CN111549041A - Ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof - Google Patents
Ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof Download PDFInfo
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Abstract
The invention provides an ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof. The nucleotide sequence of the ethylene-induced BAHD acyltransferase ERAT2 gene is shown as SEQ ID NO. 1. The invention constructs a CRISPR-Cas9 gene knockout vector of ERAT2 gene, and obtains a CRISPR-Cas9 gene knockout strain of ERAT2 gene, the strain has lower germination rate compared with seeds and shorter root length of seedlings under the conditions of drought stress and salt stress; in addition, RNAi gene knockout strains of ERAT2 genes can participate in synthesis of various lipid substances, namely lysophosphatidylcholine, and the technical scheme of the invention provides theoretical basis and experimental basis for biosynthesis of lysophosphatidylcholine and breeding of osmotic stress resistance in plants.
Description
Technical Field
The invention belongs to the field of biological genetic engineering, and particularly relates to an ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof.
Background
Ethylene is a gaseous plant hormone and widely participates in the processes of seed germination, organ senescence, fruit ripening, leaf abscission, reaction to adversity and pathogen infection and the like of plants. Arabidopsis thaliana is a model crop for molecular biology research of plant hormones, and signal transduction pathways of many plant hormones are clarified through the research of arabidopsis thaliana, which also includes ethylene signal transduction pathways. In the ethylene signal transduction pathway of arabidopsis thaliana, ethylene synthesized in a plant body is first bound to ethylene receptors located on endoplasmic reticulum and golgi apparatus, and normally, the ethylene receptors are bound to CTR1 protein to synergistically negatively regulate downstream ethylene response, thereby inhibiting downstream signal transduction and expression of corresponding genes. When ethylene is combined with a receptor thereof, the protein conformation of the ethylene receptor is changed, the combination of the ethylene receptor and CTR1 is inhibited, and the ethylene receptor is combined with a downstream positive regulatory factor EIN2, so that the EIN2 protein is activated. After dephosphorylation of the activated EIN2 protein is carried out at the S645 site, the C end of the protein is cut off, the protein enters the cell nucleus and activates downstream EIN3/EILs transcription factors, thereby leading the expression of a series of related genes such as AP2/ERFs and the like, such as growth and stress resistance. Although the signal transduction pathway of ethylene has been studied more, no report on the involvement of ethylene in the expression and regulation of acyltransferase genes has been disclosed.
Disclosure of Invention
The invention provides an ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof. The invention obtains a novel gene ERAT2 (the gene number is AT5G01210) capable of coding BAHD acyltransferase through comprehensive analysis of a plurality of experiments, and verifies the function of participating in biosynthesis and osmotic stress response to plant lysophosphatidylcholine substances through the experiments.
In order to solve the production problem, the invention adopts the following technical scheme:
the invention provides an ethylene-induced BAHD acyltransferase ERAT2 gene, wherein the nucleotide sequence of the ERAT2 gene is shown as SEQ ID NO. 1.
Further, the expression of the ERAT2 gene is regulated by the induction of ethylene and is located downstream of the ethylene signaling pathway.
Further, the ERAT2 gene functions in multiple tissue organs without tissue specificity.
Further, the ERAT2 gene has the function of participating in plant osmotic stress response.
Further, the ERAT2 gene has a function of participating in the biosynthesis of a plant lysophosphatidylcholine substance.
Further, the expression of the ERAT2 gene can be affected by drought stress and salt stress.
Further, the expression of the ERAT2 gene can influence the sensitivity of plant seedlings to stress.
The invention also provides a gene knockout vector of the ERAT2 gene, wherein the gene knockout vector is CRISPR-Cas9, and the gene knockout vector contains an ERAT2 gene shown as SEQ ID NO. 1.
The invention also provides an era 2 mutant, wherein the era 2 mutant contains an ERAT2 gene shown as SEQ ID NO. 1.
The invention also provides application of the ethylene-induced BAHD acyltransferase ERAT2 gene in response to plant osmotic stress.
Further, under osmotic stress conditions, CRISPR-Cas9 knockout lines of the ERAT2 gene have reduced seed germination compared to wild-type lines.
