CN114807177A

CN114807177A - Wild buckwheat rhizome transcription factor FdFAR1 gene and application thereof

Info

Publication number: CN114807177A
Application number: CN202210717374.XA
Authority: CN
Inventors: 王继永; 李进瞳; 曾燕; 卢翔; 林晖才; 靳云西; 矣健玲; 郑司浩
Original assignee: China Traditional Chinese Medicine Seed & Seedling Co ltd; China National Traditional Chinese Medicine Co ltd
Current assignee: China Traditional Chinese Medicine Seed & Seedling Co ltd; China National Traditional Chinese Medicine Co ltd
Priority date: 2022-06-23
Filing date: 2022-06-23
Publication date: 2022-07-29
Anticipated expiration: 2042-06-23
Also published as: CN114807177B

Abstract

The invention provides a wild buckwheat rhizome transcription factor FdFAR1 gene and application thereof, wherein the nucleotide sequence of the transcription factor FdFAR1 is shown as SEQ ID NO. 1. The wild buckwheat rhizome transcription factor FdFAR1 gene and the application thereof, which are disclosed by the invention, adopt a transgenic engineering strategy of the transgenic wild buckwheat rhizome FdFAR1 gene to obtain a transgenic hairy root system, fill up the blank of cloning the FAR transcription factor gene in the wild buckwheat rhizome of a medicinal plant and regulating and controlling the synthesis of flavonoid compounds, lay a molecular foundation for further researching the biological metabolism engineering of the flavonoid as a component of the wild buckwheat rhizome of a Chinese medicinal herb, and have important significance for the genetic engineering research and the innovative utilization of the active ingredients of the wild buckwheat rhizome of the medicinal plant.

Description

Wild buckwheat rhizome transcription factor FdFAR1 gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a wild buckwheat rhizome transcription factor FdFAR1 gene and application thereof.

Background

The wild buckwheat belongs to the genus Fagopyrum of the family Polygonaceae, is a medicinal plant originated in southern China, and has high nutritive value and economic value. Meanwhile, wild buckwheat rhizome contains rich functional components, namely flavonoid compounds, such as rutin, quercetin, kaempferol-3-O-rutinoside and the like. According to the records of Ben Cao gang mu, jin Qian Ma is used to treat scrofula and swollen sore throat, and is the main raw material of Ji Zhi Tang, jin Qian Ma Jiao and jin Qian Ma pian. Golden buckwheat as forage grass can improve the immunity of livestock and poultry and obviously improve the quality of meat and egg products, and is loaded in Chinese animal pharmacopoeia and famous records of allowed varieties of feed pharmaceutical additives. The wild buckwheat rhizome has the characteristics of strong adaptability, drought resistance, barren resistance, strong regeneration capacity, high comparative benefit, low investment, high efficiency, ecological environmental protection and the like. However, the regulation mechanism of the wild buckwheat rhizome medicinal active ingredient flavonoid compounds is still unclear at present, and the development and utilization of the plants are greatly restricted, so that the research aims to efficiently excavate and regulate the key genes of the wild buckwheat rhizome flavonoid substances by molecular biological means such as gene cloning and the like, analyze the genetic mechanism and the regulation network of the metabolic pathway of the key genes, and have important significance on the genetic engineering and the innovative utilization of the wild buckwheat rhizome.

Disclosure of Invention

Aiming at the problems, the invention provides a wild buckwheat rhizome transcription factor FdFAR1 gene, wherein the nucleotide sequence of the transcription factor FdFAR1 is shown as SEQ ID NO. 1.

The invention also provides a recombinant expression vector which comprises a nucleotide sequence shown as SEQ ID NO. 1.

The invention also provides application of the wild buckwheat rhizome transcription factor FdFAR1 gene in regulation and control of synthesis of flavonoids compounds, wherein the nucleotide sequence of the transcription factor FdFAR1 is shown as SEQ ID NO. 1.

