Gain-of-function mutants of caffeic acid-o-methyltransferase 2, recombinant expression vectors and uses thereof

Abstract

The application discloses a caffeic acid-O-methyltransferase 2 gain-of-function mutant, a recombinant expression vector and application thereof, and belongs to the technical field of crop biological breeding. It is found for the first time that after the 344th amino acid of tomato caffeic acid-O-methyltransferase 2 is changed from cysteine (C) to tryptophan (W), the content of melatonin of the tomato can be increased, the tomato can be endowed with broad-spectrum resistance to abiotic and biotic stresses, and the tomato can be used as a key target point for resistance breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of crop biological breeding, and in particular to a caffeic acid-O-methyltransferase 2 gain-of-function mutant, a recombinant expression vector and applications thereof. Background Art

[0002] Biological and abiotic stresses are one of the important issues that restrict the high-quality development of the tomato industry. Breeding broad-spectrum stress-resistant varieties is an effective way to improve the comprehensive environmental adaptability of tomatoes. Traditional hybrid breeding relies on phenotypic identification, parental recombination and resistant germplasm screening to obtain disease-resistant and stress-resistant tomato varieties. However, traditional breeding methods take a long time, are difficult, labor-intensive and time-consuming, and it is not easy to break the linkage between resistance and adverse agronomic traits. However, by finding resistance genes, studying the key targets of resistance gene functions, and transforming and utilizing related targets for biological breeding, the above problems can be effectively solved.

[0003] Melatonin, a small molecule beneficial to human health, is widely found in various plants and can enhance their broad-spectrum resistance to various biotic and abiotic stresses. Improving melatonin content is a goal of crop quality and resistance breeding. While the regulatory genes and metabolic pathways for melatonin synthesis have been described, little is known about how to modify and utilize these synthetic genes to further promote melatonin accumulation and enhance plant resistance.

[0004] Caffeic acid-O-methyltransferase (COMT) is a key enzyme in the plant phenylpropanoid biosynthesis pathway. It has a broad substrate range and can catalyze the synthesis of a variety of phenylpropanoid compounds and flavonoids. Recent studies have revealed that some COMTs also function similarly to N-acetyl-5-hydroxytryptamine methyltransferase in melatonin biosynthesis, catalyzing the conversion of N-acetyl-5-hydroxytryptamine to melatonin. Solanum lycopersicum caffeic acid O-methyltransferase 2 (SlCOMT2) is a potent melatonin synthase gene in tomatoes, promoting melatonin synthesis. However, there are currently no reports of modifying caffeic acid O-methyltransferase 2 in tomatoes to increase melatonin content and stress tolerance. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention aims to provide a gain-of-function mutant of caffeic acid-O-methyltransferase 2, a recombinant expression vector, and its use. By modifying the tomato caffeic acid-O-methyltransferase 2 site, the present invention improves protein stability and melatonin synthesis, potentially serving as a key target for tomato resistance breeding.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present application provides a caffeic acid-O-methyltransferase 2 gain-of-function mutant, wherein the amino acid at position 344 of caffeic acid-O-methyltransferase 2 is mutated; the amino acid sequence of the caffeic acid-O-methyltransferase 2 is shown in SEQ ID NO. 1.

[0008] Specifically, the caffeic acid-O-methyltransferase 2 gain-of-function mutant is a mutant in which the amino acid at position 344 of caffeic acid-O-methyltransferase 2 is mutated to an amino acid other than cysteine, such as alanine, valine, leucine, isoleucine, proline, serine, threonine, methionine, tyrosine, aspartic acid, glutamic acid, lysine, tryptophan, histidine, phenylalanine, asparagine or glutamine.

[0009] Preferably, the caffeic acid-O-methyltransferase 2 gain-of-function mutant is a mutant in which the cysteine (C) at position 344 of caffeic acid-O-methyltransferase 2 is mutated to tryptophan (W); the amino acid sequence of the caffeic acid-O-methyltransferase 2 gain-of-function mutant after mutation is shown in SEQ ID NO. 2.

[0010] In a second aspect, the present application provides a coding gene of the caffeic acid-O-methyltransferase 2 gain-of-function mutant.