Further, under osmotic stress conditions, seedlings of CRISPR-Cas9 knock-out lines of ERAT2 gene have shorter root length compared to wild type lines.
Further, seedlings of the RNAi gene knock-out line of the ERAT2 gene were more sensitive than seedlings.
Further, the plant osmotic stress includes salt stress and drought stress.
The invention also provides the application of the ethylene-induced BAHD acyltransferase ERAT2 gene in the biosynthesis of lysophosphatidylcholine in plants.
Further, the CRISPR-Cas9 gene knockout strain of the ERAT2 gene can synthesize lysophosphatidylcholine which is a lipid substance.
Compared with the prior art, the invention has the advantages and beneficial technical effects that:
1. the invention obtains and discloses a new gene ERAT2 (gene number is AT5G01210) capable of coding ethylene-induced BAHD acyltransferase for the first time through sequencing transcriptome of an ethylene mutant ctr1-8 and comprehensively analyzing the gene ChIP analysis result of the root system of Arabidopsis processed by ChIP-Seq and ACC of EIN3 transcription factor in an ethylene signal channel.
2. The invention utilizes the existing plant genetic engineering technology to obtain the ERAT2 gene Arabidopsis thaliana larvae DNA insertion mutant era 2 and CRISPR-Cas9 gene knockout vector and strains thereof, and various experiments prove that seedlings of era 2 mutant and CRISPR-Cas9 gene knockout strains are more sensitive to adversity than seedling formation, drought and salt stress can influence the growth of the root systems of the seedlings of the two strains, the growth speed of the roots of the seedlings is delayed, the germination rate of the plants is reduced, and the strain osmotic stress related genes are knocked out by analyzing the era 2 mutant and the CRISPR-Cas9 gene, so that the ERAT2 gene is determined to have the function of participating in plant osmotic stress response. The invention also proves that the era 2 mutant and the CRISPR-Cas9 gene knockout strain participate in the generation of the plant lysophosphatidylcholine through experiments, and further proves that the ERAT2 gene participates in the biosynthesis of the plant lysophosphatidylcholine.
3. The technical scheme of the invention provides theoretical basis and experimental basis for biosynthesis of lysophosphatidylcholine and breeding of osmotic stress resistance in plants.
Drawings
FIG. 1 shows the results of comprehensive analysis of transcriptome sequencing of ethylene mutant ctr1-8, ChIP-Seq of EIN3 transcription factor in ethylene signal pathway and gene ChIP analysis data of root system of Arabidopsis thaliana treated by ACC in the present invention.
FIG. 2 shows the change in expression level of ERAT2 in wild type Arabidopsis thaliana after ACC treatment, a precursor for ethylene biosynthesis, in the present invention.
FIG. 3 shows the change in expression level of ERAT2 in an ethylene-insensitive Arabidopsis mutant after ACC treatment, a precursor for ethylene biosynthesis, according to the present invention.
FIG. 4 shows the change of expression level of ERAT2 in ethylene hypersensitive Arabidopsis mutants after ACC treatment, a precursor for ethylene biosynthesis, in the present invention.
FIG. 5 shows the relative expression of ERAT2 in different tissues and organs of Arabidopsis thaliana in the present invention.
FIG. 6 shows the relative expression of ERAT2 under drought stress in the present invention.
FIG. 7 shows the relative expression of ERAT2 under salt stress in the present invention.
FIG. 8 is the relative expression of ERAT2 under cold stress in the present invention.
FIG. 9 shows the relative expression level of ERAT2 in the mutants of the present invention.
Figure 10 is the growth profile of the era 2 mutant of the invention, where a is the mutant seedling that is sown directly in soil and no longer grows after germination; and B is a mutant seedling which is cultured on 1/2MS culture medium for three weeks and then transplanted into soil to grow normally.
FIG. 11 shows the germination rates of wild type Arabidopsis thaliana and the era 2 mutant without mannitol treatment in the present invention.
FIG. 12 shows the germination rates of wild type Arabidopsis thaliana and the era 2 mutant treated with 400mM mannitol in the present invention.
FIG. 13 is a comparison of root length of wild type Arabidopsis thaliana and the era 2 mutant treated with 400mM mannitol for 10 days in the present invention.