Further, the method comprises the following steps:

cloning a transcription factor FdFAR1 gene from wild buckwheat by adopting a gene cloning method, wherein the nucleotide sequence of the transcription factor FdFAR1 is shown as SEQ ID NO. 1;

the FdFAR1 gene is operably constructed in an expression regulatory sequence to form a plant expression vector containing the FdFAR1 gene;

transforming the plant expression vector containing the FdFAR1 gene into agrobacterium rhizogenes to obtain an agrobacterium rhizogenes strain containing the FdFAR1 gene for transforming wild buckwheat rhizome;

genetically transforming the agrobacterium rhizogenes strain containing the FdFAR1 gene into a wild buckwheat rhizome tissue, and obtaining a positive wild buckwheat rhizome identified by PCR;

and (3) carrying out high performance liquid chromatography analysis on the flavone content of the transgenic hairy roots to finish the regulation and control of the FdFAR1 gene in the synthesis of the flavonoid compounds.

Further, the method for cloning the transcription factor FdFAR1 gene from the wild buckwheat rhizome by adopting a gene cloning method comprises the following steps:

fully grinding wild buckwheat rhizome seedlings by liquid nitrogen, and extracting the total RNA of the wild buckwheat rhizome seedlings;

carrying out reverse transcription by taking the total RNA as a template to obtain cDNA of the wild buckwheat rhizome seedling;

designing a specific primer FAR1 according to an open reading frame of the FdFAR1 gene, and carrying out PCR amplification by taking cDNA as a template according to the specific primer FAR1 to obtain the FdFAR1 gene, wherein the nucleotide sequence of the transcription factor FdFAR1 is shown as SEQ ID NO. 1.

Furthermore, quantitative PCR determination and analysis of the expression level of the FdFAR1 gene under the induction of jasmonic acid are also included after the transcription factor FdFAR1 gene is obtained.

Further, quantitative PCR determination and analysis of the expression level of FdFAR1 gene induced by jasmonic acid include:

processing the wild buckwheat rhizome seedlings by adopting methyl jasmonate, and extracting the total RNA of the wild buckwheat rhizome seedlings;

an internal reference gene FtH3 primer and a specific primer qFAR1 are designed, and quantitative PCR analysis is carried out by taking the same amount of cDNA as a template to determine the expression level of the FdFAR1 gene.

Further, the FdFAR1 gene can be constructed in an expression control sequence to form a plant expression vector containing the FdFAR1 gene, and the construction of 1302-FdFAR1 over-expression vector is included.

Further, the construction of 1302-FdFAR1 overexpression vector comprises:

designing a homologous recombination primer, taking an FdFAR1-T vector as a template and 1302-FdFAR 1F/R as a primer, and carrying out PCR amplification on a full-length sequence of FdFAR 1;

after enzyme digestion, recovery, connection and transformation, the full-length sequence of FdFAR1 is inserted into the downstream of CaMV35S promoter of pCAMBIA-FdFAR1 vector, and the over-expression vector pCAMBIA1302-FdFAR1 is obtained after complete sequencing.

Further, transforming the plant expression vector containing the FdFAR1 gene into agrobacterium rhizogenes to obtain the agrobacterium rhizogenes strain containing the FdFAR1 gene for transforming wild buckwheat rhizome, which comprises the following steps:

respectively transforming the pCAMBIA1302-FdFAR1 recombinant plasmid and the pCAMBIA 1302-empty vector plasmid which are verified to be correct by sequencing into agrobacterium rhizogenes by a heat shock method;

after colony PCR identification, pCAMBIA1302-FdFAR1 recombinant plasmid positive bacteria and pCAMBIA 1302-empty vector positive bacteria are obtained.

Further, the pCAMBIA1302-FdFAR1 recombinant plasmid positive bacteria and pCAMBIA 1302-empty vector positive bacteria are subjected to genetic transformation to obtain golden buckwheat rhizome tissues;

after colony PCR identification, the hairy root of the transgenic pCAMBIA1302-FdFAR1 gene is used as a positive control, and the hairy root of the transgenic pCAMBIA 1302-empty vector is used as a negative control.

Further, the flavonoids compounds produced in different hairy roots are analyzed by high performance liquid chromatography, and the chromatographic conditions are as follows: the chromatographic column was a C18 column, the detection temperature was 30 ℃ and the mobile phase was 0.1% formic acid in water and 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL/min.