[0011] Preferably, the coding gene is a nucleic acid molecule as shown in i) or ii) below:

[0012] i) the nucleotide sequence is a nucleic acid molecule as shown in SEQ ID NO. 4;

[0013] ii) a nucleic acid molecule other than i) encoding the amino acid sequence as shown in SEQ ID NO. 2.

[0014] In a third aspect, the present application provides a recombinant expression vector containing the coding gene of the caffeic acid-O-methyltransferase 2 gain-of-function mutant.

[0015] Preferably, the recombinant expression vector is a pCAMBIA1306 vector containing the coding gene of the caffeic acid-O-methyltransferase 2 gain-of-function mutant.

[0016] In a fourth aspect, the present application provides the use of the above-mentioned caffeic acid-O-methyltransferase 2 gain-of-function mutant, the coding gene of the caffeic acid-O-methyltransferase 2 gain-of-function mutant or the recombinant expression vector containing the coding gene of the caffeic acid-O-methyltransferase 2 gain-of-function mutant in at least one of the following (1)-(4):

[0017] (1) increasing the content of melatonin in tomatoes;

[0018] (2) Improve the resistance of tomatoes to biotic stress;

[0019] (3) Improve the resistance of tomatoes to abiotic stress;

[0020] (4) Tomato resistance breeding.

[0021] In the above applications, the biological stress mainly refers to the stress caused by pathogen infection on plants, for example, tomato gray mold caused by infection with Botrytis cinerea.

[0022] In the above applications, the abiotic stress includes but is not limited to: high temperature, low temperature, saline-alkali stress, etc.

[0023] A fifth aspect of the present invention provides a method for improving the ability of caffeic acid-O-methyltransferase 2 to synthesize melatonin, comprising the following steps:

[0024] The amino acid 344 of caffeic acid-O-methyltransferase 2 shown in SEQ ID NO. 1 is mutated to tryptophan (W);

[0025] Alternatively, the nucleotide 1032 of the gene encoding caffeic acid-O-methyltransferase 2 shown in SEQ ID NO. 3 is mutated to G.

[0026] Through the above mutation treatment, the stability of caffeic acid-O-methyltransferase 2 and the ability to synthesize melatonin can be improved, thereby giving tomatoes stronger broad-spectrum resistance.

[0027] Beneficial effects of the present invention:

[0028] The present invention discovered for the first time that changing the 344th amino acid of tomato caffeic acid-O-methyltransferase 2 from cysteine ​​(C) to tryptophan (W) can increase the melatonin content of tomatoes and confer broad-spectrum resistance to abiotic and biotic stresses on tomatoes, which can serve as a key target for tomato resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : SlCOMT2 and SlCOMT2 C344W Results of protein stability study.

[0030] Figure 2 :WT, SlCOMT2-OE and SlCOMT2 C344W -OE tomato plants COMT enzyme activity (A) and melatonin content (B) determination results.

[0031] Figure 3 :WT, SlCOMT2-OE and SlCOMT2 C344W-Results of resistance test of OE tomato plants to gray mold, high temperature, low temperature and saline-alkali stress. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0033] As mentioned above, Solanum lycopersicum caffeic acidO-methyltransferase 2 (SlCOMT2) is an effective melatonin synthase gene in tomato, which can promote the synthesis of melatonin.

[0034] The amino acid sequence of S1COMT2 is shown in SEQ ID NO. 1, and is as follows:

[0035] MENANGEPSNNNNNDERLAIMELANMISVPMSLNAVVKLKVTDAVWENGSNAPLSPVEILAKIRGPQGGGDAENLQRILRMLTSYGVFKEHVDDGSQRRYSLTKVGKTLVTDENGLSHGSYVLQHHQDALMKAWTMVHEAVNDSSIEPFAKANGEPAYSYYGKNSEMNSLMLNAMSGVSVP FMKAILQGYDGFQGVKTLVDVGGSGGDCLKMILEKHTDIELGINFDLPEVVEKAPQIPSIKHVGGDMFDYIPKGDAIFMKWVLTTWTDECKEIMKSCYNALPEKGKFIACEPVLPHHTDDSKRTRALLEGDIFVMTIYRAKGKHRTEEEYRQLGRAAGFNECKGFYIDHFFTILEFHKS.

[0036] The CDS sequence of the S1COMT2 gene is shown in SEQ ID NO. 3, and is as follows:

[0037]

[0038] So far, there has been no report on the modification of caffeic acid-O-methyltransferase 2 in tomatoes to increase the melatonin content and stress resistance of tomatoes.

[0039] In view of this, the present invention, through in-depth research, discovered the key target in SlCOMT2 that can increase COMT enzyme activity, namely: amino acid 344 of SlCOMT2. By mutating the cysteine ​​(C) at position 344 to tryptophan (W), a gain-of-function mutant of caffeic acid-O-methyltransferase 2 was obtained and named SlCOMT2. C344W , whose amino acid sequence is shown in SEQ ID NO.2, and is as follows:

[0040] MENANGEPSNNNNNDERLAIMELANMISVPMSLNAVVKLKVTDAVWENGSNAPLSPVEILAKIRGPQGGGDAENLQRILRMLTSYGVFKEHVDDGSQRRYSLTKVGKTLVTDENGLSHGSYVLQHHQDALMKAWTMVHEAVNDSSIEPFAKANGEPAYSYYGKNSEMNSLMLNAMSGVSVP FMKAILQGYDGFQGVKTLVDVGGSGGDCLKMILEKHTDIELGINFDLPEVVEKAPQIPSIKHVGGDMFDYIPKGDAIFMKWVLTTWTDDECKEIMKSCYNALPEKGKFIACEPVLPHHTDDSKRTRALLEGDIFVMTIYRAKGKHRTEEEYRQLGRAAGFNEWKGFYIDHFFTILEFHKS.

[0041] The gene encoding the caffeic acid-O-methyltransferase 2 gain-of-function mutant (SlCOMT2) C344W The nucleotide sequence of the gene is shown in SEQ ID NO.4, and is as follows:

[0042]

[0043] Studies have found that changing the 344th amino acid in the protein sequence of tomato caffeic acid-O-methyltransferase 2 from cysteine ​​(C) to tryptophan (W) can increase the melatonin content and broad-spectrum resistance of tomatoes to abiotic and biotic stresses, which led to the present invention.

[0044] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0045] The test materials used in the examples of the present invention are all conventional test materials in the field and can be purchased through commercial channels. Experimental methods without detailed conditions were carried out according to conventional test methods or the operating instructions recommended by the supplier.

[0046] Example 1: SlCOMT2-OE and SlCOMT2 C344W -Construction of OE tomato plants

[0047] Based on the CDS sequence information of the SlCOMT2 gene (ID: Solyc10g085830) in the tomato genome, the wild-type SlCOMT2 gene (SEQ ID NO. 3) and the mutant SlCOMT2 were obtained by in vitro gene synthesis technology. C344W gene (SEQ ID NO.4).

[0048] SlCOMT2 and SlCOMT2 C344W The gene was assembled into the pCAMBIA1306 (35S::3×FLAG) vector by gene cloning and vector construction technology, and then transformed into Agrobacterium strain LBA4404. Agrobacterium-mediated transformation of tomato was used to obtain SlCOMT2-OE and SlCOMT2 C316W -OE tomato plants. The transformation method was based on the published article "Efficient transfer of a glyphosate tolerance gene into tomato using abinary Agrobacterium tumefaciens vector" (DOI:10.1038 / nbt0787-726). The wild-type (WT) tomato variety used for LBA4404-mediated tomato transformation was Ailsa Craig (AC). Fluorescence quantitative PCR technology was used to select SlCOMT2-OE and SlCOMT2 with consistent SlCOMT2 gene expression levels. C316W -OE tomato plants were used in subsequent experimental procedures.

[0049] Example 2: SlCOMT2 and SlCOMT2 C344W Analysis of protein stability

[0050] 1. Test method:

[0051] The SlCOMT2-OE and SlCOMT2 constructed in Example 1 were treated with the protein synthesis inhibitor cycloheximide. C316W -OE tomato leaves, making SlCOMT2 and SlCOMT2 C316W The initial amount of protein was kept consistent, and SlCOMT2-OE and SlCOMT2 were extracted 0, 5 and 10 h after treatment. C316W -OE tomato leaves, and Western blotting combined with FLAG antibody was used to identify the remaining SlCOMT2 and SlCOMT2 C316W The protein content was used to evaluate the stability difference between the two proteins.

[0052] 2. Test results:

[0053] The results are as follows Figure 1 As shown, after 10 hours of treatment, SlCOMT2 C344W The protein content still remains high, indicating that SlCOMT2 C344W The stability of the protein is significantly higher than that of SlCOMT2 protein, which indicates that SlCOMT2 C344W The enzymatic activity of the protein may also be higher than that of the SlCOMT2 protein.

[0054] Example 3: SlCOMT2 and SlCOMT2 C344W Analysis of COMT enzyme activity and melatonin content in proteins

[0055] 1. Test method:

[0056] Using WT, SlCOMT2-OE and SlCOMT2 C344W -OE tomato plant leaf tissues were used to determine COMT enzyme activity and melatonin content using high-performance liquid chromatography to evaluate the enzyme activity of the two proteins and the melatonin synthesis capacity.

[0057] COMT enzyme activity was determined according to the method described in "Caffeic acid O-methyltransferase is involved in the synthesis of melatonin by methylating N-acetylserotonin in Arabidopsis. Journal of Pineal Research 57:219-227" (DOI:10.1111 / jpi.12160). S-adenosyl-l-methionine and N-acetyl-5-hydroxytryptamine were used as substrates, and COMT activity was calculated based on the initial linear rate of melatonin production.

[0058] Melatonin content determination method: First, melatonin extraction was performed: frozen leaf samples were ground into a fine powder in liquid nitrogen. 400 mg of the powder was extracted with 4 mL of 50 mM sodium phosphate buffer (pH 7.9). After centrifugation at 500 g for 5 min, 80 μL of 0.1 M KOH was added to the supernatant, followed by 2 mL of chloroform extraction. The chloroform fraction was mixed with 0.5 mL of 0.45 M sodium borate buffer (pH 10.0) and centrifuged as described above. The sample was evaporated and dissolved in 0.1 mL of water, and a 10 μL aliquot was taken for subsequent analysis. Melatonin was then analyzed using an Agilent 1200 high-performance liquid chromatography system (Agilent Technologies). An API 5000 tandem mass spectrometer (AB Sciex, Darmstadt, Germany) equipped with a turbospray ion source was operated in negative ionization mode. The parent ion of melatonin and its corresponding fragments were analyzed by multiple reaction monitoring. Data were collected and processed using ANALYST 1.6 software (Applied Biosystems, Shanghai, China). The linearity of the ionization efficiency was confirmed by analyzing serial dilutions of the standard mixture. Melatonin concentrations were determined relative to the corresponding internal standard.

[0059] 2. Test results:

[0060] The results are as follows Figure 2 As shown, SlCOMT2-OE and SlCOMT2 were found C344W -OE tomato plants had higher COMT enzyme activity and melatonin content than WT, and SlCOMT2 C344W -OE tomato plants also had higher COMT enzyme activity and melatonin content than SlCOMT2-OE.

[0061] Example 4: S1COMT2 C344W Effects of protein on broad-spectrum resistance in tomato

[0062] 1. Test method:

[0063] (1) Biological stress resistance:

[0064] WT, SlCOMT2-OE and SlCOMT2 C344W -OE tomato plants were inoculated with Botrytis cinerea (ATCC58025), and the disease resistance of different tomato plants was determined by the leaf disease phenotype and lesion area statistics. The specific method is as follows:

[0065] WT, SlCOMT2-OE and SlCOMT2 C344W -OE-primed seeds were sown in small pots containing a seedling medium (turf:vermiculite 2:1). During cultivation, the photosynthesis cycle was 12 / 12 (day / night), the temperature was 18°C / 28°C (day / night), and the humidity was 80-90%. When the plants were 4-5 weeks old, they were inoculated with Botrytis cinerea spores and the leaf lesion area was observed.