FIG. 14 is a comparison of root length of wild type Arabidopsis thaliana and era 2 mutant treated with different concentrations of mannitol for 10 days in the present invention.
FIG. 15 shows the germination rates of wild type Arabidopsis thaliana and the era 2 mutant in the absence of NaCl treatment in the present invention.
FIG. 16 shows the germination rates of wild type Arabidopsis thaliana and the era 2 mutant treated with 100mM NaCl in the present invention.
FIG. 17 is a comparison of root lengths of wild type Arabidopsis thaliana and the era 2 mutant treated with 150mM NaCl for 6 days in accordance with the present invention.
FIG. 18 is a comparison of root lengths of wild type Arabidopsis thaliana and the era 2 mutant treated with different concentrations of NaCl for 6 days in the present invention.
FIG. 19 shows the change in the expression level of HK1 gene after mannitol-simulated drought treatment of the era 2 mutant of the invention.
FIG. 20 shows the change in expression level of ERD4 gene after salt treatment of the era 2 mutant of the present invention.
FIG. 21 shows the change in the expression level of ERF11 gene after salt treatment of the era 2 mutant of the present invention.
FIG. 22 shows the change in the expression level of ERF4 gene after salt treatment of the era 2 mutant of the present invention.
FIG. 23 shows the change in the expression level of ERF105 gene after salt treatment of the era 2 mutant of the present invention.
FIG. 24 shows the change in the expression level of ERF1 gene after salt treatment of the era 2 mutant of the present invention.
FIG. 25 is the germination rates of CRISPR-Cas9 knockout lines of wild type Arabidopsis and ERAT2 of the present invention when treated with 100mM mannitol, wherein L1 and L2 are 2 independent homozygous knockout lines, respectively, screened.
FIG. 26 results of root length of CRISPR-Cas9 knockout lines of wild type Arabidopsis and ERAT2 of the present invention when treated with mannitol at different concentrations.
FIG. 27 is the germination rates of the CRISPR-Cas9 knockout lines of wild type Arabidopsis and ERAT2 of the present invention when treated with 200mM NaCl.
FIG. 28 is the result of the root length of the CRISPR-Cas9 knockout lines of wild type Arabidopsis and ERAT2 of the present invention when treated with NaCl at different concentrations.
FIG. 29 shows the change of expression of HK1 gene after mannitol-simulated drought treatment of CRISPR-Cas9 gene knockout strain of ERAT2 in the invention.
FIG. 30 shows the change of expression of ERF105 gene after salt treatment of CRISPR-Cas9 knockout strain of ERAT2 in the present invention.
Wherein a, b, c and d all represent that the individual-to-individual difference under different letter labels reaches a significance level of 0.05.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the accompanying drawings, the attached tables and the specific embodiments.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available products unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1: ethylene-induced expression of BAHD acyltransferase ERAT2
1. Comprehensive analysis of independent experiments
(1) When the result of sequencing the ethylene pathway mutant CTR1-8 by transcriptome is analyzed, the expression level of 1702 genes is found to be regulated and controlled by CTR 1.
(2) Downloading original ChIP-Seq data with data number SRA063695 and aiming at EIN3 transcription factor on NCBI;
(3) downloading the original data of the Arabidopsis root system processed by ACC for 3 hours when the data number on Arrayexpress is E-MEXP-362 and performing gene chip analysis;
(4) the data of the three independent experiments are comprehensively analyzed, and as shown in FIG. 1, 25 genes are found to be completely overlapped;
(5) further analysis of the above 25 overlapping genes revealed that the presence of an unknown novel gene encoding BAHD acyltransferase, AT5G01210, was significantly affected in all three independent experiments. The invention names AT5G01210 as ERAT2(Ethylene Regulated BAHD AcylTransferase 2), and the nucleotide sequence is shown as SEQ ID NO. 1. From the above results, it is presumed that BAHD acyltransferase ERAT2 is induced by ethylene and is located downstream of the ethylene pathway EIN3 transcription factor.
2. Bioinformatics method for analyzing promoter sequence of ERAT2
(1) The sequence 2000bp upstream of the ATG start codon of BAHD acyltransferase ERAT2 was searched by the Arabidopsis database (http:// www.arabidopsis.org) and NCBI database (http:// www.ncbi.nlm.nih.gov/guide).