The wild buckwheat rhizome transcription factor FdFAR1 gene and the application thereof adopt a transgenic engineering strategy of the wild buckwheat rhizome transcription factor FdFAR1 gene to obtain a transgenic hairy root system, fill up the blank of cloning the FAR transcription factor gene in the wild buckwheat rhizome as a medicinal plant and regulating and controlling the synthesis of flavonoid compounds, lay a molecular foundation for further researching the biological metabolic engineering of the flavonoid as the Chinese medicinal component of the wild buckwheat rhizome, and have important significance for the gene engineering research and the innovative utilization of the wild buckwheat rhizome medicinal active component.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.

FIG. 1 is a schematic diagram showing the CDS sequence amplification of wild buckwheat rhizome FdFAR1 gene in an example of the present invention;

FIG. 2 shows a phylogenetic tree of wild buckwheat rhizome FdFAR1 protein and FAR homologous proteins in other plants according to an embodiment of the present invention;

FIG. 3 shows a schematic diagram of the induction of the FdFAR1 gene in response to jasmonic acid stress in an example of the invention;

FIG. 4 is a schematic view showing a process of culturing wild buckwheat rhizome hairy roots in the example of the present invention;

FIG. 5 shows a positive identification map of hairy roots overexpressing FdFAR1 gene in the examples of the present invention;

FIG. 6 is a diagram showing an analysis of the expression level of FdFAR1 gene overexpressing hairy roots in the examples of the present invention;

FIG. 7 is a graph showing rutin content in transgenic hairy roots in an example of the present invention;

FIG. 8 shows a graphical representation of kaempferol-3-O-rutinoside content in transgenic hairy roots in an example of the invention;

FIG. 9 is a diagram showing the expression level of brass gene in FAR1 hairy roots in the example of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention clones a transcription factor, named as FdFAR1, and explores the role of the transcription factor in the metabolism pathway of the wild buckwheat rhizome flavone; and by utilizing a genetic engineering means, the transcription factor FdFAR1 gene is genetically transformed into a wild buckwheat rhizome explant to obtain an over-expressed transgenic wild buckwheat rhizome hairy root so as to efficiently obtain medicinal active ingredients such as rutin, kaempferol-3-O-rutinoside and the like.

The embodiment of the invention provides application of cloning of a wild buckwheat rhizome transcription factor FdFAR1 gene and regulation of synthesis of flavonoids.

The application of the wild buckwheat rhizome transcription factor FdFAR1 gene in regulating and controlling the synthesis of flavonoids compounds in the embodiment of the invention comprises the following steps:

(1) sterilizing wild buckwheat rhizome seeds by using a 1% sodium hypochlorite solution and 75% ethanol, and culturing by using an MS culture medium to obtain wild buckwheat rhizome aseptic seedlings;

(2) cloning a transcription factor FdFAR1 gene from wild buckwheat by adopting a gene cloning method, and analyzing gene sequence information, wherein the nucleotide sequence of the transcription factor FdFAR1 is shown as SEQ ID NO. 1;

(3) quantitative PCR determination and analysis of jasmonic acid induced expression;

(4) the FdFAR1 gene is operably constructed in an expression regulatory sequence to form a plant expression vector containing the FdFAR1 gene;

(5) transforming the plant expression vector containing the FdFAR1 gene obtained in the step (4) into agrobacterium rhizogenes A4 to obtain an agrobacterium rhizogenes strain containing the FdFAR1 gene for transforming wild buckwheat rhizome;

(6) genetically transforming the agrobacterium rhizogenes strain containing the FdFAR1 gene into a wild buckwheat rhizome tissue, and obtaining a positive wild buckwheat rhizome identified by PCR;

(7) and (3) carrying out high performance liquid chromatography analysis on the flavone content of the transgenic hairy roots to finish the regulation and control of the FdFAR1 gene in the synthesis of the flavonoid compounds.

Specifically, the step (1) of sterilizing and raising the wild buckwheat seeds comprises the following steps: the disinfection of the wild buckwheat seeds is to disinfect the wild buckwheat seeds by using a 1% sodium hypochlorite solution for 10 min; sterilizing with 75% ethanol for 2 min; then cleaning with sterile water until the water is clear; the sterile seedling is obtained by placing the seeds on sterilized filter paper to absorb water, and planting the seeds on an MS culture medium; the culture conditions are 22-25 deg.C, 16 h/8 h photoperiod, 75-80% humidity, and 2-4 weeks.

