Caffeic acid-O-methyltransferase 2 function-acquired mutant, recombinant expression vector and application thereof

Abstract

The invention discloses a caffeic acid-O-methyltransferase 2 function acquired 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 344 amino acid of tomato caffeic acid-O-methyltransferase 2 is changed into tryptophan (W) from cysteine (C), the melatonin content of tomatoes can be increased, the tomatoes can be endowed with broad-spectrum resistance to abiotic and biological stress, and the tomato caffeic acid-O-methyltransferase 2 can serve as a key target for tomato resistance breeding.

Description

Technical Field

[0001] The 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 to carry out biological breeding, the above problems can be effectively solved.

[0003] Melatonin is a small molecule that is beneficial to human health. It is widely present in various plants and can enhance the broad-spectrum resistance of plants to various biotic and abiotic stresses. Improving melatonin content is one of the goals of crop quality and resistance breeding. Although the regulatory genes and metabolic pathways of melatonin synthesis have been reported, little is known about how to modify and utilize these synthetic genes to further promote melatonin accumulation in plants and improve plant resistance.

[0004] Caffeic acid-O-methyltransferase (COMT) is a key enzyme in the biosynthetic pathway of plant phenylpropanoids. It has a wide range of substrates and can catalyze a variety of phenylpropanoid compounds and flavonoids. In recent years, studies have found that some COMTs also have a similar effect to N-acetyl-5-hydroxytryptamine methyltransferase in the biosynthesis of melatonin, and can catalyze N-acetyl-5-hydroxytryptamine to produce melatonin. Caffeic acid-O-methyltransferase 2 (Solanum lycopersicum caffeic acidO-methyltransferase 2, SlCOMT2) is an effective melatonin synthase gene in tomatoes, which can promote the synthesis of melatonin. So far, there has been no report on the modification of caffeic acid-O-methyltransferase 2 in tomatoes to improve the melatonin content and stress resistance of tomatoes. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a gain-of-function mutant of caffeic acid-O-methyltransferase 2, a recombinant expression vector and its application. The present invention improves the protein stability and the ability to synthesize melatonin by performing site modification on tomato caffeic acid-O-methyltransferase 2, and can be used as a key target for tomato resistance breeding.

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

[0007] In a first aspect of the present invention, a gain-of-function mutant of caffeic acid-O-methyltransferase 2 is provided, wherein the gain-of-function mutant of caffeic acid-O-methyltransferase 2 is obtained by mutating the 344th amino acid of caffeic acid-O-methyltransferase 2; the amino acid sequence of 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 344th amino acid of caffeic acid-O-methyltransferase 2 is mutated to other amino acids except 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 gain-of-function mutant of caffeic acid-O-methyltransferase 2 is obtained by mutating the 344th cysteine ​​(C) of caffeic acid-O-methyltransferase 2 to tryptophan (W); the amino acid sequence of the gain-of-function mutant of caffeic acid-O-methyltransferase 2 formed after the mutation is shown in SEQ ID NO.2.

[0010] In a second aspect of the present invention, a gene encoding a gain-of-function mutant of caffeic acid-O-methyltransferase 2 is provided.

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

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

[0013] ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

[0014] The third aspect of the present invention provides a recombinant expression vector containing a gene encoding a gain-of-function mutant of caffeic acid-O-methyltransferase 2.

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

[0016] In a fourth aspect of the present invention, there is provided 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) Increase the melatonin content 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 application, the biological stress mainly refers to the stress caused by pathogen infection on plants, for example, tomato gray mold caused by infection of 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] In a fifth aspect, 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 discovers for the first time that by changing the 344th amino acid of tomato caffeic acid-O-methyltransferase 2 from cysteine ​​(C) to tryptophan (W), the melatonin content of tomatoes can be increased, and tomatoes can be given broad-spectrum resistance to abiotic and biotic stresses, which can be used as a key target for tomato resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : SlCOMT2 and SlCOMT2 C344W The 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 are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those 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 is 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 has discovered through in-depth research the key target in SlCOMT2 that can increase the activity of COMT enzyme, namely: the 344th amino acid of SlCOMT2. By mutating the 344th cysteine ​​(C) to tryptophan (W), a gain-of-function mutant of caffeic acid-O-methyltransferase 2 can be obtained, which is 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 gene) is shown in SEQ ID NO.4, and is as follows:

[0042]

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

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

[0046] Example 1: SlCOMT2-OE and SlCOMT2 C344W -OE tomato plant construction

[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 a binary Agrobacterium tumefaciens vector" (DOI: 10.1038 / nbt0787-726). The wild-type (WT) tomato variety used in 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 the same, and SlCOMT2-OE and SlCOMT2 were extracted 0, 5 and 10 h after treatment. C316W -OE tomato leaves, total protein, and Western blotting combined with FLAG antibody to identify remaining SlCOMT2 and SlCOMT2 C316W The protein content was used to evaluate the difference in stability between the two proteins.

