Application of TaARS1 gene in regulation and control of plant alkyl resorcinol and / or plant resistance

Abstract

The invention belongs to the technical field of genetic engineering and plant disease prevention and control, and particularly relates to application of a TaARS1 gene in regulation and control of plant alkylresorcinol and / or plant resistance. The invention provides an application of the TaARS1 gene. The amino acid sequence of the protein coded by the TaARS1 gene is shown as SEQ ID NO: 3. The TaARS1 gene is developed and utilized, the variety and content of alkylresorcinol in wheat and other crops can be effectively increased, the resistance of plants to fusarium, especially fusarium graminearum, is enhanced, and the nutritional value of related crops is increased.

Description

Technical Field

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

[0002] Alkylresorcinol (ARs) is a special class of phenolic lipids, which is a general term for a class of derivatives in which the 5-position of the benzene ring of 1,3-resorcinol is substituted by an alkyl group with an odd number of carbon atoms. It is amphiphilic, including a hydrophilic resorcinol ring synthesized through the polyketide synthesis pathway and a hydrophobic alkyl chain synthesized through the fatty acid synthesis pathway. ARs are very important components in wheat bran and have a variety of physiological and biological activities, such as anti-cancer, antioxidant, antibacterial, as a biomarker, and enzyme inhibition. Therefore, it is of great significance to apply ARs to crop disease resistance breeding and improve the nutritional quality of crops.

[0003] Fusarium graminearum is a type of plant pathogenic fungus that mainly infects wheat, barley, corn and other cereal economic crops, causing wheat fusarium head blight and corn stem 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, with the greatest harm to wheat ears in the flowering period, and in severe cases, causing wheat crop failure. Therefore, it is particularly important to explore 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 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 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 comprises: overexpressing the TaARS1 gene to increase the types of plant alkylresorcinols; the types of plant alkylresorcinols comprise 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 comprises: overexpressing TaARS1 gene to improve plant pathogen resistance.

[0015] Preferably, the Fusarium 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 plant alkylresorcinols and improving plant resistance, which comprises 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 an application of the TaARS1 gene; the amino acid sequence of the protein encoded by the TaARS1 gene is shown in SEQ ID NO: 3. The present invention develops and utilizes the TaARS1 gene to effectively increase the types and contents of alkylresorcinol in wheat and other crops, enhance the resistance of plants 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 to be used in the embodiments are briefly introduced below.

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

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

[0026] Figure 3 This is the expression calorimeter of TaARS candidate genes in samples of all growth periods;

[0027] Figure 4 The diagram shows the differentially expressed alkylresorcinols in the TaARS1-expressing recombinant yeast and the wild-type yeast and the biosynthetic pathway of alkylresorcinols; wherein A is the differentially expressed alkylresorcinols; and B is 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 corn;

[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 is the alkylresorcinol content, B is the TaARS1 gene expression, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001;

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

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

[0033] Fig.10 The phenotype of wheat resistance to Fusarium graminearum under different alkylresorcinols;

[0034] Fig.11 The 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 resistance to plant pathogens; the pathogens include Fusarium; the amino acid sequence of the protein encoded by the TaARS1 gene is shown in SEQ ID NO: 3.

[0036] As an embodiment, the regulating plant alkylresorcinol species of the present invention includes: overexpressing the TaARS1 gene to increase the plant alkylresorcinol species. As an embodiment, the plant alkylresorcinol species of the present invention include one or more of C17:0, C19:0, C21:0 and C23:0; as another embodiment, the plant alkylresorcinol species of the present invention include C17:0, C19:0, C21:0 and C23:0. As an embodiment, the plant alkylresorcinol species of the present invention include alkylresorcinol species in plant seeds.

[0037] As an embodiment, the regulating plant alkylresorcinol content of the present invention includes: overexpressing TaARS1 gene to increase the plant alkylresorcinol content. As an embodiment, increasing the plant alkylresorcinol content of the present invention includes increasing the content of one or more of plant C17:0, C19:0, C21:0 and C23:0; as another embodiment, increasing the plant alkylresorcinol content of the present invention includes increasing the content of plant C17:0, C19:0, C21:0 and C23:0. As an embodiment, the plant alkylresorcinol content of 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] As an embodiment, the regulation of plant pathogen resistance of the present invention includes: overexpressing TaARS1 gene to improve plant pathogen resistance. As an embodiment, the plant diseases of the present invention include wheat head blight and / or corn stalk rot. As an embodiment, the Fusarium of the present invention includes Fusarium graminearum. As an embodiment, the plant of the present invention includes wheat and / or corn.

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

[0041] The invention provides a method for increasing the types and contents of plant alkylresorcinols and improving plant resistance, and overexpressing 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 in conjunction with 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 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 and 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 the alkylresorcinol synthase involved in the biosynthesis of ARs was mainly expressed in the grains.

[0048] (2) Six characterized alkylresorcinol synthases (ARS) were used for local Blast analysis with the whole genome sequence of Chinese spring wheat. Genes with E values ​​close to zero and amino acid sequence similarity of more than 50% were screened out through homology comparison of amino acid sequences. After removing duplicates, 38 candidate genes were initially screened out from the six Blast results, of which 12 candidate genes were found through the comparison of 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 Batch CD-search provided by NCBI. The results showed that these 38 genes all belonged to the superfamily PLN03171, which is the same as the superfamily of 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 58 wheat throughout its growth period. Based on RNA-seq data and combined with local Blast results, genes with expression patterns similar to the metabolite accumulation patterns were identified. The results are shown in Figure 3 .

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

[0051]

[0052]

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

[0054] 2. Amplification of TaARS1 gene

[0055] According to the sequence information of TaARS1 gene in the genome of wheat "Chinese Spring", in order to obtain the full-length cDNA sequence of TaARS1 gene, total RNA of wheat variety Chinese Spring (CS) was extracted, reverse transcribed into cDNA, and amplified using primers (TaARS1-F: 5'-ATGGGAAGCATCGGAACCGC-3', SEQ ID NO: 4; TaARS1-R: 5'-CTAGCGTGGACAGCGGAGCA-3', SEQID NO: 5). The PCR amplified target fragment was connected with the blunt-end vector, and the reaction product was transformed into Escherichia coli DH5α, and 1mL LB was added for recovery for 1h. 150μL of the recovered bacterial solution was applied to a plate containing LB medium with resistance Amp and cultured in an incubator at 37℃ for 12h. Single clones were detected by PCR, and plasmids were extracted for sequencing. The PCR product was sequenced and analyzed to obtain the cDNA sequence of TaARS1 gene, and a 1236bp 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 heterologous expression vector of TaARS1 in yeast

[0059] The 1236bp TaARS1 gene fragment amplified from CS in Example 1 was subjected to homologous recombination reaction with the target vector pYES2. The reaction product was transformed into Escherichia coli DH5α, and 1mL LB was added for 1h of recovery. 150μL of the recovered bacterial solution was applied to a plate containing LB medium with resistance Amp and cultured in an incubator at 37°C for 12h. Single clones were tested by PCR, and plasmids were extracted to obtain recombinant vectors.

