CN117660485A - Application of Arabidopsis ERF012 gene in regulation of seed germination - Google Patents
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Abstract
The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an arabidopsis ERF012 gene in regulating seed germination. The invention converts wild arabidopsis thaliana by a genetic engineering technology and an agrobacterium inflorescence infection method to obtain the overexpression and mutant strain of the ERF012 gene, and the function of regulating seed germination of the ERF012 gene is verified and disclosed for the first time. According to the invention, the CRISPR vector for inhibiting the ERF012 gene is introduced into a target plant to obtain a mutant transgenic plant, and the tolerance to ABA is higher than that of a wild type plant line in a seed germination stage, so that the suppression of the ERF012 gene expression can improve the tolerance to ABA in the plant seed germination stage, and gene resources can be provided for regulating and controlling the plant ABA tolerance molecular breeding.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an arabidopsis ERF012 gene in regulating seed germination.
Background
Seed germination is one of the most environmentally sensitive stages in the plant life cycle and is critical to agricultural production. How to control the germination time under the adversity to enable the seeds to sprout regularly and timely is the basis of high and stable yield of crops, and is also an important research direction and leading edge problem in the field of plant adversity biology.
The germination process of the seeds is regulated and controlled by various factors such as internal and external factors, wherein Abscisic acid (ABA) is a main signal substance for regulating the germination of the seeds. ABA anabolism, signaling and related transcriptional regulatory networks play an important role in the seed germination process.
Ethylene response factors (ethylene response factor, ERF) belong to the AP2/ERF transcription factor superfamily and are widely distributed in plants. Many members of ERF have been found to be involved in regulating plant growth (flower and fruit development, etc.) and various environmental stress responses (high salt, drought, etc.), but it is unclear whether members of ERF are involved in regulating seed germination and related mechanisms. The early-stage research of the team topic group of the invention finds that the ERF012 gene (ERF family DREBA-5 subfamily member) is expressed in the overground part and root of Arabidopsis thaliana, and further researches find that the ERF012 gene participates in regulating and controlling various biological processes such as root growth and development, hormone and stress response in the seedling stage, but whether the ERF012 gene participates in regulating and controlling seed germination process is not clear. Based on the method, the team of the invention continuously explores the influence of the ERF012 gene on the seed germination process, which has important significance for timely and orderly germination of seeds and ensuring high and stable yield of crops.
Disclosure of Invention
The invention aims at providing an application of an arabidopsis ERF012 gene in regulating seed germination.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the application of an Arabidopsis ERF012 gene in regulating seed germination, the application of the Arabidopsis ERF012 gene in promoting or inhibiting seed germination in the presence of abscisic acid, and the nucleotide sequence of the Arabidopsis ERF012 gene is shown as SEQ ID NO. 1.
Further, the amino acid sequence of the ERF012 gene encoding protein is shown in SEQ ID NO. 2.
Further, in the presence of abscisic acid, inhibiting the expression of the Arabidopsis ERF012 gene promotes seed germination.
Further, the overexpression of the Arabidopsis ERF012 gene inhibits seed germination in the presence of abscisic acid.
The second purpose of the invention is to provide a cultivation method for improving the germination capacity of plant seeds.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a cultivation method for improving germination capacity of plant seeds comprises inhibiting ERF012 gene expression in target plants in the presence of abscisic acid.
Further, the plant of interest is arabidopsis thaliana.
Compared with the prior art, the invention has the beneficial effects that:
the invention converts wild arabidopsis thaliana by a genetic engineering technology and an agrobacterium inflorescence infection method to obtain an over-expression strain for promoting the ERF012 gene and a mutant strain for inhibiting the ERF012 gene, and the function of regulating seed germination of the ERF012 gene is verified and disclosed for the first time. According to the invention, the ERF012 gene is introduced into a target plant to obtain the over-expressed transgenic plant, and the tolerance of the over-expressed transgenic plant to ABA is lower than that of a wild type plant line in a seed germination stage, which indicates that the over-expressed ERF012 inhibits seed germination. According to the invention, the CRISPR vector for inhibiting ERF012 gene expression is introduced into a target plant through experiments, so that a mutant transgenic plant is obtained, and the tolerance to ABA in the seed germination stage is higher than that of a wild type plant, which shows that inhibiting ERF012 gene expression can improve the tolerance to ABA in the plant seed germination stage, and the invention can provide gene resources for regulating and controlling plant ABA tolerance molecular breeding.