The cloning of the transcription factor FdFAR1 gene in the step (2) is specifically to extract RNA of wild buckwheat seeds and synthesize cDNA through reverse transcription, the transcription factor FdFAR1 gene obtained by cloning wild buckwheat seedlings is obtained by selecting seedlings of four weeks old, taking 50-100 mg of the seedlings, adding liquid nitrogen, fully grinding and then extracting total RNA by using a Trizol method. Using the RNA as a template, cDNA of seedlings were obtained by reverse transcription using HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) Kit (Nanjing Novozam Biotechnology Co., Ltd.).

The method adopting gene cloning is to clone a transcription factor FdFAR1 gene from cDNA of wild buckwheat rhizome seedlings according to the opening of the FdFAR1 geneReading Frame (ORF, Open Reading Frame) design specific primer FAR1, the primer sequence is shown in Table 1, PCR amplification is carried out by taking cDNA as a template according to specific primer FAR1 to obtain FdFAR1 gene, the transcription factor FdFAR1 nucleotide sequence is shown in SEQ ID NO.1, and concretely, PCR amplification is carried out by taking wild buckwheat rhizome 'LJS-1' strain cDNA as a template to obtain CDS sequence of target gene. The PCR program is 95 ℃ for 3 min; 30 s at 95 ℃, 30 s at 58 ℃, 90s at 72 ℃ and 31 cycles. The PCR-purified product was ligated to pTOPO-Blunt Simple Blunt-ended cloning vector to obtainFdFAR1-a T-vector plasmid. Obtained after sequencing, analysis and splicingFdFAR1Full-length sequence. Through the above steps, the full-length coding sequence (SEQ ID NO. 1) of the transcription factor in wild buckwheat was obtained, and the protein coding sequence (SEQ ID NO. 2) thereof was deduced.

TABLE 1 PCR amplification primers

Carrying out PCR amplification by using wild buckwheat rhizome 'LJS-1' strain cDNA as a template (the coded amino acid sequence is shown in SEQ ID NO. 2) to obtain a CDS sequence of a target gene, wherein FIG. 1 shows a schematic amplification diagram of the wild buckwheat rhizome FdFAR1 gene CDS sequence in the embodiment of the invention, and in FIG. 1, M: DL2000 marker, through DNAMAN software alignment, the obtained CDS sequence is basically consistent with the sequence screened by transcriptome sequencing.

After cloning to obtain the transcription factor FdFAR1 gene, gene sequence Information analysis is also carried out, the FdFAR1 gene amino acid sequence is compared with the other 14 amino acid sequences of the reported FAR transcription factor family found from NCBI (National Center for Biotechnology Information), and FdFAR1 (FD 04G 006530) is found to be a typical FAR transcription factor by MEGA 7.0 construction phylogenetic tree, and FIG. 2 shows a phylogenetic tree diagram of wild buckwheat FdFAR1 protein and FAR homologous protein in other plants in the embodiment of the invention.

To determine the expression level of FdFAR1 gene induced by jasmonic acid stress, the method is further describedThe application of wild buckwheat rhizome FdFAR1 in response to jasmonic acid stress induction is researched, seedling treatment is carried out by using 50mM methyl jasmonate, and total RNA of the wild buckwheat rhizome in a seedling stage is extracted. And using the RNA as a template and using a HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) Kit to perform reverse transcription to obtain cDNA. FtH3 gene constitutively expressed by buckwheat is used as internal reference FtActin-Q, a gene specific primer qFAR1 is designed at the same time, FdFAR1 expression quantity detection primers are shown in table 2, 3 biological repeated tests are carried out, and Ft FAR1 expression quantity is detected on a BAI 7500 real-time fluorescent Quantitative PCR instrument by using real-time fluorescent Quantitative PCR (Quantitative real-time PCR qRT-PCR). In this example, RQ (Relative Expression) =2 ^-ΔