Application of TaHQT1 gene in regulation and control of chlorogenic acid content of plants

Abstract

The invention provides application of a TaHQT1 gene in regulating and controlling the content of chlorogenic acid in plants, and belongs to the technical field of gene engineering. The invention provides an application of a TaHQT1 gene in regulating and controlling the content of chlorogenic acid in a plant. The invention also proves that a recombinant protein coded by the TaHQT1 gene can generate a product chlorogenic acid by taking caffeoyl-coenzyme A and quinic acid as substrates; meanwhile, overexpression is carried out on the TaHQT1 gene, and it is found that the content of chlorogenic acid in mature grains and leaves is remarkably increased, so that the TaHQT1 gene can be developed and utilized, and the content of wheat chlorogenic acid is increased through overexpression of the TaHQT1 gene, so that the nutritional quality of wheat is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of the TaHQT1 gene in regulating the chlorogenic acid content of plants. Background Art

[0002] Wheat is the world's leading staple food crop, providing approximately 20% of the world's calories and protein, and providing a significant amount of energy and abundant nutrients for human survival. With the continuous improvement of people's living standards, the pursuit of high crop yields is no longer the sole goal of crop breeders. Improving crop quality traits while maintaining a certain yield has become increasingly important. Wheat quality can be broadly categorized into three categories: processing quality, appearance quality, and nutritional quality. Wheat nutritional quality primarily refers to the balance of protein content and its amino acid profile. It also encompasses the nutritional value, distribution, and factors influencing the accumulation of trace elements, resistant starch, phytobioactive substances, dietary fiber, carotenoids, and other substances.

[0003] Chlorogenic acids are a class of phenylpropanoid secondary metabolites synthesized in plants via the shikimic acid pathway. They are esterified derivatives of hydroxycinnamic acid and quinic acid. Studies have shown that this family of compounds possesses multiple biological activities, including antioxidant, antibacterial, anti-inflammatory, antiviral, and lipid metabolism regulation. In plants, chlorogenic acid typically exists as a complex of caffeoylquinic acid isomers, with significant species-specific composition and content. Chlorogenic acid is widely distributed in the vegetative and reproductive organs of higher dicots and ferns. Limited by complex stereoisomerization characteristics and the presence of multiple chiral centers, the chemical synthesis of chlorogenic acids faces challenges in controlling regioselectivity and stereoselectivity. Existing synthetic routes are generally plagued by technical bottlenecks such as low yield and high cost, and the establishment of an industrial production system has yet to be achieved.

[0004] In the chlorogenic acid biosynthesis pathway, hydroxycinnamoyl-CoA:quinic acid hydroxycinnamoyltransferase (HQT), quinic acid / shikimic acid hydroxycinnamoyltransferase (HCT), and p-coumarate 3-hydroxylase (C3H) serve as core catalytic enzymes, participating in the hydroxycinnamoyl transfer reaction and phenylpropane backbone modification, respectively. Currently, the metabolic regulatory network analysis, physiological and ecological function exploration, and resource utilization development of chlorogenic acid compounds in wheat, a major food crop, are still in the early stages of systematic research, and the relevant molecular mechanisms and metabolic engineering strategies urgently need to be further explored. Summary of the Invention

[0005] The present invention provides an application of the TaHQT1 gene in regulating the chlorogenic acid content in plants. The TaHQT1 can serve as a key gene in the chlorogenic acid synthesis pathway, regulate the chlorogenic acid content in plants, and improve crop quality.

[0006] The present invention provides an application of the TaHQT1 gene in regulating the chlorogenic acid content of plants. The protein encoded by the TaHQT1 gene includes the amino acid sequence shown in SEQ ID No. 1.

[0007] In a preferred embodiment of the present invention, the TaHQT1 gene includes the genomic sequence shown in SEQ ID No. 2.

[0008] In a preferred embodiment of the present invention, the TaHQT1 gene comprises the CDS sequence shown in SEQ ID No. 3.

[0009] In a preferred embodiment of the present invention, the regulation includes overexpressing the TaHQT1 gene, thereby increasing the chlorogenic acid content in the plant.

[0010] In a preferred embodiment of the present invention, the plants include plants of the family Poaceae.

[0011] The present invention also provides a biomaterial comprising a TaHQT1 gene and increasing the expression level of the TaHQT1 gene. The protein encoded by the TaHQT1 gene comprises the amino acid sequence shown in SEQ ID No. 1.

[0012] In a preferred embodiment of the present invention, the biological material includes an overexpression vector.