Drawings
FIG. 1 is a graph showing the results of GUS staining of Pro ERF012, GUS homozygous seeds germination treated to different stages;
FIG. 2 shows ERF012 gene expression after ABA treatment;
FIG. 3 is a seed germination and seedling morphogenic phenotype of CRISPR mutant lines, overexpressing lines after ABA treatment;
FIG. 4 shows seed germination rate for CRISPR mutant lines and overexpressing lines after ABA treatment;
FIG. 5 is a plot of seed green leaf rate for CRISPR mutant lines and overexpressing lines after ABA treatment;
FIG. 6 shows the fresh weight of the aerial parts of CRISPR mutant lines and overexpressing lines after ABA treatment.
Detailed Description
The technical scheme of the invention is further explained below by combining specific embodiments and drawings.
In the following examples, materials and methods of preparation are conventional in the art, unless otherwise indicated.
The following examples are presented in brief to illustrate some of the biological materials, reagents, etc:
primer synthesis and sequencing were performed by the company Pengham and Zhengzhou qingke;
t4 DNA ligase was purchased from Sieimer, inc. Thermo Scientific, USA TM T4 DNA ligase (5U/. Mu.L), EL0011;
homologous recombinase One Step Seamless Cloning kit is purchased from beijing ideley biology company, 2×onesite Cloning, CV1901;
total RNA extraction kit was purchased from Eastep, promega, shanghai TM Super Total RNA Extraction Kit，LS1040；
Reverse transcription kit was purchased from Nor Renzan IncIIQ RT SuperMix for qPCR(+g DNA wiper)，R223-01；
Fluorescent quantification kits were purchased from beijing ideley biology company 2x SYBR Green qPCR Mix (PC 3302);
MS media was purchased from beijing cool pacing technologies limited;
GUS staining kit was purchased from Biosharp company (BL 622A-03);
the nucleotide/amino acid sequences of the Arabidopsis ERF012 genes in the examples are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively:
SEQ ID NO.1
ATGGTGAAACAAGAACGCAAGATCCAAACCAGCAGCACAAAAAAGGAAATGCCTTTGTCATCATCACCATCTTCTTCTTCTTCTTCATCTTCTTCCTCGTCTTCGTCTTCGTGTAAGAACAAGAACAAGAAGAGTAAGATTAAGAAGTACAAAGGAGTGAGGATGAGAAGTTGGGGATCATGGGTCTCTGAGATTAGGGCACCAAATCAAAAGACAAGGATTTGGTTAGGTTCTTACTCAACAGCTGAAGCAGCTGCTAGAGCTTACGATGTTGCACTCTTATGTCTCAAAGGCCCTCAAGCCAATCTCAACTTCCCTACTTCTTCTTCTTCTCATCATCTTCTTGATAATCTCTTAGATGAAAATACCCTTTTGTCCCCCAAATCCATCCAAAGAGTAGCTGCTCAAGCTGCCAACTCATTTAACCATTTTGCCCCTACTTCATCAGCCGTCTCGTCACCGTCCGATCATGATCATCACCATGATGATGGGATGCAATCTTTGATGGGATCTTTTGTGGACAATCATGTGTCTTTGATGGATTCAACATCTTCATGGTATGATGATCATAATGGGATGTTCTTGTTTGATAATGGAGCTCCATTCAATTACTCTCCTCAACTAAACTCGACGACGATGCTCGATGAATACTTCTACGAAGATGCTGACATTCCGCTTTGGAGTTTCAATTAA
SEQ ID NO.2
MVKQERKIQTSSTKKEMPLSSSPSSSSSSSSSSSSSSCKNKNKKSKIKKYKGVRMRSWGSWVSEIRAPNQKTRIWLGSYSTAEAAARAYDVALLCLKGPQANLNFPTSSSSHHLLDNLLDENTLLSPKSIQRVAAQAANSFNHFAPTSSAVSSPSDHDHHHDDGMQSLMGSFVDNHVSLMDSTSSWYDDHNGMFLFDNGAPFNYSPQLNSTTMLDEYFYEDADIPLWSFN
example 1
Example 1 the expression pattern of the ERF012 gene was investigated as follows:
(1) The expression of GUS gene is started by ERF012 self promoter, and Pro ERF012 is obtained, wherein the specific process is as follows:
(1) construction of an expression vector for GUS Gene driven by the promoter of ERF012 gene (Pro ERF012: GUS): primers were designed based on the promoter nucleotide sequence of the ERF012 gene, and the primer sequences were as follows:
pERF012-F：GGTCGACGGATCCCCGTCATTTGTTGGGAACTGGTACGGA(SEQ ID NO:3)；
pERF012-R：AGGGACTGACCACCCTGTGTACGTACAGGCTTTGTAGAGTG(SEQ ID NO:4)；