(2) A network promoter analysis website plantare is used for analyzing a promoter sequence of ERAT2, the nucleotide sequence of the promoter sequence is shown as SEQ ID NO.2, the promoter sequence of ERAT2 contains an ethylene response element (-ATTTCAAA-), and the expression of ERAT2 is presumed to be controlled by ethylene.
3. qRT-PCR demonstrated that expression of ERAT2 is regulated by ethylene
(1) The wild type Arabidopsis thaliana Col, the ethylene insensitive mutants etr1-1 and ein2, and the ethylene super sensitive mutants rte1-3 and rte5 were selected as experimental materials.
(2) Wild type Arabidopsis thaliana Col and ethylene pathway mutants etr1-1, rte1-3, ein2 and rte5 seeds were cultured on 1/2MS medium for 2 weeks, then transferred to 1/2MS liquid medium supplemented with 100. mu.M ACC, and cultured for 6 hours under sealed illumination.
(3) qRT-PCR is used for detecting the change of the relative expression quantity of ERAT2 in each sample before and after ethylene treatment, wherein the qRT-PCR primer sequence used for detecting ERAT2 gene is as follows:
AtERAT2-FP：5’-CGTCTCCGATCTCCGTCTCT-3’(SEQ ID NO.3)；
AtERAT2-RP：5’-GTCGTCGAAGGAGAAGGAAGG-3’(SEQ ID NO.4)。
the experimental results show that the expression level of ERAT2 is significantly increased by the treatment of ethylene in wild type Arabidopsis thaliana (FIG. 2); in the ethylene insensitive mutants etr1-1 and ein2, the expression level of ERAT2 did not change significantly before and after ethylene treatment (FIG. 3); in the ethylene hypersensitive mutants rte1-3 and rte5, the expression level of ERAT2 increased even more after ethylene treatment than that of the wild type Arabidopsis control (FIG. 4). Taken together, it is demonstrated that expression of BAHD acyltransferase ERAT2 is regulated by ethylene and is located downstream of the ethylene signaling pathway, thus further demonstrating that ethylene induces expression of the BAHD acyltransferase ERAT2 gene.
Example 2: effect of ERAT2 mutants on osmotic stress and lysophosphatidylcholine biosynthesis
1. Tissue expression pattern and osmotic stress-induced expression pattern of ERAT2 gene
(1) Collecting roots, leaves, stems, flowers and pods of wild arabidopsis Co1, grinding with liquid nitrogen, extracting RNA, and performing reverse transcription to obtain cDNA; qRT-PCR detected the relative expression of ERAT2 in these several tissue organs. The results are shown in fig. 5, where ERAT2 is expressed in several organs, indicating that ERAT2 functions in multiple tissue organs without tissue specificity.
(2) After 2 weeks of culturing wild type Arabidopsis thaliana Col seeds on MS culture medium, transferring the seeds to MS liquid culture medium added with 200mM mannitol (simulating drought) or 200mM NaCl, culturing for 0h, 3h, 6h, 9h, 12h and 24h in sealed illumination, and sampling.
qRT-PCR detects the change trend of the expression level of ERAT2 after different times of drought and salt stress treatment. The results show that the expression of ERAT2 after mannitol simulated drought treatment increases continuously, reaches the highest peak at 6h, then decreases temporarily, but increases for the second time from 9h (FIG. 6). In contrast, ERAT2 was expressed in increasing amounts after salt treatment (FIG. 7).
(3) Seedlings cultured for 2 weeks were transferred to MS liquid medium. After sealing, the petri dish was placed in an ice water mixture and subjected to 0 ℃ cold treatment for 0h, 3h, 6h, 9h, 12h and 24h, and a sample was taken.
qRT-PCR detects the change trend of the expression level of ERAT2 after cold stress treatment for different time. The results showed that ERAT2 showed little change in expression level within 12h after cold treatment (FIG. 8).
Taken together, ERAT2 was shown to be involved in the response to osmotic stress (drought and salt).
