Application of TaARS1 gene in regulating plant alkylresorcinol and / or plant resistance

Abstract

The present invention belongs to the technical fields of genetic engineering and plant disease control, and specifically relates to the use of the TaARS1 gene in regulating plant alkylresorcinol production and / or plant resistance. The present invention provides uses of the TaARS1 gene; the amino acid sequence of the protein encoded by the TaARS1 gene is shown in SEQ ID NO: 3. The development and utilization of the TaARS1 gene in the present invention can effectively increase the types and content of alkylresorcinols in wheat and other crops, enhance plant resistance to Fusarium, particularly Fusarium graminearum, and improve the nutritional value of related crops.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and plant disease prevention and control, and specifically relates to the application of the TaARS1 gene in regulating plant alkylresorcinol and / or plant resistance. Background Art

[0002] Alkylresorcinols (ARs) are a special class of phenolic lipids. They are a general term for derivatives of 1,3-resorcinol in which the 5-position of the benzene ring is substituted with an odd-numbered alkyl group. These are amphiphilic, consisting of a hydrophilic resorcinol ring synthesized via the polyketide synthesis pathway and a hydrophobic alkyl chain synthesized via the fatty acid synthesis pathway. ARs are important components of wheat bran and possess a variety of physiological and biological activities, including anticancer, antioxidant, antibacterial, biomarker, and enzyme inhibition. Therefore, the application of ARs in crop disease resistance breeding and improving crop nutritional quality is of great significance.

[0003] Fusarium graminearum is a plant pathogenic fungus that mainly infects wheat, barley, corn and other cereal economic crops, causing wheat scab and corn stalk rot, reducing the yield and quality of grain crops (Chen Y, Kistler HC, Ma Z (2019) Fusarium graminearum Trichothecene Mycotoxins: Biosynthesis, Regulation, and Management. Annu Rev Phytopathol 57: 15-39.). The conidia produced by asexual reproduction of Fusarium graminearum are spread by wind, rain, etc., fall on wheat leaves and ears, and can infect all developmental stages of wheat. The most harmful thing is to infect wheat ears during the flowering period, and in severe cases, it causes wheat crop failure. Therefore, it is particularly important to excavate and identify resistance substances of Fusarium graminearum and analyze its regulatory mechanism in this field. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of the TaARS1 gene in regulating plant alkylresorcinol and / or plant resistance, regulating the type and content of alkylresorcinol in plants, regulating the resistance of plants to Fusarium, especially Fusarium graminearum, and regulating the nutritional value of plants.

[0005] The present invention provides the use of the TaARS1 gene in one or more of the following:

[0006] (1) Regulate the types of plant alkylresorcinols;

[0007] (2) regulating the content of plant alkylresorcinols;

[0008] (3) regulating plant pathogen resistance; the pathogens include Fusarium;

[0009] The amino acid sequence of the protein encoded by the TaARS1 gene is shown in SEQ ID NO: 3.

[0010] Preferably, the regulating the types of plant alkylresorcinols includes: overexpressing the TaARS1 gene to increase the types of plant alkylresorcinols; the types of plant alkylresorcinols include one or more of C17:0, C19:0, C21:0 and C23:0.

[0011] Preferably, the regulating the content of plant alkylresorcinol comprises: overexpressing TaARS1 gene to increase the content of plant alkylresorcinol.

[0012] Preferably, the plant alkylresorcinol species include alkylresorcinol species in plant seeds;

[0013] The plant alkylresorcinol content includes the alkylresorcinol content in plant seeds.

[0014] Preferably, the regulating plant pathogen resistance includes: overexpressing TaARS1 gene to improve plant pathogen resistance.

[0015] Preferably, the Fusarium species includes Fusarium graminearum.

[0016] Preferably, the plants include wheat and / or corn.

[0017] Preferably, the nucleotide sequence of the coding region of the TaARS1 gene is shown in SEQ ID NO: 2.

[0018] Preferably, the full-length genomic nucleotide sequence of the TaARS1 gene is shown in SEQ ID NO: 1.

[0019] The present invention provides a method for increasing the types and contents of alkylresorcinols in plants and improving plant resistance, which involves overexpressing the TaARS1 gene in plants; the amino acid sequence of the protein encoded by the TaARS1 gene is shown in SEQ ID NO: 3;

[0020] The pathogenic bacteria include Fusarium.

[0021] Beneficial effects:

[0022] The present invention provides applications of the TaARS1 gene; the amino acid sequence of the protein encoded by the TaARS1 gene is shown in SEQ ID NO: 3. The development and utilization of the TaARS1 gene in the present invention can effectively increase the types and contents of alkylresorcinols in wheat and other crops, enhance plant resistance to Fusarium, especially Fusarium graminearum, and improve the nutritional value of related crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0024] Figure 1 This is a heat map of the content of alkylresorcinol in the tissues of Aikang 58 wheat throughout its growth period;

[0025] Figure 2 The phylogenetic tree of candidate genes for the amino acid sequence of alkylresorcinol synthase;

[0026] Figure 3 This is the expression calorimetry of TaARS candidate genes in samples from all growth stages;

[0027] Figure 4 Figure 2 shows the differentially expressed alkylresorcinols in TaARS1-expressing recombinant yeast and wild-type yeast, as well as the biosynthetic pathway diagram of alkylresorcinols; A represents the differentially expressed alkylresorcinols; B represents the biosynthetic pathway diagram;

[0028] Figure 5 Schematic diagram of the genetic transformation vector structure for overexpressing TaARS1 in wheat;

[0029] Figure 6 Schematic diagram of the genetic transformation vector structure for overexpressing TaARS1 in maize;

[0030] Figure 7 The results of alkylresorcinol content and TaARS1 gene expression in wild-type wheat and transgenic wheat overexpressing TaARS1 are shown in Figure 2; A represents the alkylresorcinol content, B represents the TaARS1 gene expression, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001;

[0031] Figure 8 is the alkylresorcinol content in the kernels of wild-type corn and transgenic corn overexpressing TaARS1;

[0032] Figure 9 The in vitro antibacterial activity test results of different alkylresorcinols; A is the plate map; B is the statistical result of mycelial area; ** indicates P < 0.01, *** indicates P < 0.001;

[0033] Figure 10 This is the phenotype of wheat resistance to Fusarium graminearum under different alkylresorcinols;

[0034] Figure 11 Statistical results of the infection rate of Fusarium graminearum on wheat under different alkylresorcinols; ** indicates P < 0.01, *** indicates P < 0.001. DETAILED DESCRIPTION

[0035] The present invention provides the use of the TaARS1 gene in one or more of the following: (1) regulating the types of plant alkylresorcinols; (2) regulating the content of plant alkylresorcinols; (3) regulating plant pathogen resistance; the pathogens include Fusarium spp.; the amino acid sequence of the protein encoded by the TaARS1 gene is shown in SEQ ID NO: 3.

[0036] In one embodiment, the present invention regulates the types of plant alkylresorcinols, including: overexpressing the TaARS1 gene to increase the types of plant alkylresorcinols. In one embodiment, the types of plant alkylresorcinols include one or more of C17:0, C19:0, C21:0, and C23:0. In another embodiment, the types of plant alkylresorcinols include C17:0, C19:0, C21:0, and C23:0. In one embodiment, the types of plant alkylresorcinols include those found in plant seeds.

[0037] In one embodiment, regulating the alkylresorcinol content of plants according to the present invention includes: overexpressing the TaARS1 gene to increase the alkylresorcinol content of plants. In one embodiment, increasing the alkylresorcinol content of plants according to the present invention includes increasing the content of one or more of C17:0, C19:0, C21:0, and C23:0 in plants. In another embodiment, increasing the alkylresorcinol content of plants according to the present invention includes increasing the content of C17:0, C19:0, C21:0, and C23:0 in plants. In one embodiment, the alkylresorcinol content of plants according to the present invention includes the alkylresorcinol content in plant seeds.

[0038] As an embodiment, the CAS numbers of C17:0, C19:0, C21:0 and C23:0 described in the present invention are 41442-57-3, 35176-46-6, 70110-59-7 and 70110-60-0, respectively.

[0039] In one embodiment, the regulating plant pathogen resistance described herein includes overexpressing the TaARS1 gene to increase plant pathogen resistance. In one embodiment, the plant diseases described herein include wheat head blight and / or corn stalk rot. In one embodiment, the Fusarium described herein includes Fusarium graminearum. In one embodiment, the plants described herein include wheat and / or corn.

[0040] In one embodiment, the coding region nucleotide sequence of the TaARS1 gene of the present invention is shown as SEQ ID NO: 2. In one embodiment, the full-length genomic nucleotide sequence of the TaARS1 gene of the present invention is shown as SEQ ID NO: 1.

[0041] The present invention provides a method for increasing the type and content of plant alkylresorcinols and improving plant resistance, which involves overexpressing the TaARS1 gene in plants; the amino acid sequence of the protein encoded by the TaARS1 gene is shown in SEQ ID NO: 3; and the pathogenic bacteria include Fusarium.

[0042] As an embodiment, the plant of the present invention is wheat and / or corn. The present invention has no strict requirements on the method of overexpressing the TaARS1 gene, and conventional methods in the art can be used.

[0043] To further illustrate the present invention, the application of the TaARS1 gene provided by the present invention in regulating plant alkylresorcinol and / or plant resistance is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Acquisition of the TaARS1 gene

[0046] 1. TaARS1 gene identification and structural analysis

[0047] (1) Two replicate samples of 16 tissues of Ayikang 58 wheat, including roots at the one-leaf-one-heart stage, leaves at the one-leaf-one-heart stage, leaves at the three-leaf-one-heart stage, leaves at the rising stage, leaves at the jointing stage, flag leaf sheaths at the flowering stage, stem nodes at the flowering stage, husks, stamens, and grains (filling weeks I-V, maturity), were used as materials. The freeze-dried samples were ground into powder and extracted with 100% methanol to obtain the content of alkylresorcinols (ARs) at different stages of wheat. The results showed that the main accumulation period of ARs in wheat was during the grain development stage ( Figure 1 ), it was speculated that alkylresorcinol synthase involved in ARs biosynthesis was mainly expressed in grains.

[0048] (2) Six characterized alkylresorcinol synthases (ARS) were compared with the whole genome sequence of Chinese spring wheat for local blast analysis. By comparing the amino acid sequences, genes with E values ​​close to zero and amino acid sequence similarity of more than 50% were selected. After removing duplicates, 38 candidate genes were initially screened from the six blast results, of which 12 candidate genes were found by searching the six characterized genes. The Pfam database annotated these 38 genes as Chalcone and stilbene synthases (PF00195 / PF02797). The conserved domain analysis of the candidate genes was further performed using the NCBI Batch CD-search. The results showed that these 38 genes all belonged to the superfamily PLN03171, which is the same superfamily as the characterized ARS, but different from the CHS (belonging to the superfamily PLN03169 / PLN03170). The amino acid sequences of the 38 wheat ARS candidate genes were compared with the amino acid sequences of ARS, CHS, and STS published in other species, and 5 pseudogenes with large sequence deletions were manually screened out. The remaining 33 candidate genes were subjected to phylogenetic analysis using MEGA11 software. The results are shown in Figure 2 .

[0049] (3) Transcriptome sequencing was performed on multiple tissue samples of Dwarf Anti-58 wheat throughout its growth period. Based on RNA-seq data and combined with local Blast results, genes with similar expression patterns to metabolite accumulation patterns were identified. The results are shown in the figure. Figure 3 .

[0050] (4) Based on previous research on ARs in wheat resistance to Fusarium graminearum and the transcriptome data of wheat infected with Fusarium graminearum from the WheatOmics website, the structural gene was selected from the wheat genome data, with the number TraesCS6B01G022500.1, and named TaARS1. The nucleotide sequence of the full-length TaARS1 genome is 1713 bp (see SEQ ID NO: 1 in the sequence listing), the nucleotide sequence of the coding region is 1236 bp (see SEQ ID NO: 2 in the sequence listing), and the amino acid sequence of the coding region is 411 (see SEQ ID NO: 3 in the sequence listing). The gene contains one intron.

[0051]

[0052]

[0053] SEQ ID NO: 3: MGSIGTANGNGNGIGHGSAPVAAGRQHAEGPAAMLGI GTANPTGVEVPQNVFAENLFRVTKSDHLTELQQKLTRICEKTGIDKRHFHLTEETLVAHPELYDHDAQSLDNRLAMTVDAVPKLAQCAAAKAIAEWGRPASEITHLVFSTYSAWGAPSADLRLATLLGLRPTVSRTILSLHGCYGGGRALGLARELAENNRGARVLVACAEITLVCFGGPDGGNLVGH ALFGDGAGAVIVGAGPFRDGEQSPIFEMVHATQTTVPKTEHALGMQVSGSGVDFHLAIQVPTLIGQNVERCLLHAFRGGDDDDNDDGGAHLPSPLSGNGKWNDLFWAVHPGGRPILDNIDMVLKLEPEKLAASRHVLREYGNMSGATIVFVLDELRRRRSLLPEWGAMLAFGPGVTIETMVLRCPR*.

[0054] 2. Amplification of the TaARS1 gene

[0055] Based on the sequence information of the TaARS1 gene in the wheat "Chinese Spring" genome, to obtain the full-length cDNA sequence of the TaARS1 gene, total RNA was extracted from the wheat variety "Chinese Spring" (CS), reverse transcribed into cDNA, and amplified using primers (TaARS1-F: 5'-ATGGGAAGCATCGGAACCGC-3', SEQ ID NO: 4; TaARS1-R: 5'-CTAGCGTGGACAGCGGAGCA-3', SEQ ID NO: 5). The PCR-amplified target fragment was ligated with a blunt-ended vector. The reaction product was transformed into Escherichia coli DH5α and revived in 1 mL of LB for 1 hour. 150 μL of the revived bacterial suspension was plated on a plate containing LB medium containing Amp-resistant resistant bacteria and incubated at 37°C for 12 hours. Single clones were detected by PCR, and the plasmid was extracted and sequenced. The PCR product was sequenced to obtain the cDNA sequence of the TaARS1 gene. A 1236-bp TaARS1 gene sequence was amplified from CS. After the sequence alignment is correct, homologous recombination is performed with the target vector respectively.

[0056] Example 2

[0057] Functional verification of TaARS1 by heterologous expression in yeast

[0058] 1. Construction of TaARS1 heterologous expression vector in yeast

[0059] The 1236 bp TaARS1 gene fragment amplified from CS in Example 1 was subjected to homologous recombination with the target vector pYES2. The reaction product was transformed into Escherichia coli DH5α and revived in 1 mL of LB for 1 hour. 150 μL of the revived bacterial suspension was plated on a plate containing LB medium containing Amp resistance and incubated in a 37°C incubator for 12 hours. Single clones were detected by PCR, and the plasmid was extracted to obtain the recombinant vector.

[0060] 2. Yeast heterologous expression

[0061] The recombinant vector containing the target gene in step 1 was transferred into the yeast INVSc1. The yeast monoclone containing the recombinant vector was picked and placed in SD / -Ura liquid medium, cultured at 28°C and 200rpm for 1 day, and then transferred to SD / -Ura medium without glucose to consume glucose. Finally, the yeast was collected and protein induced in SD / -Ura liquid medium containing 2% galactose and raffinose, and cultured at 28°C and 200rpm for 2 days. After induction, the yeast was collected and glass beads and methanol were added to crush the yeast and extract ARs. After ultrasonication for 30 minutes, the mixture was centrifuged, the supernatant was collected and filtered, and LC-MS / MS showed that the recombinant enzyme encoded by TaARS1 produced several ARs in the yeast that did not exist in the control expressing the empty vector, confirming that TaARS1 was involved in the biosynthesis pathway of ARs and had the activity of alkylresorcinol synthase ( Figure 4 ).

[0062] Example 3

[0063] Analysis of ARs content in the genetic material of TaARS1

[0064] 1. Construction of TaARS1 genetic transformation vector and genetic transformation

[0065] The 1236 bp TaARS1 gene fragment amplified from CS in Example 1 was homologously recombined with the target vectors pZZ (for gene overexpression in corn) and pLGY (for gene overexpression in wheat) to obtain a recombinant vector. The vector map is shown in Figure 5 and Figure 6 .

[0066] 2. TaARS1 genetic transformation

[0067] Through the Agrobacterium EHA105-mediated genetic transformation system of wheat and corn, the correctly cloned plasmid was introduced into Fielder wheat and KN5585 corn. After pre-culture, infection, co-cultivation, screening of callus tissue with corresponding resistance, differentiation, rooting, seedling hardening and transplanting, transgenic plants were obtained.

[0068] 3. Analysis of alkylresorcinol synthase gene expression and alkylresorcinol content in TaARS1 genetic material

[0069] DNA levels of T0 transgenic wheat and maize plants were tested to confirm the construction of TaARS1-positive plants. RNA and metabolic samples were collected from grains of T2-positive transgenic wheat one week after filling. It was found that the expression of TaARS1 was significantly upregulated in both overexpression lines, and LC-MS / MS detection showed that the content of ARs also increased ( Figure 7 A), where the expression level and metabolic content showed a consistent upward trend ( Figure 7 Mature grains of T2-generation positive transgenic corn were used to extract ARs. LC-MS / MS analysis revealed that the ARs content in the three overexpression lines was significantly increased ( Figure 8 The increased ARs content in transgenic wheat and corn further indicated that TaARS1 is a key gene for the synthesis of ARs.

[0070] Example 4

[0071] Analysis of ARs-enhanced resistance to wheat scab

[0072] 1. In vitro antibacterial activity of alkylresorcinol

[0073] Four pure ARs (C17:0, C19:0, C21:0, C23:0) were selected for in vitro antibacterial experiments. Using DMSO as the solvent, when the final concentration of pure ARs was 0.01mmol / L, the length of hyphae on the culture medium was recorded after 4 days of growth of Fusarium graminearum. The repeated data of no less than 6 groups for each AR were statistically analyzed for one-way analysis of variance. It was found that after the addition of the four ARs, the growth rate of Fusarium graminearum on the culture medium showed a significant inhibitory effect, and there was no significant difference in the inhibition level among the four ARs. Taking AR (C21:0) with the most significant plate resistance compared with the control as an example, it was found that when the final concentration was 0.01mmol / L, the inhibition rate was 13.78±8.87%, indicating that under this in vitro experimental method, the treatment of ARs inhibited the growth of Fusarium graminearum ( Figure 9 ).

[0074] 2. Resistance of ARs to Fusarium graminearum in living plants

[0075] Purified ARs (C17:0, C19:0, C21:0, and C23:0) were mixed with Fusarium graminearum spores and inoculated into wheat ears during the flowering stage using the single flower inoculation method (see Liu S, Zhang X, Pumphrey MO, et al. Complex microcolinearity among wheat, rice, and barley revealed by fine map** of the genomic region harboring amajor QTL for resistance to Fusarium head blight in wheat[J]. Functional & Integrative Genomics, 2006, 6:83-89.). The pathogenicity of Fusarium head blight under different treatments was analyzed using DMSO as the solvent. The morphology of wheat ears infected with F. graminearum was observed and the infection rate was calculated. The results showed that the infected part of the wheat ear was wrapped by hyphae, dark brown to yellow-white spots appeared on the spikelets, some wheat awns were twisted, and the whole ear of wheat in the control group (DMSO) was almost damaged ( Figure 10 The evaluation of resistance to Fusarium spp. showed that the addition of ARs to the spore solution could significantly reduce the number of diseased spikelets. C17:0, C19:0, C21:0, and C23:0 could reduce the infection rate from 97.8±2.9% to 34.17±5.1%, 67.0±6.1%, 48.8±7.5%, and 72.2±15.6%, respectively. Among them, AR17:0 had the best antibacterial effect against Fusarium spp. ( Figure 11 ).

[0076] Example 5

[0077] (1) A homozygous mutant of the TaARS1 gene in maize (TaARS1-KO) was constructed and inoculated with Fusarium graminearum in a greenhouse at the same time as wild-type maize (WT) and transgenic maize overexpressing the TaARS1 gene in Example 3 (TaARS1-OE). The phenotypic differences were later observed to verify the antibacterial activity of ARs synthesized by TaARS1 in wheat. The results showed that the degree of susceptibility was: TaARS1-KO > WT > TaARS1-OE.

[0078] (2) In a plot specifically designed to identify disease phenotypes, wild-type maize (WT) and transgenic maize overexpressing the TaARS1 gene (TaARS1-OE) were inoculated with Fusarium spp., and the severity of stalk rot was observed to verify the antibacterial activity of ARs synthesized by TaARS1 in maize. The results showed that compared with the wild-type, the maize overexpressing the TaARS1 gene had a more disease-resistant phenotype.

[0079] Based on the above content, it can be seen that overexpression of the TaARS1 gene can effectively increase the content of alkylresorcinol in plants (especially wheat and corn) and enhance the tolerance of plants to pathogens such as Fusarium graminearum.

[0080] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of the TaARS1 gene for one or more of the following: (1) Regulating the content of plant alkylresorcinols; (2) regulating plant pathogen resistance; the pathogen is Fusarium graminearum; The amino acid sequence of the protein encoded by the TaARS1 gene is shown in SEQ ID NO: 3; The regulating of the plant alkylresorcinol content comprises: overexpressing the TaARS1 gene to increase the plant alkylresorcinol content; The regulating plant pathogen resistance is: overexpressing TaARS1 gene to improve plant pathogen resistance; The plants are wheat and / or corn.

2. The use according to claim 1, characterized in that The plant alkylresorcinol includes one or more of C17:0, C19:0, C21:0 and C23:

0.

3. The use according to claim 1, characterized in that The plant alkylresorcinol content includes the alkylresorcinol content in plant seeds.

4. The use according to any one of claims 1 to 3, characterized in that The nucleotide sequence of the coding region of the TaARS1 gene is shown in SEQ ID NO:

2.

5. The use according to claim 4, characterized in that The full-length genomic nucleotide sequence of the TaARS1 gene is shown in SEQ ID NO:

1.

6. A method for increasing the content of alkylresorcinol in plants and improving the resistance of plant pathogens, characterized in that: Overexpressing the TaARS1 gene in a plant; the amino acid sequence of the protein encoded by the TaARS1 gene is shown in SEQ ID NO: 3; The pathogen is Fusarium graminearum; The plants are wheat and / or corn.

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