[0060] 2. Yeast heterologous expression

[0061] The recombinant vector containing the target gene in step 1 was transferred into the yeast INVSc1. A single clone of yeast containing the recombinant vector was picked and placed in SD / -Ura liquid medium, cultured at 28°C and 200rpm for 1 day, then transferred to SD / -Ura medium without glucose to consume glucose, and finally the yeast was collected for protein induction 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 break the yeast and extract ARs. After ultrasonication for 30 minutes, the supernatant was collected and filtered. LC-MS / MS showed that the recombinant enzyme encoded by TaARS1 produced several ARs in yeast that were not present 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] The DNA level of T0 transgenic wheat and corn plants was tested to confirm the construction of TaARS1 positive plants. RNA samples and metabolic samples of grains of T2 positive transgenic wheat were taken after 1 week of filling. It was found that the expression of TaARS1 was significantly upregulated in both overexpression lines, and the content of ARs was also increased by LC-MS / MS. Figure 7 A), where the expression level and metabolic content showed a consistent upward trend ( Figure 7 Mature grains of T2 positive transgenic corn were used to extract ARs. LC-MS / MS analysis revealed that the content of ARs in the three overexpression lines increased significantly ( Figure 8 The increase in 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 fusarium head blight

[0072] 1. In vitro antibacterial activity of alkylresorcinol

[0073] Four pure ARs (C17:0, C19:0, C21:0, and 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 Fusarium graminearum had grown for 4 days. The repeated data of no less than 6 groups for each AR were statistically analyzed for one-way analysis of variance, and 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), which had 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 ( Fig. 9 ).

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

[0075] After the pure ARs (C17:0, C19:0, C21:0, C23:0) were mixed with the spores of Fusarium graminearum, the wheat ears were inoculated with the single flower drip method (refer to 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.) at the flowering stage of wheat, and the pathogenicity of Fusarium under different treatments was statistically analyzed, using DMSO as the solvent. The morphology of wheat ears after infection with Fusarium graminearum was observed by inoculating the spore suspension containing 0.5mmol / L ARs, 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 basically damaged ( Fig.10 ). The evaluation of resistance to Fusarium showed that the number of diseased spikelets could be significantly reduced by adding ARs to the spore solution. 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 on Fusarium ( Fig.11 ).

[0076] Example 5

[0077] (1) A homozygous mutant of the maize TaARS1 gene (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 for identifying disease phenotypes, wild-type corn (WT) and transgenic corn overexpressing the TaARS1 gene (TaARS1-OE) were inoculated with Fusarium solani and the severity of corn stalk rot was observed to verify the antibacterial activity of ARs synthesized by TaARS1 in corn. The results showed that compared with the wild type, corn materials 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 describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. Use of TaARS1 gene in one or more of the following; (1) Regulate the types of plant alkylresorcinols; (2) regulating the content of plant alkylresorcinols; (3) regulating plant pathogen resistance; the pathogens include Fusarium; The amino acid sequence of the protein encoded by the TaARS1 gene is shown in SEQ ID NO:

3.

2. The use according to claim 1, characterized in that: The regulating types of plant alkylresorcinols comprises: overexpressing the TaARS1 gene to increase the types of plant alkylresorcinols; the types of plant alkylresorcinols comprise one or more of C17:0, C19:0, C21:0 and C23:

0.

3. The use according to claim 1, characterized in that: The method for regulating the content of plant alkylresorcinol comprises: overexpressing TaARS1 gene to increase the content of plant alkylresorcinol.

4. The use according to claim 1, characterized in that: The plant alkylresorcinol species include alkylresorcinol species in plant seeds; The plant alkylresorcinol content includes the alkylresorcinol content in plant seeds.

5. The use according to claim 1, characterized in that: The regulating plant pathogen resistance includes: overexpressing TaARS1 gene to improve plant pathogen resistance.

6. The use according to claim 1, characterized in that: The Fusarium species include Fusarium graminearum.

7. The use according to claim 1, characterized in that: The plants include wheat and / or corn.

8. The use according to any one of claims 1 to 7, characterized in that: The nucleotide sequence of the coding region of the TaARS1 gene is shown in SEQ ID NO:

2.

9. The use according to claim 8, characterized in that: The full-length genomic nucleotide sequence of the TaARS1 gene is shown in SEQ ID NO:

1.

10. A method for increasing the types and contents of plant alkylresorcinols 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 pathogenic bacteria include Fusarium.

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