2. Acquisition of an ERAT2T-DNA insertion mutant
(1) Arabidopsis thaliana T-DNA insertion mutants of the BAHD acyltransferase ERAT2 were purchased from the tair website, mutant SALK no: SALKseq _ 129231.
(2) And detecting the homozygosity of the mutant by using an RT-PCR single plant. Individuals in which only the 2-LP and 2-RP amplified bands are present are wild-type; individuals with only Lba and 2-RP amplified bands are homozygous for the T-DNA insertion mutant of the gene; individuals with both 2-LP and 2-RP amplified bands, as well as Lba and 2-RP amplified bands, are heterozygous individuals for wild-type and T-DNA insertion mutants. The detection structure shows that all the purchased mutants are homozygous individuals of the gene T-DNA insertion mutant and accord with the mark on a website.
Wherein, the PCR primer sequence used for detecting the homozygosity of the era 2T-DNA insertion mutant is as follows:
2-LP：5’-AAACCTGGTTTGAACTTTGGC-3’(SEQ ID NO.5)；
2-RP：5’-AGTTACACAGGAACGTGGTGG-3’(SEQ ID NO.6)；
Lba：5’-TGGTTCACGTAGTGGGCCATCG-3’(SEQ ID NO.7)。
(3) qRT-PCR was used to determine the relative expression level of ERAT2 in the mutants. Extracting RNA of wild type and era 2T-DNA insertion mutant, and performing reverse transcription to obtain cDNA; qRT-PCR was used to determine the relative expression level of ERAT2 in the mutants. The results are shown in fig. 9, where the expression level of ERAT2 in the mutant was significantly reduced relative to the wild type, indicating that the purchased mutant was acceptable and could be used in subsequent experiments.
3. era 2 mutant seedling is sensitive to adverse circumstances
(1) The mutant seeds were sown in nutrient soil and it was found that the era 2 mutant did not grow and eventually died after germination in soil (fig. 10A).
(2) After sowing the mutant seeds on thicker 1/2MS medium for three weeks, they were transferred into soil and found to grow normally (FIG. 10B).
The above results indicate that mutant era 2 seedlings are more sensitive to stress than to seedling.
4. era 2 mutant sensitive to drought stress
(1) Seeds of the era 2 mutant were sown on 1/2MS medium with mannitol at final concentrations of 0 and 400mM, respectively. After the seeds were cold-treated at 4 ℃ for 3 days, they were cultured under light and the germination percentage was recorded every day. The results are shown in fig. 11 and 12, where the era 2 mutant seeds had significantly reduced germination rates under mannitol-simulated drought stress as compared to the wild type arabidopsis thaliana Col.
(2) Seeds of the mutant era 2 were sown on 1/2MS medium and were chilled at 4 ℃ for 3 days. Taken out, transferred to 1/2MS medium with mannitol at final concentrations of 0, 100, 200, 300 and 400mM, respectively, and cultured in the light for 3 days, and the change in root length was observed and measured. Results are shown in fig. 13-14, after 10 days of culture on media containing mannitol of different concentrations, roots of era 2 mutant seedlings were significantly shorter than those of wild arabidopsis thaliana, indicating that drought stress affects the growth of era 2 mutant seedling roots, and also indicating that ERAT2 mutants are susceptible to drought stress.
5. era 2 mutant sensitive to salt stress
(1) Seeds of the era 2 mutant were sown on 1/2MS medium with final NaCl concentrations of 0 and 100 mM. After the seeds were cold-treated at 4 ℃ for 3 days, they were cultured under light and the germination percentage was recorded every day. The results are shown in fig. 15-16, and the germination rates of the seeds of the era 2 mutant are obviously reduced under the salt stress of different concentrations compared with the seeds of the wild type arabidopsis thaliana Col.
(2) Seeds of the mutant era 2 were sown on 1/2MS medium and were chilled at 4 ℃ for 3 days. Taking out, culturing for 3 days under light, transferring to 1/2MS culture medium containing NaCl with final concentration of 0, 50, 100, 150 and 200mM, culturing for 6 days under light, and observing the change of root length. The results are shown in fig. 17-18, after 6 days of culture on media containing salts with different concentrations, the roots of era 2 mutant seedlings are obviously shorter than those of wild arabidopsis thaliana, which indicates that salt stress influences the growth of era 2 mutant seedling roots and also indicates that ERAT2 mutant is sensitive to salt stress.