[0066] (2) Resistance to abiotic stress:

[0067] WT, SlCOMT2-OE and SlCOMT2 C344W -OE tomato plants were used as experimental objects, and four treatments were set up in the experiment: control, high temperature stress, low temperature stress and salt stress. C344W -OE seeds were germinated and sown, and when they had three fully expanded leaves, the tomato seedlings were transplanted into plastic pots (length 6.5 cm, width 6.5 cm, height 9.8 cm). After acclimatization for 7 days, the plants were placed in the above four environments, with each treatment of WT, SlCOMT2-OE and SlCOMT2 C344W -OE 20 plants each. The temperature of the control treatment group was set at 18℃ / 28℃ (day / night), and the wild type and SlCOMT2 C344W Tomato plants (pTRV-WRKY50) were treated at 36℃ / 31℃ (day / night) for 72h. C344W Tomato plants (pTRV-WRKY50) were treated at 25°C / 4°C (day / night) for 48 h. The wild type and SlCOMT2 plants were irrigated with 100 ml of 200 mM NaCl solution on days 1, 3, 5, and 7 of salt stress treatment. C344W Tomato plants were treated for 12 days.

[0068] 2. Test results:

[0069] The results are as follows Figure 3 As shown, WT, SlCOMT2-OE and SlCOMT2 C316W-OE tomato plants were inoculated with Botrytis cinerea or placed under different abiotic stress environments, and the stress resistance of different tomato plants was determined by leaf disease phenotype, lesion area, and mortality of tomato seedlings.

[0070] The results showed that SlCOMT2 C316W -OE tomato plants inoculated with Botrytis cinerea showed no obvious disease phenotype ( Figure 3 A), the lesion area is smaller than that of SlCOMT2-OE( Figure 3 B) Description of SlCOMT2 C316W -OE showed higher resistance to biotic stress than SlCOMT2-OE.

[0071] SlCOMT2 C316W -OE tomato plant mortality test results under high temperature, low temperature, saline-alkali stress Figure 3 As shown in C, the results showed that SlCOMT2 C316W -OE showed higher resistance to abiotic stress than SlCOMT2-OE.

[0072] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A caffeic acid-O-methyltransferase 2 gain-of-function mutant, characterized in that The amino acid sequence of the caffeic acid-O-methyltransferase 2 gain-of-function mutant is shown in SEQ ID NO.

2.

2. The gene encoding the caffeic acid-O-methyltransferase 2 gain-of-function mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The coding gene is a nucleic acid molecule as shown in the following i) or ii): i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.4; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 2 except i).

4. A recombinant expression vector comprising a gene encoding the caffeic acid-O-methyltransferase 2 gain-of-function mutant according to claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that The recombinant expression vector is a pCAMBIA1306 vector containing a gene encoding a gain-of-function mutant of caffeic acid-O-methyltransferase 2.

6. Use of the caffeic acid-O-methyltransferase 2 gain-of-function mutant of claim 1, the gene encoding the caffeic acid-O-methyltransferase 2 gain-of-function mutant of claim 2 or 3, or the recombinant expression vector containing the gene encoding the caffeic acid-O-methyltransferase 2 gain-of-function mutant of claim 4 in at least one of the following (1) to (4): (1) Increase the melatonin content in tomatoes; (2) Improve the resistance of tomatoes to biotic stress; (3) Improve the resistance of tomatoes to abiotic stress; (4) Tomato resistance breeding; The biological stress is tomato gray mold caused by infection of Botrytis cinerea; The abiotic stress is high temperature, low temperature and / or saline-alkali stress.

7. A method for improving the ability of caffeic acid-O-methyltransferase 2 to synthesize melatonin, characterized in that: The following steps are involved: The amino acid 344 of caffeic acid-O-methyltransferase 2 shown in SEQ ID NO.1 is mutated to tryptophan; Alternatively, the nucleotide 1032 of the gene encoding caffeic acid-O-methyltransferase 2 shown in SEQ ID NO. 3 is mutated to G.

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