FNS protein, its encoding gene and application thereof in increasing apigenin content of plants

Abstract

The application discloses a FNS protein, a coding gene thereof and application of the FNS protein in increasing apigenin content of plants, and relates to the technical field of biotechnology.The amino acid sequence of the FNS protein is shown as SEQ ID NO.1.A brand new FNS I type protein and a coding gene thereof are cloned from plants of the Umbelliferae family, and it is first confirmed that the FNS I type protein can be stably expressed in non-Umbelliferae plants and can play a catalytic function, and a cross-family apigenin synthesis metabolic pathway is effectively opened up.Experimental data show that the apigenin content of the FNS gene transformed Arabidopsis thaliana can reach 115.75 mu g / g, is significantly improved compared with a wild type, and meanwhile, total flavonoid accumulation of the plant is also increased, and the whole flavonoid synthesis pathway is positively activated.The application fills a technical blank of cross-family heterologous application of the FNS I of the Umbelliferae family, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an FNS protein, its encoding gene, and its application in increasing the apigenin content of plants. Background Technology

[0002] Apigenin, a natural flavonoid compound, is widely found in fruits, vegetables, and medicinal plants. It possesses multiple physiological activities, including antioxidant, anti-inflammatory, antiviral, antitumor, and metabolic regulation, making it highly valuable in functional foods, pharmaceutical intermediates, and plant nutrition improvement. Currently, industrial-scale apigenin production mainly relies on natural plant extraction. However, this process is limited by the growth cycle, origin, and harvesting season of the raw materials, resulting in complex extraction processes, high purification costs, and low product yields, hindering large-scale and stable supply. Modifying the endogenous flavonoid metabolic pathways in plants through genetic engineering to directionally increase apigenin accumulation is a mainstream research direction for low-cost and sustainable apigenin production.

[0003] The FNS (Flavone synthase) gene catalyzes the conversion of naringenin to apigenin (C). 15 H 10 The key enzyme of 5,4',5,7-trihydroxyflavone. In plant evolution, two types of FNS genes exist: Apiaceae plants are all of type FNSI, while other plants are of type FNSII (Gebhardt Yvonne, et al. Molecular evolution of flavonoid dioxygenases in the family Apiaceae. Phytochemistry. 2005, 66(11):1273-1284). Type FNSI consists of soluble 2-oxoglutaric acid and Fe... 2+ The dioxygenase-dependent FNS II is a NADPH and molecular oxygen-dependent membrane-bound cytochrome P-450 monooxygenase (Xiao-Juan Han, et al. Functional characterization of a plagiochasma appendiculatum flavone synthase I showing flavanone 2-hydroxylase activity. FEBS Lett, 2014, 588: 2307–2314.).

[0004] To date, there is no research on whether the FNS I type FNS gene can catalyze the conversion of naringenin to apigenin in non-umbelliferous plants. Summary of the Invention

[0005] The purpose of this invention is to provide an FNS protein, its encoding gene, and its application in increasing the apigenin content of plants, thereby addressing the problems existing in the prior art. This FNS protein can be stably expressed and exert catalytic functions in non-umbelliferous plants, effectively opening up cross-family apigenin biosynthetic pathways, thereby increasing the apigenin and total flavonoid content in non-umbelliferous plants.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides an FNS protein for catalyzing the synthesis of apigenin, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a gene encoding the above-mentioned FNS protein.

[0008] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0009] The present invention also provides a gene expression cassette, comprising the above-described encoding gene.

[0010] The present invention also provides a recombinant expression vector comprising the gene expression cassette described above.

[0011] The present invention also provides a recombinant host cell comprising the above-described recombinant expression vector.

[0012] The present invention also provides the application of the above-mentioned FNS protein in increasing the content of apigenin and / or total flavonoids in non-umbelliferous plants.

[0013] The present invention also provides the application of the above-mentioned coding gene, gene expression cassette, recombinant expression vector or recombinant host cell in increasing the content of apigenin and / or total flavonoids in non-umbelliferous plants.

[0014] Furthermore, the non-umbelliferous plant is Arabidopsis thaliana.

[0015] The present invention also provides a method for increasing the content of apigenin and / or total flavonoids in non-umbelliferous plants, comprising the step of genetically transforming the above-mentioned encoding gene into the non-umbelliferous plant to construct a transgenic plant overexpressing the encoding gene.

[0016] The present invention discloses the following technical effects: This invention cloned a novel FNS type I protein and its encoding gene from Apiaceae plants, demonstrating for the first time that this gene can be stably expressed and exert catalytic function in non-Apiaceae plants, effectively opening up a cross-family apigenin biosynthesis pathway. Experimental data show that the apigenin content in FNS-transgenic Arabidopsis thaliana can reach 115.75 μg / g, a significant increase compared to the wild type. Simultaneously, the total flavonoid accumulation in the plant also increases, indicating positive activation of the overall flavonoid biosynthesis pathway.

[0017] This invention fills the technological gap in the cross-family heterologous application of apigenin FNS I, and provides a complete set of gene, vector, host and plant improvement methods. It can be widely applied to the flavonoid quality improvement of various non-Apiaceae economic crops and medicinal plants, significantly reducing the raw material and production costs of apigenin large-scale preparation, and has good prospects for industrial application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Electrophoresis diagram of the PCR cloned FNS gene; where M: Marker; 1-2: PCR clone fragments of the FNS gene; Figure 2 This is a diagram showing the domain structure analysis of the FNS protein; Figure 3 This is a schematic diagram of the three-dimensional structure of the FNS protein; Figure 4 Staining diagrams of wild Arabidopsis thaliana and FNS transgenic Arabidopsis thaliana; Figure 5 A statistical graph showing the apigenin content in wild Arabidopsis thaliana (WT) and FNS-transgenic Arabidopsis thaliana; Figure 6 A statistical graph showing the total flavonoid content of wild Arabidopsis thaliana (WT) and FNS transgenic Arabidopsis thaliana. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] This invention cloned a novel FNS gene from plants of the Apiaceae family. Functional verification revealed that overexpression of this gene in plants can increase the content of apigenin.

[0026] The amino acid sequence of the FNS protein involved in this invention is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene (FNS gene) is shown in SEQ ID NO.2.

[0027] SEQ ID NO.1: MAPTTITALSQEKTLNLDFVRDEDERPKVAYNQFSNEIPIISLAGLDDDSNGRRAEICRKIVEAFEEWGIFQVVDHGIDSGLISEMSRLSREFFALPAEEKLVYDTTGGKKGGFTISTHLQGDDVRDWREFVTYFSYPISARDYSRWPKKPEGWRRSTTEVYSEKLMVLGAKLLEV LSEAMGLEKEALTKACVEMEQKVLINYYPTCPEPDLTLGVRRHTDPGTITILLQDMVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHYLSNGRFRNADHQAVVNSTSTRLSIATFQNPAQNAIVYPLKIREGEKAILDEAITYAEMYKKNMTKHIAVATQKKLAKEKRLQDEKAKMKI.

[0028]

[0029] Example 1 This invention cloned a novel FNS gene with a length of 1068 bp, which encodes an FNS protein with an amino acid length of 355 aa.

[0030] Gel electrophoresis analysis of the FNS gene showed that its length was within the expected range. Figure 1 ).

[0031] The cloned FNS gene was translated into its corresponding amino acid sequence using BioXM 2.6 software, and its domains were analyzed using the online software SMART (https: / / smart.embl.de / ). The results are shown below. Figure 2 This indicates that it contains structural domains of type FNS I (DIOX_N and 2OG-FeⅡ_Oxy).

[0032] The three-dimensional structure of the FNS protein was analyzed using the online SWISS-MODEL software (https: / / swissmodel.expasy.org / interactive). Its structure (see...) Figure 3 It is identical to the published Flavonone synthase I (i.e., FNS I) reference 3D model (model number: B3RFV8.1.A).

[0033] Example 2 1. Construction of transgenic Arabidopsis thaliana Homologous transgenic primer pairs were designed based on the FNS gene: forward primer pair: TTTCAATTACCATGGGATCCATGGCTCCTACAACTATAACTGC (SEQ ID NO.3), reverse primer pair: ACCGATGATACGAACGAGCTCTCATATCTTCATCTTGGCCTTCT (SEQ ID NO.4).

[0034] The FNS gene was amplified by PCR using homologous transgenic primers, yielding the amplification product. The amplification product and the pY7736 vector containing the CaMV 35S promoter (which contains the GUS reporter gene) were then subjected to... Sca I and Bam After HI digestion, a ligation reaction was performed to recombinant the FNS gene into the pY7736 vector containing the CaMV 35 S promoter, resulting in a recombinant plasmid.