6. qRT-PCR detection of relative expression change of osmotic stress related gene in mutant
Seedlings of Arabidopsis thaliana wild type and era 2 mutant cultured for 2 weeks were transferred onto MS liquid medium. Then adding 200mM mannitol or 200mM NaCl solution to treat for 0h, 3h, 6h, 9h, 12h, 24h, 48h and 72h, sampling and extracting RNA, and performing reverse transcription to obtain cDNA; and qRT-PCR is used for detecting the expression quantity change trend of the osmotic stress related gene. The results are shown in FIGS. 19-24, which show that the expression levels of the genes related to osmotic stress of HK1, ERD4, ERF1, ERF4, ERF11 and ERF105 were decreased in the era 2 mutant compared with the wild type Arabidopsis COL, whether the mutant was treated with mannitol to simulate drought or NaCl.
Taken together, the ERAT2 gene plays a role in plant response to osmotic stress.
7. Effect of the era 2 mutant on the biosynthesis of lysophosphatidylcholine
In the invention, the mutant era 2 seedling is considered to be more sensitive to stress than seedling formation, and two-week-old seedlings on a culture medium are selected as experimental materials to detect the function of ERAT2 in plant secondary metabolism.
(1) The wild arabidopsis thaliana Col is used as a control, the mutant era 2 seedling aged for two weeks is frozen by liquid nitrogen, placed in dry ice, and sent to Wuhan Miteville Biotechnology Limited to carry out extensive targeted metabonomics analysis on the sample. The results are shown in table 1, that BAHD acyltransferase ERAT2 is primarily involved in the production of the lipid substance lysophosphatidylcholine, suggesting that ERAT2 gene plays a role in the biosynthesis of the lipid substance lysophosphatidylcholine in plants.
TABLE 1 BAHD acyltransferase ERAT2 is involved in the production of lysophosphatidylcholine from lipid substances
Example 3: effect of CRISPR-Cas9 knockout strain of ERAT2 on osmotic stress and lysophosphatidylcholine biosynthesis
1. CRISPR-Cas9 gene knockout strain of ERAT2 is obtained
(1) A kit is constructed by using a Baige CRISPR-Cas9 vector, and a gRNA target sequence (http:// www.biogle.cn/index/excrispr) of ERAT2 is designed and generated. The PAM sequence CGG was found over the CDS sequence of ERAT 2. One CGG site was selected at each of the front, middle and rear ends of the CDS sequence of ERAT 2.
(2) Inputting a sequence of 19bp at the front end of each CGG site into a website www.biogle.cn, designing a primer, preparing an Oligo dimer, and constructing the Oligo dimer onto a CRISPR-Cas9 vector BGK 01.
(3) Transforming the constructed CRISPR-Cas9 vector into escherichia coli;
(4) after PCR verification, positive strain plasmids are extracted and transferred into agrobacterium strain GV 3101.
(5) Arabidopsis thaliana was transformed by the floral dip method. After PCR identification, the transformed lines were determined by sequencing.
The PCR primer sequence for detecting CRISPR-Cas9 gene knockout strain of ERAT2 is as follows:
ERAT2CRI-Ff：5’-ATAACTCCTACTCATCACCAATAC-3’(SEQ ID NO.8)；
ERAT2CRI-Rf：5’-TCGTTGCAGACAATGGAAAT-3’(SEQ ID NO.9)；
ERAT2CRI-Fm：5’-CGTTAGTTACAACGGTCATC-3’(SEQ ID NO.10)；
ERAT2CRI-Rm：5’-CTGAGAGATTGGAACGATGA-3’(SEQ ID NO.11)。
(6) 2 independent homozygous CRISPR-Cas9 gene knockout strains L1 and L2 of ERAT2 obtained by continuous selfing are used as experimental materials.
2. CRISPR-Cas9 gene knockout strain of ERAT2 is sensitive to stress
(1) Seeds of CRISPR-Cas9 knockout strains of ERAT2 are directly sown in nutrient soil, and the seeds are found not to grow any more after germinating in the soil and finally die.
