain. SmERF1 mainly interacts with the corresponding kinases and transcription factors in the process of plant growth and signal transduction, and the promoter of SmERF1 gene contains cis-acting elements related to plant growth and development and hormone signal transduction process. It shows that SmERF1 gene is mainly involved in the hormone signal transduction process of plant growth and development and abiotic stress.

Example 7

Expression pattern analysis of eggplant SmERF1 gene in response to different abiotic stresses

1. Processing of plant material

Using March nightshade grown in pots in the greenhouse (light 16h, 27°C/darkness 8h, 19°C) for about 60 days and with the same size as materials, they were subjected to low temperature (4°C), High temperature (40°C) and drought stress treatments (20% PEG), and leaf tissues without any stress treatment were used as the control group. After treatment for different time (low temperature and high temperature treatment 1, 3, 6, 12 and 24h, drought treatment 3, 6 and 12h). Collect the second and third true leaves from the top of March eggplant, wrap the samples in aluminum platinum paper, put them into liquid nitrogen immediately, and then transfer them to a -80°C ultra-low temperature freezer for storage until use.

2. Extraction of RNA

Refer to the instructions of the Plant RNA Extraction Kit (HiPure HP Plant RNA Kit) to extract the processed RNA from the leaves of March nightshade, and use ordinary agarose gel electrophoresis (gel concentration 1.2%; 1 × TAE electrophoresis buffer; 150v, 15min) to detect the integrity sex. The maximum rRNA brightness in the electrophoresis strip should be 1.5-2.0 times the brightness of the second rRNA, otherwise it indicates the degradation of the rRNA sample. For RNA with good purity, A260/A280 and A260/A230 are about 2.0. The OD value was measured with a spectrophotometer and the RNA content was calculated.

3. Acquisition of cDNA

Using 1 μg of total RNA as a template, reverse transcription was performed using PrimeScript ^™ RT reagent Kit with gDNA Eraser to obtain cDNA for future use.

4. Design specific primers for real-time fluorescent quantitative PCR analysis of gene expression under different stresses. According to the obtained eggplant SmERF1 gene sequence, the primer design software Snapgene was used to design specific primers for the quantitative analysis of SmERF1 gene in Real-time PCR,

ERF1-F: 5'-AGTGCCACCTTTGTCACCAT-3' (SEQ ID NO.5)

ERF1-R: 5'-TCATGGATTGTAACAGCAAGATGA-3' (SEQ ID NO.6)

The internal reference gene is APRT, and its primers are

APRT-F: 5'-GAGATGCATGTAGGTGCTGTGCAA-3' (SEQ ID NO.7)

APRT-R: 5'-GGCCCTTCAATTCTGGCAACTCAA-3' (SEQ ID NO.8)

5. Real-time fluorescence quantitative analysis of the target gene in the sample to be tested. Using the first strand of the synthesized cDNA as a template, the target gene and the internal reference gene were amplified with specific primers for fluorescence quantitative analysis. The qRT-PCR test was performed on a Roche Cobaz480 real-time fluorescent quantitative PCR instrument, and the reaction system was 20 μL.

The reaction adopts a three-step method, denaturation at 95°C for 10 minutes, followed by 40 cycles: 95°C for 10s; 58°C for 10s; 72°C for 45s.

6. Use 2 ^-ΔΔCt method for relative quantitative analysis. The results showed that during the treatment of low temperature stress (4℃), the relative expression of SmERF1 gene was induced by low temperature, and the overall expression first increased and then decreased. Among them, the SmERF1 gene reached the most significant level (about 34.86 times that of the control) when treated at 4°C for 3 hours, and its relative expression was the lowest (about 9.97 times that of the control) when it was treated for 24 hours, reaching a significant level of difference from the control. Under the stress of 40°C, the expression level of SmERF1 gene showed a trend of first decreasing, then increasing and then decreasing, and the expression level was significantly induced at 6h, which was about 1.26 times that of the control, while the expression of SmERF1 gene was significantly inhibited at 1h, 12h and 24h (respectively about 0.79 times, 0.53 times and 0.26 times of the control). In the drought treatment, the SmERF1 gene showed a trend of increasing first and then decreasing, and its expression was induced at 3h (about 1.82 times of the control) and inhibited at 6h and 12h (about 0.30 times and 0.07 times of the control, respectively), but None of them reached a significant level of difference (Figure 8). It indicated that SmERF1 gene plays an important regulatory role in the response of eggplant to low temperature stress.

Example 8

Functional verification of eggplant SmERF1 gene

1. Construction of SmERF1 gene expression vector

(1) In this experiment, TaKaRa conventional restriction enzymes (TaKaRa, Japan) were used to carry out enzyme digestion reaction experiments on the transformation vector pBI121. The specific reaction system is as follows:

10×K Buffer 5μL pBI121 32 μL BamHI 1μL ddH₂O 12μL Total volume 50μL

After the solutions in the system were mixed, they were centrifuged instantaneously, incubated in a water bath at 37°C for 16 hours, and then the enzyme digestion reaction was completed. The enzyme-cut bands were observed by agarose gel electrophoresis, and then the carrier fragments were cut and recovered for subsequent carrier ligation reactions. .

(2) Referring to the operation manual of Trelief ^TM SoSoo Cloning Kit Ver.2 (Qingke Bio, China), the expression vector pBI121 recovered after the enzyme digestion reaction was connected to the product of the target gene fragment. The system is as follows:

components Dosage Target gene fragment 1.5μL pBI121(BamH I) 1.0 μL 2×SoSoo Mix Ver.2 5.0 μL ddH₂O 2.5μL Total volume 10.0 μL

The positive control system is as follows

components Dosage Control Vector (5ng/μL) 2.0 μL Control Template (10ng/μL) 3.0 μL 2×SoSoo Mix Ver.2 5.0 μL Total volume 10.0 μL

Mix the solutions in the system in a microtube and react at 50°C for 15 minutes. E.coli DH5α was transformed, and positive clones were identified by PCR and sequenced, confirming that the overexpression vector containing the gene SmERF1 was successfully constructed.

2. Transformation of Agrobacterium

a. Take frozen Agrobacterium GV3101 competent cells and place them on ice to slowly thaw.

b. Add 7 μL of extracted and purified recombinant plasmid DNA to the above 100 μL of competent cells that have not completely melted, mix well, and place on ice for 45 minutes.

c. Seal the centrifuge tube, freeze it in liquid nitrogen for 3 minutes, and quickly transfer it to a 37°C water bath for 5 minutes;

d. Repeat operation c and place on ice for 5 minutes.

e. Add 900 μL of fresh YEB liquid medium to the centrifuge tube, shake and culture at 28° C. and 225 rpm for 4 hours.

f. Centrifuge at 4000rpm for 5min at 4°C, carefully absorb the supernatant with a pipette gun, leave about 150μl, and gently pipette with the tip of the pipette to resuspend the bacteria.

g. Spread the bacterial solution evenly on the YEB solid plate containing Kan (50mg/L), Str (50mg/L), Rif (80mg/L) and Cb (100mg/L), and cultivate it in a thermostat at 28°C for 2 sky.

h. Pick the single clone on the plate, add appropriate amount of YEB liquid medium, culture at 28°C, 220rpm for 48h, detect positive clones by PCR, and then use 1.0% agarose gel electrophoresis to detect the correctness of the PCR amplified fragment, and obtain Agrobacterium containing the SmERF1 gene expression vector.

3. Eggplant SmERF1 gene overexpression

The eggplant material is March eggplant. Culture the Agrobacterium GV3101 containing the SmERF1 gene expression vector with an OD value of 0.4-0.6, centrifuge at 4000rpm for 5min, discard the supernatant, resuspend the bacterial cell pellet with MS solution, infect the cotyledon of March Eggplant for 18min, and shake it continuously, sterile Absorb excess bacterial solution on the filter paper, put it back into the callus induction medium for dark culture (Figure 9), until the callus grows. The calli were cut into small pieces and transferred to Kan selection medium for differentiation culture. Subculture until the buds are differentiated, and when the buds grow to 2-3 cm, transfer them to the rooting medium to induce rooting. After about a month, a complete root system grows and is transplanted into the greenhouse to obtain resistant plants. Plants were harvested as T1 generation seeds. Select the T1 generation seeds with plump grains and the same size, first soak them in 75% alcohol for 90 seconds, perform preliminary surface disinfection, rinse with sterile water for 1-2 times; then use 20% sodium hypochlorite for surface disinfection for 42 minutes, rinse with sterile water for 5-7 Second-rate. Inoculate it in the seed germination medium (MS + 3% sucrose) by the pendulum method, and cultivate it under the light condition of 25°C. After the seeds germinate, transfer to the stress treatment at 4±0.5°C for 4 weeks, and observe its growth and measure its root length. It was found that the transgenic plants (O2 and O5) had long root systems with prominent lateral roots, while the control plants (WT) had shorter root systems with indistinct lateral roots (Fig. 10 and Fig. 11). It indicated that the overexpression of SmERF1 gene could promote the root growth and development of eggplant seedlings under low temperature stress.

Claims (9)

1. An eggplant cold resistance regulatory gene SmERF1, characterized in that its base sequence is as shown in SEQ ID NO.1. 2. An eggplant cold resistance regulatory protein SmERF1 encoded by the eggplant cold resistance regulatory gene SmERF1 according to claim 1, wherein the amino acid sequence of the protein is as shown in SEQ ID NO.2. 3. a pair of primers for amplifying the eggplant cold-resistant regulatory gene SmERF1 according to claim 1, wherein the base sequence of the primer pair is as shown in SEQ ID NO.3, SEQ ID NO.4 . 4. An application of the eggplant cold resistance regulating gene SmERF1 according to claim 1 or the eggplant cold resistance regulating protein SmERF1 according to claim 2 in improving plant quality. 5. The application according to claim 4, characterized in that the improvement of plant quality is the application of improving poor growth of plants in low temperature and giving plants good growth in low temperature environment. 6. The application of the eggplant cold resistance regulatory gene SmERF1 according to claim 1 or the eggplant cold resistance regulatory protein SmERF1 according to claim 2 in cultivating plant stress-resistant varieties. 7. The application according to claim 6, characterized in that, the application is an application in cultivating cold-resistant plant varieties. 8. The application according to claims 4-7, characterized in that the plant is preferably eggplant or Arabidopsis. 9. an expression analysis method of eggplant cold resistance regulation gene SmERF1 as claimed in claim 1 when eggplant responds to low temperature, high temperature and drought, is characterized in that, described method comprises the steps: S1. Obtain the leaf tissues of the plants treated with low temperature, high temperature and drought stress for different periods of time, and extract their total RNA; S2. Using the total RNA as a template, obtain cDNA by reverse transcription; S3. Using the first strand of the cDNA as a template, amplify the specific primers of the SmERF1 gene and the internal reference gene APRT (JX448345) respectively for fluorescence quantitative analysis to obtain the expression levels of the SmERF1 gene under different stresses in eggplant; The base sequence of the specific primer for the amplification of the SmERF1 gene is shown in SEQ ID NO.5 and SEQ ID NO.6; the base sequence of the specific primer for the amplification internal reference gene APRT (JX448345) is as in SEQ ID NO .7, as shown in SEQ ID NO.8.

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