An NtCOMT gene and its application in increasing the secretion amount of methyl ferulate in roots and disease resistance

Abstract

The present invention provides an NtCOMT gene and its application in increasing the secretion amount of methyl ferulate in roots and disease resistance. The nucleotide sequence of the NtCOMT gene is shown as SEQ ID NO.1. After overexpression of the NTCOMT10 gene improved in the present invention, the content of methyl ferulate in root exudates can be increased, and the disease resistance of plants can be improved, which can provide very good ideas and theoretical basis for biological control and disease-resistant breeding.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to an NtCOMT gene and its application in increasing the secretion amount of methyl ferulate in roots and disease resistance. Background Art

[0002] Using plant primary and secondary metabolites to control plant diseases is one of the hot research contents in current biological control and disease-resistant breeding. For soil-borne diseases, plant root exudates play an important bridging and regulatory role in the entire disease triangle. Screening and identifying key active components with antibacterial functions from root exudates and increasing their secretion amount have far-reaching significance for exploring new ways of biological control of diseases.

[0003] Caffeic acid O-methyltransferases (COMT) are a class of multifunctional enzymes and are enzymes in the biosynthesis pathways of lignin and flavonoids. In Arabidopsis thaliana, it has been reported to be involved in the conversion of caffeic acid to ferulic acid, and in the conversion of coniferaldehyde / coniferyl alcohol to sinapaldehyde / sinapyl alcohol to form G and S units of lignin (Guo et al., 2001). Overexpression of COMTs can help plant growth and improve drought resistance because it can catalyze the production of melatonin from n-acetylserotonin (Goujon et al., 2003; Yang et al., 2019). In sorghum, the COMT gene has been reported to be involved in the synthesis of luteolin and syringin (Eudes et al., 2017). In blueberries, the COMT gene has been reported to be involved in fruit growth and development by regulating lignin synthesis (Liu et al., 2021).

[0004] Therefore, it is necessary to seek a method that can increase the secretion amount of methyl ferulate in roots and disease resistance. Summary of the Invention

[0005] The object of the present invention is to provide an NtCOMT gene and its application in increasing the secretion amount of methyl ferulate in roots and disease resistance.

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

[0007] In the first aspect of the present invention, an NtCOMT gene is provided, and the nucleotide sequence of the NtCOMT gene is as shown in SEQ ID NO.1.

[0008] In the second aspect of the present invention, an NtCOMT protein is provided, and the NtCOMT protein is encoded by the gene, and the amino acid sequence is as shown in SEQ ID NO.2.

[0009] In the third aspect of the present invention, there is provided an expression cassette of the NtCOMT gene, and the expression cassette contains the NtCOMT gene.

[0010] In the fourth aspect of the present invention, there is provided an expression vector of the NtCOMT gene, and the expression vector contains the expression cassette.

[0011] As an optional embodiment, the construction method of the expression vector of the NtCOMT gene is as follows:

[0012] The primers shown in SEQ ID NO.3 - SEQ ID NO.4 are used to amplify a DNA fragment containing the complete coding region of the NtCOMT10 gene using tobacco genomic cDNA as a template, and then ligated into the pRI101-AN expression vector digested with restriction enzymes KpnI and BamH1 (pRI101-AN dicotyledonous plant hairy root agrobacterium plasmid product number: P0652) to obtain the expression vector of the NtCOMT gene.

[0013] Further, the vector selected for constructing the recombinant vector is a vector that can direct the expression of foreign genes in plants.

[0014] The vector selected for constructing the recombinant expression vector is a Ti plasmid or a plant virus vector.

[0015] In the fifth aspect of the present invention, there is provided a transformant of the NtCOMT gene, and the transformant contains the expression vector.

[0016] Preferably, the host of the transformant is a plant.

[0017] The plant is tobacco or other plants such as rice.

[0018] In the sixth aspect of the present invention, there is provided the application of the NtCOMT gene, the NtCOMT protein, the expression cassette, the expression vector, or the transformant in increasing the content of methyl ferulate in root exudates.

[0019] In the seventh aspect of the present invention, there is provided the application of the NtCOMT gene, the NtCOMT protein, the expression cassette, the expression vector, or the transformant in increasing the resistance of tobacco to Phytophthora parasitica var. nicotianae.

[0020] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0021] The present invention provides an NtCOMT gene and its application in increasing the secretion amount of methyl ferulate in roots and disease resistance. The applicant analyzed the changes in the expression levels of these family members in disease-resistant and disease-susceptible varieties and found that the expression level of NtCOMT10, a member of the second subfamily, changed the most significantly. It was significantly up-regulated after inoculation in the disease-resistant variety (R) and its expression intensity was higher than that in the disease-susceptible variety (S) (as Figure 1 ). Subsequently, the applicant constructed overexpression materials of NtCOMT10 in tobacco and obtained four overexpression materials ( Figure 2 ). Two of them were selected for LC-MS detection of the content of methyl ferulate in root exudates. The detection results showed that after overexpression of the enhanced NTCOMT10 gene, the content of methyl ferulate in root exudates could be increased, and the disease resistance of plants could be improved, which could provide very good ideas and theoretical basis for biological control and disease-resistant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 It is for the analysis of the COMT gene family and expression level analysis in tobacco; among them, Figure 1 A is the phylogenetic analysis and gene structure analysis of COMT family members in the tobacco genome, Figure 1 B is the gene expression levels of COMT family members before and 60 h after inoculation in disease-resistant and disease-susceptible varieties.

[0024] Figure 2 It is for the positive detection of NtCOMT10 overexpression materials;

[0025] Figure 3 It is for the detection results of the content of methyl ferulate in root exudates;

[0026] Figure 4 It is the result that overexpression of the tobacco NtCOMT10 gene can improve the resistance to tobacco black shank. A is the disease-resistant phenotype, B is the disease index, C is the number of pathogenic bacteria in the soil, * represents p < 0.05, and ** represents p < 0.01. DETAILED DESCRIPTION OF THE INVENTION

[0027] The following will specifically describe the present invention in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than limiting the present invention.

[0028] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains. In case of any contradiction, this specification shall prevail.

[0029] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchase or can be obtained by existing methods.

[0030] The following will, in conjunction with examples and experimental data, elaborate in detail on a NtCOMT10 gene of the present application and its application in increasing the secretion amount of methyl ferulate in roots and disease resistance.

[0031] Example 1. Discovery of the NtCOMT gene

[0032] 1. Detection and analysis of gene expression levels

[0033] The applicant conducted a family analysis on the members of the caffeic acid O-methyltransferase family in tobacco and found that the COMT family in tobacco can be divided into three subfamilies. To further study their functions, the applicant analyzed the changes in the expression levels of these family members in a disease-resistant variety and a disease-susceptible variety (disease-resistant variety Innovation No. 3, disease-susceptible variety Xiaohuangjin 1025, sourced from the germplasm preserved by the Tobacco Research Institute of the Chinese Academy of Agricultural Sciences. For the specific disease resistance phenotype identification, see the literature: Zhang, C.S., Feng, C., Zheng, Y.F., Wang, J., and Wang, F.L. (2020). Root Exudates Metabolic Profiling Suggests Distinct Defense Mechanisms Between Resistant and Susceptible Tobacco Cultivars Against Black Shank Disease. Frontiers in Plant Science 11 ARTN 559775 10.3389 / fpls.2020.559775. The transcriptome data before and after inoculation have been entered into NCBI). The expression level data before and 60 h after inoculation were downloaded from the database for analysis.

[0034] 2. Results

[0035] The results are as Figure 1As shown in the figure, it was found that the expression level of NtCOMT10, a member of the second subfamily, changed the most significantly up-regulated after inoculation with the pathogen in the disease-resistant variety (R), and the expression intensity was higher than that in the disease-susceptible variety (S). Through sequencing analysis, the nucleotide sequence of the NtCOMT10 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0036] Example 2. Construction of an overexpression vector of the genetically transformed NtCOMT10 gene

[0037] 1. Creation and identification of transgenic materials

[0038] Vector construction: Use the NtCOMT10 gene primers

[0039] NtCOMT10-F: 5’-GGggtaccATGGGTTCAACAAGCGAGAG-3’ (SEQ ID NO.3),

[0040] NtCOMT10-R: 5’-CGggatccCTTGTTGAATTCCATGACCCAA-3’ (SEQ ID NO.4) to amplify a DNA fragment containing the complete coding region of the NtCOMT10 gene;

[0041] The PCR reaction conditions were: pre-denaturation at 94°C for 3 min; 94°C for 30 sec, 55°C for 30 sec, 72°C for 2 min, for 30 cycles; extension at 72°C for 5 min.

[0042] The obtained PCR product was ligated into the pRI101-AN expression vector digested with the restriction enzymes KpnI and BamH1 by the Gibson Assembly method, and the vector was sequenced and confirmed. Finally, an overexpression vector of the NtCOMT10 gene available for genetic transformation was obtained.

[0043] 2. Genetic transformation: Remove the leaf margins and veins of sterile tobacco seedlings, cut them into appropriate sizes, place them in MS medium, and pre-culture for 2 d; culture the Agrobacterium tumefaciens solution containing the target gene until OD 600 = 0.6 - 0.8, centrifuge to discard the supernatant, and resuspend the cells with pre-cooled MS0 to make its OD 600≈0.8; Place the pre-cultured leaves in the bacterial solution, take them out after 8 min of infection, blot the excess bacterial solution with sterilized filter paper, place the infected leaves with the veins facing up, spread them flat on medium G, and conduct dark culture for 2 - 3 d; Wash the co-cultured explants 3 times with sterilized water containing Cef, then blot them dry with sterilized filter paper, inoculate the leaves with the veins facing down onto the S1 differentiation medium, and conduct dark culture for 2 - 3 weeks; When adventitious buds grow from the leaf margins and the bud length is 0.1 - 0.5 cm, cut the S1 adventitious buds and transfer them to the S2 differentiation medium, conduct light culture for 1 - 2 weeks, and the adventitious buds grow into young seedlings; Break off the S2 seedlings onto the S3 medium, conduct light culture for 1 - 2 weeks, and the seedlings gradually become robust; Remove the swollen part at the bottom and the yellowed leaves at the lower part of the S3 robust seedlings, inoculate them onto the rooting medium, and conduct light culture for 1 - 2 weeks at 28 °C; Identify positive seedlings using specific primers and conduct relevant experiments.

[0044] F: TGGCGGAATCCCATTCAA; R: GGATCTGTGCCATGGTACTCAA.

[0045] 3. Results: Overexpression materials of NtCOMT10 were constructed in tobacco, and four overexpression materials were obtained as Figure 2 shown. Next, two of them were selected for LC-MS detection of the content of methyl ferulate in root exudates.

[0046] Example 3. Overexpression of tobacco NtCOMT10 gene increases the content of methyl ferulate in root exudates

[0047] 1. Collection of root exudates

[0048] The root exudates of the tested tobacco seedlings were collected by the root immersion method. Carefully take out the tobacco seedlings from the pots, carefully wash the soil particles on the roots, rinse the roots with deionized water, and then place the roots into 100 mL of distilled water to collect root exudates for 12 h;

[0049] 2. Determination of the content of methyl ferulate

[0050] The content of methyl ferulate was accurately qualitatively and quantitatively determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS).

[0051] The detection results are as Figure 3 shown. Compared with the wild type, the methyl ferulate content in the root exudates of these three overexpression families can be increased.

[0052] Example 4. Determination of the resistance of tobacco to Phytophthora parasitica var. nicotianae

[0053] Identify the disease-resistant phenotypes of the above two overexpression families. Conduct the inoculation treatment with the pathogen of Phytophthora parasitica var. nicotianae 14 days after transplantation. The specific operation is as follows:

[0054] Pot seedling stage: Inoculation was carried out when the tobacco plants grew to 6 true leaves. Each plant was inoculated with 10 mL of Phytophthora parasitica var. nicotianae zoospores by root injury (inoculating an equal volume of sterile water as a control). 7 days after inoculation, the disease incidence was investigated and the disease index was calculated. There were at least 30 tobacco plants in each treatment, with 3 replicates.

[0055] The results are as Figure 4 shown, indicating that the disease resistance index of the overexpression materials was significantly reduced, and the disease resistance to Phytophthora parasitica var. nicotianae was significantly improved.

[0056] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

[0057] Finally, it should also be noted that the term "comprising", "including" or any other variation thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and variations.

Claims

1. NtCOMT A gene or an expression cassette containing the NtCOMT gene, or an expression vector containing the expression cassette, or a transformant containing the expression vector is used for increasing the content of methyl ferulate in the root exudates of tobacco plants, characterized in that The said NtCOMT The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. NtCOMT The application of a gene, an NtCOMT protein, or an expression cassette containing the NtCOMT gene, or an expression vector containing the expression cassette, or a transformant containing the expression vector in enhancing the resistance of tobacco to Phytophthora parasitica var. nicotianae, characterized in that The NtCOMT nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the NtCOMT protein is shown in SEQ ID NO.2.

Images