(2) Seeds of CRISPR-Cas9 gene knockout strains of ERAT2 are sown on a thicker 1/2MS culture medium and cultured for three weeks, and then transferred into soil, and normal growth is found, which indicates that L1 and L2 seedlings are more sensitive to stress than mature seedlings.
3. CRISPR-Cas9 gene knockout strain of ERAT2 sensitive to drought stress
(1) Seeds of ERAT2 CRISPR-Cas9 gene knockout strain were sown on 1/2MS medium with 100mM final mannitol concentration. After the seeds were cold-treated at 4 ℃ for 3 days, they were cultured under light and the germination percentage was recorded every day. The result is shown in fig. 25, compared with wild arabidopsis thaliana Col, the germination rate of CRISPR-Cas9 gene knockout strain seeds is obviously reduced under mannitol simulated drought stress.
(2) Seeds of the CRISPR-Cas9 gene knockout strain are sown on 1/2MS culture medium and are subjected to cold treatment at 4 ℃ for 3 days. Taken out, transferred to 1/2MS medium with mannitol at final concentrations of 0, 100, 200, 300 and 400mM, respectively, and cultured in the light for 3 days, and the change in root length was observed and measured. The results are shown in fig. 26, after the seedlings of the CRISPR-Cas9 gene knockout line are cultured for 10 days on the culture medium containing mannitol with different concentrations, the roots of the seedlings of the CRISPR-Cas9 gene knockout line are obviously shorter than those of wild arabidopsis thaliana, which indicates that drought stress affects the growth of the roots of the seedlings of the CRISPR-Cas9 gene knockout line, and also indicates that the CRISPR-Cas9 gene knockout line of ERAT2 is sensitive to drought stress.
4. CRISPR-Cas9 gene knockout strain sensitive to salt stress
(1) Seeds of the CRISPR-Cas9 knockout line were sown on 1/2MS medium with a final NaCl concentration of 200 mM. After the seeds were cold-treated at 4 ℃ for 3 days, they were cultured under light and the germination percentage was recorded every day. The result is shown in fig. 27, compared with wild arabidopsis thaliana Col, the germination rate of seeds of the CRISPR-Cas9 gene knockout line is obviously reduced under salt stress of different concentrations.
(2) Seeds of the CRISPR-Cas9 gene knockout strain are sown on 1/2MS culture medium and are subjected to cold treatment at 4 ℃ for 3 days. Taking out, and culturing under light for 3 days. The cells were transferred to 1/2MS medium containing NaCl at final concentrations of 0, 100, 200 and 400mM, and cultured under light for 6 days, and the change in root length was observed. The results are shown in fig. 28, after being cultured on culture media containing salts with different concentrations for 6 days, the roots of the seedlings of the CRISPR-Cas9 gene knockout line are obviously shorter than those of wild arabidopsis thaliana, which indicates that salt stress influences the growth of the roots of the seedlings of the CRISPR-Cas9 gene knockout line, and also indicates that the CRISPR-Cas9 gene knockout line of ERAT2 is sensitive to salt stress.
5. qRT-PCR detection of relative expression change of osmotic stress related gene in mutant
Seedlings of Arabidopsis wild-type and CRISPR-Cas9 knockout lines cultured for 2 weeks were transferred onto MS liquid medium. Then adding 200mM mannitol or 200mM NaCl solution for treating for 0h, 3h, 6h, 9h, 12h, 24h, 48h and 72h, sampling and extracting RNA, and performing reverse transcription to obtain cDNA. And qRT-PCR is used for detecting the expression quantity change trend of the osmotic stress related gene. The results are shown in FIGS. 29-30, and compared with wild type Arabidopsis COL, the expression levels of genes related to osmotic stress of HK1 and ERF105 are reduced in CRISPR-Cas9 gene knockout strains L-1 and L-2 after mannitol simulated drought treatment or NaCl treatment.
Taken together, the ERAT2 gene plays a role in plant response to osmotic stress.
6. Biological effect of ERAT2 on lysophosphatidylcholine
Considering that CRISPR-Cas9 gene knockout line seedlings are more sensitive to stress than mature seedlings, two-week-old seedlings on a culture medium are selected as experimental materials to detect the function of ERAT2 in plant secondary metabolism.