[0013] The present invention also provides the use of the TaHQT1 gene or the above-mentioned biological material in cultivating plants with high chlorogenic acid content. The protein encoded by the TaHQT1 gene includes the amino acid sequence shown in SEQ ID No. 1.

[0014] The present invention also provides a method for cultivating plants with high chlorogenic acid content, comprising overexpressing the TaHQT1 gene in the plant genome, wherein the protein encoded by the TaHQT1 gene includes the amino acid sequence shown in SEQ ID No. 1.

[0015] In a preferred embodiment of the present invention, the plants include plants of the family Poaceae.

[0016] Beneficial effects: The present invention provides an application of the TaHQT1 gene in regulating the chlorogenic acid content of plants, and the present invention also proves that the recombinant protein encoded by the TaHQT1 gene can use caffeoyl-CoA and quinic acid as substrates to generate the product chlorogenic acid; at the same time, the TaHQT1 gene is overexpressed, and it is found that the chlorogenic acid content in mature grains and leaves is significantly increased. Therefore, the TaHQT1 gene can be developed and utilized, and by overexpressing the TaHQT1 gene, the chlorogenic acid content of wheat can be increased, thereby improving the nutritional quality of wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Manhattan plot of TaHQT1 for genome-wide association analysis of relative chlorogenic acid content in leaves of natural wheat populations;

[0018] Figure 2 Correlation analysis between the two haplotypes generated by the TaHQT1 promoter region mutation and the relative content of chlorogenic acid in natural populations;

[0019] Figure 3 This is the analysis of the transcriptional levels of two haplotypes of TaHQT1 in leaves of different varieties in natural populations;

[0020] Figure 4 This is the peak diagram of the in vitro enzyme activity reaction of TaHQT1 protein;

[0021] Figure 5 Schematic diagram of the genetic transformation vector structure for overexpressing TaHQT1 in wheat;

[0022] Figure 6 The expression levels of TaHQT1 and the content of chlorogenic acid in leaves of wild-type wheat and transgenic wheat overexpressing TaHQT1 are shown; ** indicates P < 0.01, *** indicates P < 0.001;

[0023] Figure 7 represents the content of chlorogenic acid in mature grains of wild-type wheat and transgenic wheat overexpressing TaHQT1; *** indicates P < 0.001. DETAILED DESCRIPTION

[0024] The present invention provides an application of the TaHQT1 gene in regulating the chlorogenic acid content of plants. The protein encoded by the TaHQT1 gene includes the amino acid sequence shown in SEQ ID No. 1.

[0025] The TaHQT1 gene described in the present invention is a candidate gene annotated as shikimate / quinate hydroxycoumaryltransferase, with a gene number of TraesCS3B01G002000. The TaHQT1 gene contains one intron and two exons, and its full-length genomic sequence is 5388bp, as specifically shown in SEQ ID No.2; the nucleotide sequence of the CDS region of the TaHQT1 gene is shown in SEQ ID No.3, totaling 1311bp, encoding 436 amino acids, and the amino acid sequence is shown in SEQ ID No.1.

[0026] The plants described in the present invention include plants of the Poaceae family. In one embodiment, wheat is used as an example. In the wheat, after the expression of the TaHQT1 gene is regulated, the chlorogenic acid content also changes significantly. For example, after expression, the chlorogenic acid content in the plant is increased.

[0027] The present invention also provides a biomaterial comprising a TaHQT1 gene and increasing the expression level of the TaHQT1 gene. The protein encoded by the TaHQT1 gene comprises the amino acid sequence shown in SEQ ID No. 1.

[0028] In a preferred embodiment of the present invention, the biological material includes an overexpression vector, such as an Agrobacterium-mediated genetic transformation vector carrying a maize ubiquitin gene promoter with constitutive and overexpression characteristics as a backbone vector. In one embodiment, the pLGY-OE3 vector is used as a backbone vector, and the nucleotide sequence shown in SEQ ID No. 3 is connected to the linearized pLGY-OE3 vector formed by single enzyme digestion using BamHI.

[0029] The present invention also provides the use of the TaHQT1 gene or the above-mentioned biological material in cultivating plants with high chlorogenic acid content. The protein encoded by the TaHQT1 gene includes the amino acid sequence shown in SEQ ID No. 1.

[0030] The TaHQT1 gene described herein is overexpressed in plants, or the aforementioned biological material is transformed into target plants using genetic transformation methods, such that the TaHQT1 gene is overexpressed in the target plants, thereby increasing the chlorogenic acid content in the plants. The plants described herein include grasses, and in one embodiment, wheat. Verification has shown that the chlorogenic acid content in the leaves and mature grains of overexpressed wheat is significantly increased.

[0031] The present invention also provides a method for cultivating plants with high chlorogenic acid content, comprising overexpressing the TaHQT1 gene in the plant genome, wherein the protein encoded by the TaHQT1 gene includes the amino acid sequence shown in SEQ ID No. 1.

[0032] In a preferred embodiment of the present invention, the plant includes a grass plant. The present invention is not particularly limited to the overexpression method. The above-mentioned biological material can be genetically transformed with reference to conventional methods in the art. For example, the method described in the reference (Hayta S, Smedley MA, Demir SU, et al. An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticumaestivum L.) [J]. Plant Methods, 2019, 15: 1-15.) in the embodiment is introduced into the wheat variety through Agrobacterium EHA105 mediation and wheat genetic transformation system.

[0033] To further illustrate the present invention, the application of the TaHQT1 gene provided by the present invention in regulating the chlorogenic acid content in plants is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] Acquisition of the TaHQT1 gene

[0036] 1. TaHQT1 gene identification and structural analysis

[0037] Using the sequencing data and sequence analysis results of previously published wheat variety resources (Gao et al2023, Deciphering genetic basis of developmental and agronomic traits by integrating high-throughput optical phenotyping and genome-wide association studies in wheat), 374 wheat germplasms were sequenced using the MGI2000 platform. The data were corrected using the FastQC tool (De Sena Brandine and Smith 2019, Falco: high-speed FastQC emulation for quality control of sequencing data.) and aligned to the wheat reference genome IWGSC_ref1.0 (IWGSC 2018) using the Burrows-Wheeler Aligner (BWA) software (Li and Durbin 2009, Fast and accurate short read alignment with Burrows-Wheeler transform). SNP annotation was based on the reference genome IWGSC_ref 1.0 (IWGSC et al 2018, International Wheat Genome Sequencing Consortium (IWGSC). Shifting the limits in wheat research and breeding using a fully annotated reference genome. doi:10.1126 / science.aar7191) and SnpEff (Cingolani et al 2012, A program for annotating and predicting the effects of single nucleotide polymorphisms). polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3) were obtained.After screening, 12,253,596 valid markers were finally obtained for GWAS analysis. The mixed model FaST-LMM (Lippert et al 2011, FaST linear mixed models for genome-wide association studies. Nat Methods) was used for genome-wide association analysis, and the P value was less than 1×10. -7 Significantly associated sites were filtered out using Plink (Purcell et al 2007, PLINK: a tool set for whole-genome association and population-based linkage analyses) to remove tightly linked SNPs (LD>0.2) and these SNPs were converted into QTL intervals (Zhang et al 2016, Genome-wide association studies of drought-related metabolic changes inmaize using an enlarged SNP panel).

[0038] In order to find genes associated with the variation of chlorogenic acid content in leaves of natural wheat populations, a total of 12,253,596 effective SNP markers were used for genome-wide association analysis. Figure 1 As shown, there is a candidate gene annotated as shikimate / quinate hydroxycoumaryltransferase within 1.1Mb before and after the lead SNP of this substance. The gene number is TraesCS3B01G002000 and it is named TaHQT1. Through the public data of the WheatOmics website (WheatOmics) and the previous resequencing results of this team, the SNPs information of a single gene including 2.5kb upstream of the gene, exons, and introns was obtained. The R language was used to perform T-TEST analysis on these SNPs and metabolite contents. In addition, hapviewer software was used to perform genotyping analysis on the significantly associated SNPs in each gene. The analysis found that the two haplotypes produced by the variation in the promoter region of this gene had significant differences in chlorogenic acid content ( Figure 2 ), and the transcription level of TaHQT1 in different varieties of natural populations is extremely significantly correlated with the content of chlorogenic acid. The inventors further overexpressed the gene in wheat Fielder through in vitro enzyme activity experiments and genetic transformation experiments to determine that the gene has the activity of synthesizing chlorogenic acid and is a key gene related to the variation of chlorogenic acid content in natural populations ( Figure 3The full-length genomic sequence of TaHQT1 is 5388 bp (SEQ ID No. 2), and the coding sequence is 1311 bp (SEQ ID No. 3), encoding 436 amino acids (SEQ ID No. 1). The gene has one intron and two exons.

[0039] 2. Amplification of the TaHQT1 gene

[0040] Total RNA was extracted from the wheat line Fielder (Full-Form Gold AE301-02), reverse transcribed into cDNA, and amplified using specific primers. PCR amplification was performed using Fly Fast High-Fidelity DNA Polymerase and specific primers according to the manufacturer's instructions to obtain the desired fragment product.

[0041] TaHQT1-F (SEQ ID No. 4): 5'-ATGGGCATCACGGTGACGGT-3';

[0042] TaHQT1-R (SEQ ID No. 5): 5'-TTACAGTGCCAAGATCAACTGTCG-3'.

[0043] The target fragment was amplified by PCR and ligated with a blunt-ended vector (pTOPO-T simple 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 culture was plated on a plate containing LA medium (50-100 μg / ml) of the resistance AMP and incubated at 37°C for 12 hours. Single clones were detected by PCR, and plasmids were extracted and sequenced. Sequencing analysis of the PCR product revealed the cDNA sequence of the TaHQT1 gene. An 1174-bp TaHQT1 gene sequence amplified from Fielder was identical to that shown in SEQ ID No. 3. After correct sequence alignment, the recombinant protein vector (pGEX-6P-1) and the overexpression vector (pLGY-OE3) were used for homologous recombination.

[0044] Example 2

[0045] Regulation of chlorogenic acid in wheat grains by TaHQT1 genetic material

[0046] 1. The recombinant protein TaHQT1 expressed in Escherichia coli can use caffeoyl-CoA and quinic acid as substrates to produce chlorogenic acid.

[0047] Prokaryotic protein expression: Transform the constructed protein expression vector into the expression strain BL21, pick the colony in 5mL LB (containing corresponding resistance) medium, and culture at 37℃ overnight. Add the bacterial solution after overnight culture to 100mL LB (containing corresponding resistance) medium and culture at 37℃ until OD600 =0.4-1.0, then add 100 μL 1M IPTG (final concentration 0.1 mM), induce overnight at 18°C, 4000 rpm, collect the cells for 15 min, discard the culture medium, add 20 mL Lysis buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol, 400 mM NaCl) to resuspend, place on ice, add 100 μL 100 mM PMSF and 10 μL β-mercaptoethanol, and use a high-pressure disruptor to disrupt the suspended cells. The disrupted cells are always kept on ice and centrifuged at 12000 g for 30 min at 4°C. The supernatant is transferred to a new 50 mL centrifuge tube, and 10 μL of the sample is labeled SP (supernatant).

[0048] Purification of GST-tagged fusion proteins: Resuspend the beads and transfer them to an empty chromatography column. Wash off the 20% ethanol with approximately 10 mL of ddH2O. Equilibrate the beads with approximately 10 mL of Lysis Buffer. Add the equilibrated beads to the 50 mL centrifuge tube containing the supernatant and mix in a silent mixer at 4°C for 1.5-2 hours. Add the supernatant containing the beads to the original chromatography column. Sample 10 μL of the flow-through, labeled FT (flow-through), and wash away contaminants with Lysis Buffer until the effluent does not turn blue in a Bradford assay (usually approximately 20 mL is required). Elute the protein with 15 mM reduced glutathione solution in separate tubes, labeled E1, E2, E3, etc., until the Bradford assay does not turn blue (you can start testing after elution in tube 8). Sample the eluted sample, 10 μL per tube. Use the remaining reduced glutathione solution to wash the beads. Wash the beads with approximately 10 mL of dH2O and store in 20% ethanol. Add 10 μL of 5× SDS loading buffer to the sample tube, boil at 95°C for 5 minutes, and run on SDS-PAGE to confirm protein expression and purification.

[0049] The enzyme activity reaction system was 10 μL, including 0.1 M Tris-HCl (pH = 7.5), 20 μM caffeoyl-CoA, and 5 μL of 0.1 mM protein. The reaction was allowed to proceed at 37°C for 30 minutes, then terminated with methanol, and the product was detected on an analyzer.

[0050] The results of in vitro enzyme activity analysis were as follows Figure 4As shown, compared to the empty carrier protein control group, which only contained the characteristic peak of the substrate quinic acid, the characteristic chromatographic peak of the specific product chlorogenic acid was detected in the in vitro enzymatic reaction system containing the TaHQT1-GST recombinant protein. This experimental result confirms that the TaHQT1-GST recombinant protein can catalyze the acylation reaction of quinic acid with caffeoyl in vitro to synthesize chlorogenic acid, confirming its caffeic acid acyltransferase function.

[0051] 2. Construction of TaHQT1 genetic transformation vector

[0052] The 1174 bp TaHQT1 gene fragment amplified from Fielder in Example 1 was subjected to homologous recombination reaction with the target vector pLGY-OE3 to obtain the following map: Figure 5 The recombinant vector shown is an Agrobacterium-mediated genetic transformation vector carrying a maize ubiquitin gene promoter with constitutive and overexpression characteristics.

[0053] 3. TaHQT1 genetic transformation

[0054] Referring to the prior art (Hayta S, Smedley MA, Demir SU, et al. An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.) [J]. Plant Methods, 2019, 15: 1-15.), the plasmid correctly cloned in step 1 was introduced into the wheat variety Fielder through Agrobacterium EHA105 mediation and wheat genetic transformation system. After pre-culture, infection, co-cultivation, screening of callus tissue with glufosinate resistance, differentiation, rooting, seedling hardening and transplanting, transgenic plants were obtained.

[0055] 4. Analysis of gene expression in TaHQT1 overexpressing transgenic materials and the content of chlorogenic acid, a wheat lignin precursor

[0056] The chlorogenic acid extraction method was modified based on the method described in the article (Chen et al. 2020, Metabolite-based genome-wide association study enables dissection of the flavonoid decoration pathway of wheat kernels). This laboratory's plant metabolism extraction method was modified. Sample homogenization: Wheat leaves were first freeze-dried in a vacuum oven and then ground into a powder using a grinder (MM400, Retsch) at 50 Hz for 0.5-1 min. 0.05 g of sample powder was weighed and placed in a 2 mL EP tube. 0.5 mL of 70% methanol (0.1 g of fresh sample was ground with liquid nitrogen and added to 0.3 mL of 70% methanol) was added. The sample was vortexed vigorously for 15 seconds until the powder was completely suspended. Ultrasonic extraction was performed at maximum power for 30 minutes, followed by centrifugation at 12,000 rpm at 4°C for 10 minutes. The supernatant was aspirated with a syringe, filtered through a 0.22 μm filter, and stored in a -20°C freezer until analysis was completed.

[0057] Samples were analyzed using a liquid chromatography-electrospray ionization (ESI)-MS / MS system (HPLC, Shim-pack UFLC SHIMADZUCBM20A system; MS, Applied Biosystems 5500Q-TRAP). Chlorogenic acid standards (purity ≥98%) were purchased, and their characteristic ion pairs were identified using full scan (Q1 MS) and product ion scan (Q3 MS). The declustering potential (DP) and collision energy (CE) were optimized. Target peaks were identified based on the retention time and characteristic ion pairs of the standard. Quantification was performed using the descriptive multiple reaction monitoring (MRM) method (Chen et al., 2013, Genome-wide association analyses provide genetic and biochemical insights into natural variation in rice metabolism).

[0058] The DNA level of T0 transgenic wheat plants was tested to confirm the construction of TaHQT1 positive plants. Metabolic extraction was performed on the leaves and mature grains of TaHQT1 T3 positive transgenic wheat plants and wild-type wheat plants, and the content of chlorogenic acid in the grains was analyzed. Figure 6 and Figure 7 As shown, compared with the wild-type control Fielder, the chlorogenic acid content in the leaves and mature grains of the TaHQT1-overexpressing positive transformed plants was significantly increased.

[0059] 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. The application of TaHQT1 gene in regulating the content of chlorogenic acid in plants is characterized by: The protein encoded by the TaHQT1 gene includes the amino acid sequence shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that The TaHQT1 gene includes the genome sequence shown in SEQ ID No.

2.

3. The use according to claim 1 or 2, characterized in that: The TaHQT1 gene includes the CDS sequence shown in SEQ ID No.

3.

4. The application according to claim 1, characterized in that The regulation includes overexpressing the TaHQT1 gene, thereby increasing the chlorogenic acid content in the plant.

5. The application according to claim 4, characterized in that: The plants include plants of the grass family.

6. A biomaterial comprising a TaHQT1 gene and increasing the expression level of the TaHQT1 gene, characterized in that: The protein encoded by the TaHQT1 gene includes the amino acid sequence shown in SEQ ID No.

1.

7. The biomaterial according to claim 6, characterized in that: The biological material includes an overexpression vector.

8. Use of the TaHQT1 gene or the biomaterial according to claim 6 or 7 in cultivating plants with high chlorogenic acid content, characterized in that: The protein encoded by the TaHQT1 gene includes the amino acid sequence shown in SEQ ID No.

1.

9. A method for cultivating plants with high chlorogenic acid content, characterized in that: The method comprises overexpressing the TaHQT1 gene in the plant genome, wherein the protein encoded by the TaHQT1 gene comprises the amino acid sequence shown in SEQ ID No.

1.

10. The method according to claim 9, characterized in that: The plants include plants of the grass family.

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