[0052] 2. Test results:

[0053] The results are as follows Figure 1 As shown, after 10 h of treatment, SlCOMT2 C344W The protein content still remained 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 of proteins

[0055] 1. Test method:

[0056] Using WT, SlCOMT2-OE and SlCOMT2 C344W -OE tomato plant leaf tissues were used to measure COMT enzyme activity and melatonin content in combination with HPLC to evaluate the enzyme activity of the two proteins and the melatonin synthesis capacity.

[0057] The COMT enzyme activity was determined according to the method of “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: frozen leaf samples were ground into fine powder in liquid nitrogen. 400 mg of powder was extracted with 4 mL of 50 mM sodium phosphate buffer (pH 7.9). After centrifugation at 13 500 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 then centrifuged as above. The sample was evaporated and dissolved in 0.1 mL of water, and 10 μL aliquots were taken for subsequent analysis. Then, melatonin analysis was performed using an Agilent 1200 high-performance liquid chromatography system (Agilent Technologies). An API 5000 tandem mass spectrometer (AB Sciex, Darmstadt, Germany) was equipped with a turbospray ion source and operated in negative ionization mode. The parent ions of melatonin and their corresponding fragments were analyzed by multiple reaction monitoring. The 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. The concentration of melatonin was determined relative to the corresponding internal standard.

[0059] 2. Test results:

[0060] The results are as follows Figure 2 As shown, it was found that SlCOMT2-OE and SlCOMT2 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 Effect of protein on broad-spectrum resistance of 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 leaf disease phenotype and lesion area statistics. The specific method is as follows:

[0065] WT, SlCOMT2-OE and SlCOMT2 C344W -OE germinated seeds were sown in small pots with seedling medium (turf: vermiculite 2:1). During the cultivation period, the photoperiod was 12 / 12 (day / night), the temperature was 18℃ / 28℃ (day / night), and the humidity was 80-90%. When the plants were 4-5 weeks old, they were inoculated with gray mold spores and the leaf spot area was observed.

[0066] (2) Resistance to abiotic stress:

[0067] Based on WT, SlCOMT2-OE and SlCOMT2 C344W -OE tomato plants were used as the 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 (6.5 cm long, 6.5 cm wide, and 9.8 cm high). 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 under salt stress. C344W Tomato plants, 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 in different abiotic stress environments, and the resistance of different tomato plants to stress was determined by the 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 SlCOMT2 C316W -OE has higher resistance to biotic stress than SlCOMT2-OE.

[0071] SlCOMT2 C316W -OE tomato plant mortality test results under high temperature, low temperature, salinity and 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 only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A gain-of-function mutant of caffeic acid-O-methyltransferase 2, characterized in that The caffeic acid-O-methyltransferase 2 gain-of-function mutant is obtained by mutating the 344th amino acid of caffeic acid-O-methyltransferase 2; the amino acid sequence of the caffeic acid-O-methyltransferase 2 is shown in SEQ ID NO.

1.

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

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

4. The coding gene according to claim 3, characterized in that The coding gene is a nucleic acid molecule as shown in i) or ii) below: 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).

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

6. The recombinant expression vector according to claim 5, 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.

7. Use of the caffeic acid-O-methyltransferase 2 gain-of-function mutant according to claim 1 or 2, the coding gene of the caffeic acid-O-methyltransferase 2 gain-of-function mutant according to claim 3 or 4, or the recombinant expression vector containing the caffeic acid-O-methyltransferase 2 gain-of-function mutant coding gene according to claim 5 in at least one of the following (1)-(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.

8. The use according to claim 7, characterized in that: The biological stress is tomato gray mold caused by infection of Botrytis cinerea.

9. The use according to claim 7, characterized in that: The abiotic stress includes: high temperature, low temperature and / or saline-alkali stress.

10. 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 the 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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