(2) performing PCR amplification by taking Arabidopsis wild Col-0 DNA as a template, purifying and recovering a PCR product (restriction enzyme Sma I is used for carrying out enzyme digestion on a Dx2181 vector, and 2x one step cloning Mix homologous recombinase is used for connecting a target fragment and the Dx2181 vector) to obtain Pro ERF 012;
(3) transforming the Pro ERF012 GUS recombinant vector into the competence of agrobacterium GV3101, and transforming the recombinant vector into the arabidopsis wild type Col-0 by an inflorescence infection method to obtain Pro ERF012 GUS expression strain;
(4) seeds of the infected Pro ERF 012/GUS-expressing strain were used as T 0 Instead, T is 0 Culturing the seed of the generation on a culture medium containing hygromycin (50 mg/L), selecting long-root seed, culturing in a nutrient soil culture medium, and collecting the seed of T as single plant 1 Instead, T is 1 Culturing the generation seeds on a culture medium containing hygromycin, and selecting offspring long roots: the separation ratio of the non-long roots is 3:1, the seeds received by the single plant are T in a nutrient soil culture medium of the long-root seedlings in the plant line 2 Instead, T is 2 The generation seeds are cultivated on a culture medium containing hygromycin, the strain with long roots as the offspring is selected and planted in a nutrient soil culture medium, and the received seeds are the homozygous transgenic seeds of Pro ERF012 and GUS expression strain, and are named Pro ERF012 and GUS.
(2) Pro ERF012, GUS homozygous seeds are germinated to an imbibition stage (soaking 3 d), a germination stage and seedlings which germinate for 1-3 days old, and are dyed by using a GUS kit, and the result is shown in figure 1.
FIG. 1 is a graph of GUS staining results of germination of Pro ERF012, GUS homozygous seeds to different stages, on a scale of 100. Mu.m. As is clear from FIG. 1, the GUS gene driven by Pro ERF012 was expressed in low amounts in the submerged imbibition seed and the embryo at the germination stage, but the expression amount was significantly increased after germination, which suggests that ERF012 participates in regulating seed germination and seedling growth process after germination.
Example 2
Example 2 the effect of ABA treatment on the transcriptional level of ERF012 genes was investigated as follows:
(1) Sowing the sterilized and cold-treated wild Col-0 seeds in a 1/2MS culture medium for normal growth for 7 days, transferring the seeds into the 1/2MS culture medium containing 100 mu M ABA, and sampling the seeds in the culture for 0h, 1h, 6h and 12h respectively;
(2) Total RNA of each sample was extracted using a total RNA extraction kit, synthesis of cDNA was performed using a reverse transcription kit, and the expression level of ERF012 in each sample was detected using a fluorescent quantitative kit (qRT-PCR detection), wherein the primer sequences for the qRT-PCR detection were as follows:
Actin qRT-PCR-F：ACAGTGTCTGGATCGGTGGTTC(SEQ ID NO:5)；
Actin qRT-PCR-R：TGCCTCATCATACTCAGCCTTG(SEQ ID NO:6)；
ERF012 qRT-PCR-F：ACTCATTTAACCATTTTGCCCC(SEQ ID NO:7)；
ERF012 qRT-PCR-R: GATCCCATCAAAGATTGCATCC (SEQ ID NO: 8); the results are shown in FIG. 2.
FIG. 2 shows ERF012 gene expression in each sample after ABA treatment. As can be seen from fig. 2, the expression level of ERF012 gene was reduced under ABA treatment compared to the control group, which suggests that ABA treatment suppresses expression of ERF012 gene.