[0035] The constructed recombinant plasmid was transformed into *E. coli* DH5a strain using a 42 °C heat shock method. The transformed plasmid was plated on LB solid medium containing 50 mg / L kanamycin (Kan) and incubated upside down in a dark incubator at 37 °C. Single colonies were picked and inoculated into LB liquid medium containing 50 mg / L Kan, and cultured on a shaker at 37 °C and 230 rpm for 24 h. After PCR identification, the bacterial culture was sent to a biosequencing company for sequencing to confirm successful vector construction. The recombinant plasmid was extracted using the Axygen plasmid extraction kit.

[0036] The recombinant plasmid was transformed into Agrobacterium GV3101 using an electric shock method (1.8 kV, 5 s). After the shock, 700 μL of pre-cooled, antibiotic-free LB broth was quickly added to the shock vessel. The bacterial culture in the shock vessel was then pipetted into a 1.5 ml sterile centrifuge tube. The tube was incubated for 1 h at 28°C and 230 rpm on a shaker. The incubated Agrobacterium was then placed on a clean bench, and 100 μL of the bacterial culture was pipetted onto LB broth containing 50 mg / L Kan and 50 mg / L rifampin. The plate was sealed and incubated upside down in a dark incubator at 28°C.

[0037] After 2 days of cultivation, single colonies of the grown Agrobacterium were picked and cultured in LB liquid medium containing 50 mg / L Kan and 50 mg / L Rif. PCR was used to identify the presence of the target gene. The bacterial cultures that were correctly identified by PCR were sent to a biotechnology company for sequencing. This confirmed that the recombinant plasmid containing the FNS gene had been successfully transformed into Agrobacterium, resulting in recombinant Agrobacterium.

[0038] During the flowering period of Arabidopsis thaliana, recombinant Agrobacterium was transfected into Arabidopsis using the flower infection method. After seed maturity, the seeds were sterilized and screened on solid MS medium containing 250 mg / L carbenicillin (Cb) and 50 mg / L hygromycin (Hn). The selected seeds were planted in nutrient soil and collected. After three generations of positive transgenic Arabidopsis identification, seeds were harvested for transgenic identification and subsequent experimental verification. Transgenic Arabidopsis were identified using GUS staining (…). Figure 4 ).

[0039] 2. Effects of the FNS gene on the content of apigenin and total flavonoids in Arabidopsis thaliana High-performance liquid chromatography (HPLC) was used to determine the contents of apigenin and total flavonoids in wild-type and transgenic Arabidopsis thaliana. The determination of apigenin followed the previous method (Tan Guofei, et al. Analysis of anthocyanin and apigenin content and gene expression in purple and non-purple apigenin. Journal of Horticulture, 2017, 44(7): 1327-1334). The determination of total flavonoids followed the previous method (Su Jiaojiao, et al. Optimization of HPLC method for determining total flavonoid content in Eucommia ulmoides leaf extract. Applied Chemical Industry, 2023, 52(3): 950-954). The results showed that the apigenin content in transgenic Arabidopsis thaliana was 115.75 μg / g, significantly higher than that in wild-type Arabidopsis thaliana (i.e., non-transgenic) which was 75.11 μg / g. Figure 5 The total flavonoid content in transgenic Arabidopsis thaliana (3.78 mg / g) was higher than that in wild-type Arabidopsis thaliana (3.19 mg / g), as detailed in [link to relevant documentation]. Figure 6 .

[0040] The above experimental results show that the FNS gene obtained in this invention can catalyze the synthesis of apigenin in non-umbelliferous plants, increasing the content of apigenin and total flavonoids.

[0041] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An FNS protein that catalyzes the synthesis of apigenin, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the FNS protein as described in claim 1.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

4. A gene expression cassette, characterized in that, Includes the coding gene as described in claim 2 or 3.

5. A recombinant expression vector, characterized in that, Includes the gene expression cassette as described in claim 4.

6. A recombinant host cell, characterized in that, Includes the recombinant expression vector as described in claim 5.

7. The use of the FNS protein as described in claim 1 in increasing the content of apigenin and / or total flavonoids in non-umbelliferous plants.

8. The use of the encoding gene as described in claim 2 or 3, the gene expression cassette as described in claim 4, the recombinant expression vector as described in claim 5, or the recombinant host cell as described in claim 6 in increasing the content of apigenin and / or total flavonoids in non-umbelliferous plants.

9. The application as described in claim 7 or 8, characterized in that, The non-umbelliferous plant in question is Arabidopsis thaliana.

10. A method for increasing the content of apigenin and / or total flavonoids in non-umbelliferous plants, characterized in that, The method includes the step of genetically transforming the encoding gene as described in claim 2 or 3 into the non-umbelliferous plant to construct a transgenic plant that overexpresses the encoding gene.

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