(1) And (3) freezing the seedlings of the CRISPR-Cas9 gene knockout line with two weeks old by using wild type Arabidopsis thaliana Col as a control through liquid nitrogen, and placing the seedlings in a refrigerator at the temperature of-80 ℃ for later use.
(2) And detecting the difference of lipid substance lysophosphatidylcholine in the CRISPR-Cas9 gene knockout strain by using LC-MS. The results are shown in table 2, which verifies that BAHD acyltransferase ERAT2 is involved in the production of lipid-based material lysophosphatidylcholine, indicating that BAHD acyltransferase ERAT2 plays a role in the biosynthesis of plant lysophosphatidylcholine.
TABLE 2 BAHD acyltransferase ERAT2 is involved in the production of lysophosphatidylcholine from lipid substances
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> Qingdao agricultural university
<120> ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof
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caaaaaggcg tcctcctcac cagccctcct ccttccttct ccttcgacga ccttgtctcc 180
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ctgcttttcc gtgagttttt cgtcttcgag cgtctcgtta gttacaacgg tcatcataag 420
cctctcgccg ccgttcaagt gacggagctc cacgacggtg tcttcatcgg atgtaccgtg 480
aatcattccg ttactgacgg aacttccttc tggcacttct tcaacacctt tgctgacgtc 540
actagcggtg cttgtaagat caaacacctt ccagatttct cccgccacac cgtcttcgat 600
tctccggtcg ttcttccagt ccctcccggt ggtccacgtg tcactttcga cgccgaccaa 660
cctctacggg agagaatttt tcatttcagc agagaggcga ttaccaaact gaaacagagg 720
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cggcaccggc ttgagccgaa gatggatccg tactacttcg gaaacgcgat acagagcata 1020
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aacggaagcg tggatctgga agtagttctg gcgccggaga ctatgactgg gattgagaac 1380
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caaggaagca agcaaacaaa aaaaccaaaa catttcacac ataaaagtcc gaaattgggt 300
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ctaaaacaaa acattttgct cgtgaaagtg atgttaacac ttgctataaa tattcatcta 420
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Claims (9)
1. An ethylene-induced BAHD acyltransferase ERAT2 gene, wherein the nucleotide sequence of the ERAT2 gene is shown as SEQ ID NO. 1.
2. The BAHD acyltransferase ERAT2 gene according to claim 1 wherein the expression of the ERAT2 gene is under the induction regulation of ethylene.
3. A gene knockout vector of ERAT2 gene, characterized in that: the gene knockout vector is CRISPR-Cas9 and contains ERAT2 gene shown as SEQ ID NO. 1.
4. Use of the ethylene-induced BAHD acyltransferase ERAT2 gene of claim 1 in participating in a plant osmotic stress response.
5. Use of the ethylene-induced BAHD acyltransferase ERAT2 gene in participating in plant osmotic stress response according to claim 4 characterized in that under osmotic stress conditions the CRISPR-Cas9 knock-out strain of the ERAT2 gene has a reduced seed germination compared to wild type strains.
6. Use of the ethylene-induced BAHD acyltransferase ERAT2 gene according to claim 4 in participating in a plant osmotic stress response, characterized in that under osmotic stress conditions seedlings of the CRISPR-Cas9 knock-out line of the ERAT2 gene have a shorter root length compared to wild type lines.
7. Use of the ethylene-induced BAHD acyltransferase ERAT2 gene according to claim 4 wherein the plant osmotic stress comprises salt stress and drought stress.
8. Use of the ethylene-inducible BAHD acyltransferase ERAT2 gene of claim 1 involved in the biosynthesis of lysophosphatidylcholine in plants.
9. The use of the ethylene-induced BAHD acyltransferase ERAT2 gene as claimed in claim 8 in the participation in the biosynthesis of lysophosphatidylcholine in plants, characterized in that the CRISPR-Cas9 knockout strain of the ERAT2 gene is capable of synthesizing the lipid substance lysophosphatidylcholine.
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| CN114940998B (en) * | 2022-06-20 | 2023-06-06 | 四川农业大学 | Corn transcription factor ZmEREB92 and application thereof |
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