Example 3
Example 3 construction of CRISPR mutant strains inhibiting expression of ERF012 genes, and ERF012 gene overexpression strains, the specific procedure is as follows:
(1) Constructing a recombinant vector for inhibiting ERF012 gene expression to obtain a CRISPR mutant for inhibiting ERF012 gene expression:
(1) screening two targets containing enzyme cutting sites (3 bp away from PAM/NGG) by using a website http:// www.genome.arizona.edu/crispr/CRISPRsearch, wherein target sequences are GTTGAGGAGAGTAATTGAA respectively; TTACACGAAGACGAAGACG four primers were designed using two targets, the primer sequences were as follows:
ERF012-DT1-BsF：ATATATGGTCTCGATTGGTTGAGGAGAGTAATTGAAGTT(SEQ ID NO:9)；
ERF012-DT1-F0：TGGTTGAGGAGAGTAATTGAAGTTTTAGAGCTAGAAATAGC(SEQ ID NO:10)；
ERF012-DT2-R0：AACCGTCTTCGTCTTCGTGTAACAATCTCTTAGTCGACTCTAC(SEQ ID NO:11)；
ERF012-DT2-BsR：AACCGTCTTCGTCTTCGTGTAACAATCTCTTAGTC(SEQ ID NO:12)；
(2) four-primer PCR amplification was performed using the intermediate vector pCBC-DT1T2 (containing guid RNA Scaffold +U6-26 terminator+U6-29 promoter element) as a template. Wherein, the concentration of the primer ERF012-DT1-BsF/ERF012-DT2-BsR is 10 mu M, and the concentration of the primer ERF012-DT1-F0/ERF012-DT2-R0 is 0.5 mu M;
(3) purifying and recycling the PCR product to obtain a recombinant vector for inhibiting ERF012 gene expression; configuring an enzyme digestion connection system according to table 1, and setting a reaction procedure according to table 2;
TABLE 1
TABLE 2
(4) The CRISPR recombinant vector for inhibiting the ERF012 gene expression is transformed into the competence of agrobacterium GV3101, and then is transformed into the arabidopsis wild type Col-0 by an inflorescence infection method, so as to obtain a mutant strain for inhibiting the ERF012 gene expression;
(5) seeds of the CRISPR mutant strain which is used for inhibiting ERF012 gene expression after being infected are taken as T 0 Instead, T emitting red light is selected by using simple green light lamp and red light filter glass 0 And (3) replacing seeds, namely planting the seeds containing red light in a nutrient soil culture medium, extracting DNA from the seeds, carrying out PCR amplification on sequences containing targets, sequencing the PCR fragments, and detecting mutation types. The seeds received by the single plant of the edited plant are T 1 Instead of T 1 Observing the seed generation, namely, red seed: the non-luminescent seed separation ratio is 3:1, sowing non-red light emitting seeds in a nutrient soil culture medium, and detecting mutation types of the seedlings. For pure and single plant-received seeds of editingThe son is T 2 The generation, the seed is the seed of the homozygous transgene mutant strain which inhibits ERF012 gene expression, named CR2 and CR7;
(6) the primer sequences for mutation type detection of the transgenic lines inhibiting ERF012 gene expression are as follows:
ERF012 edit detection-F: CATTACCATGGTGAAACAAGAACG (SEQ ID NO: 13);
ERF012 edit detection-R: CCGTCGGATTAATTGAAACTCCA (SEQ ID NO: 14);
(2) Constructing an ERF012 gene overexpression vector to obtain an ERF012 gene overexpression strain:
(1) construction of a 35S-driven ERF012 overexpression vector (35S:: ERF 012): primers were designed based on the ERF012 nucleotide sequence, and the primer sequences were as follows:
ERF012-F：TCCCCCCGGGATGGTGAAACAAGAACGCAAGATCC(SEQ ID NO:15)
ERF012-R：CCGCTCGAGTTAATTGAAACTCCAAAGCGGAATGT(SEQ ID NO:16)
(2) performing PCR amplification by taking Arabidopsis wild Col-0cDNA as a template, purifying and recovering PCR products (restriction enzymes EcoRI and XbaI are subjected to enzyme digestion, a target fragment and a pBin35SRed vector are connected by T4 DNA ligase), and obtaining an ERF012 gene overexpression recombinant vector;
(3) the over-expression recombinant vector is transformed into the competence of agrobacterium GV3101, and then transformed into the wild type Col-0 of arabidopsis thaliana by an inflorescence infection method, so as to obtain an ERF012 gene over-expression strain;
(4) taking the seeds containing ERF012 gene over-expression strain after infection as T 0 Instead, T emitting red light is selected by using simple green light lamp and red light filter glass 0 Seed generation is carried out, the seed containing red light is planted in nutrient soil culture medium, and the seed received by single plant is T 1 Instead of T 1 Observing the seed generation, namely, red seed: the non-luminescent seed separation ratio is 3:1, sowing red light seeds in the strain of the plant line in a nutrient soil culture medium, wherein the seeds received by the single plant are T 2 Instead of T 2 Observing the generation seeds by red light, selecting strains with the offspring being the red light seeds, and planting the strains in nutrient soil culture medium, wherein the received seeds areSeeds of homozygous transgenic lines overexpressed by ERF012 genes are designated OE4-6 and OE7-6, respectively;
(5) qRT-PCR detection is carried out on the homozygous transgenic line with the ERF012 gene over-expression, and the primer sequences are as follows:
Actin qRT-PCR-F：ACAGTGTCTGGATCGGTGGTTC(SEQ ID NO:5)；
Actin qRT-PCR-R：TGCCTCATCATACTCAGCCTTG(SEQ ID NO:6)；
ERF012 qRT-PCR-F：ACTCATTTAACCATTTTGCCCC(SEQ ID NO:7)；
ERF012 qRT-PCR-R：GATCCCATCAAAGATTGCATCC(SEQ ID NO:8)；
example 4
Example 4 explores the germination capacity of CRISPR mutant lines and ERF012 gene overexpression lines that inhibit the ERF012 gene under ABA treatment, the specific procedure was as follows:
seeds of full, uniform-size and same-period harvested wild-type Col-0, over-expressed strains (OE 4-6 and OE 7-6) and CRISPR mutant strains (CR 2 and CR 7) were selected, sterilized and washed, and then subjected to low-temperature treatment at 4 ℃ for 3 days, and respectively sown into a control group medium (Mock), a medium containing 0.5 mu M ABA and a medium containing 1 mu M ABA, the seed germination number was recorded from day 2, the number of cotyledons appeared was recorded from day 3, and the fresh weight of the aerial parts of seedlings of each experimental group was counted from day 11, and the results are shown in FIGS. 3 to 6.
