阅读说明：本技术 一种乙烯诱导bahd酰基转移酶erat2基因及其应用 (Ethylene-induced BAHD acyltransferase ERAT2 gene and application thereof ) 是由 马倩 金尚卉 于 2020-04-22 设计创作，主要内容包括：本发明提供了一种乙烯诱导的BAHD酰基转移酶ERAT2基因及其应用。所述乙烯诱导的BAHD酰基转移酶ERAT2基因的核苷酸序列如SEQ ID NO.1所示。本发明构建了ERAT2基因的CRISPR-Cas9基因敲除载体,并获得了ERAT2基因的CRISPR-Cas9基因敲除株系,该株系在干旱胁迫和盐胁迫的条件下,野生型株系相比种子的发芽率降低,幼苗的根长变短；并且ERAT2基因的RNAi基因敲除株系能够参与合成多种脂质类物质溶血磷脂酰胆碱,本发明的技术方案为植物中对溶血磷脂酰胆碱的生物合成和抗渗透胁迫育种提供了理论依据和实验基础。(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.)

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.

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.