As can be seen from FIGS. 3-5, on the control medium, there was no significant difference in germination rate and green leaf rate of Col-0 strain, over-expressed strain and mutant strain seeds, and on the medium of 0.5. Mu.M, 1. Mu.M ABA, over-expressed strain seeds had significantly lower germination rate and green leaf rate than Col-0 strain, whereas mutant strain seeds had significantly higher germination rate and green leaf rate than Col-0 strain, indicating that the mutant strain was insensitive to ABA stress.
As can be seen from FIG. 6, there was no significant difference in the fresh aerial parts of the Col-0, over-expressed and mutant strains on the control medium, whereas the fresh aerial parts of the over-expressed strain were significantly lower than the Col-0 strain and the fresh aerial parts of the mutant strain were significantly higher than the Col-0 strain on the 0.5. Mu.M ABA medium, indicating that the mutant strain is insensitive to ABA stress. It can be seen that the ERF012 gene negatively regulates seed germination under ABA stress.
In conclusion, the invention is constructed by a genetic engineering technology, and the agrobacterium inflorescence infection method is adopted to transform the wild arabidopsis thaliana, so that the over-expression strain for promoting the ERF012 gene and the mutant strain for inhibiting the ERF012 gene are obtained, and the function of regulating seed germination of the ERF012 gene is verified for the first time. According to the invention, the ERF012 gene is introduced into a target plant to obtain the over-expressed transgenic plant, and the tolerance of the over-expressed transgenic plant to ABA is lower than that of a wild type plant line in a seed germination stage, which indicates that the over-expressed ERF012 inhibits seed germination. According to the invention, the CRISPR vector for inhibiting the expression of the ERF012 gene is introduced into a target plant through experiments, so that a transgenic plant is obtained, the tolerance to ABA in the seed germination stage is higher than that of a wild type plant, and the inhibition of the expression of the ERF012 gene can improve the tolerance to ABA in the plant seed germination stage, so that the gene resource can be provided for regulating and controlling the molecular breeding of the ABA tolerance of the plant.
The foregoing is illustrative of the preferred embodiments of the present invention and is not to be construed as limiting thereof, and various modifications and variations may be made by those skilled in the art without departing from the principles of the invention. Any modification, improvement, etc. should be considered as being within the scope of the present invention.
Claims (6)
1. The application of the Arabidopsis ERF012 gene in regulating seed germination is characterized in that the application of the Arabidopsis ERF012 gene in promoting or inhibiting seed germination in the presence of abscisic acid is realized, and the nucleotide sequence of the Arabidopsis ERF012 gene is shown as SEQ ID NO. 1.
2. The application of the Arabidopsis ERF012 gene in regulating seed germination according to claim 1, wherein the amino acid sequence of the ERF012 gene encoded protein is shown in SEQ ID NO. 2.
3. The use of the arabidopsis thaliana ERF012 gene according to claim 1 for regulating seed germination, wherein inhibiting expression of the arabidopsis thaliana ERF012 gene in the presence of abscisic acid promotes seed germination.
4. The use of the arabidopsis thaliana ERF012 gene according to claim 1 for regulating seed germination, wherein overexpression of the arabidopsis thaliana ERF012 gene inhibits seed germination in the presence of abscisic acid.
5. A cultivation method for improving germination capacity of plant seeds, which is characterized in that the method comprises inhibiting expression of an ERF012 gene in a target plant in the presence of abscisic acid.
6. The method for improving germination capacity of plant seeds according to claim 5, wherein said target plant is Arabidopsis thaliana.
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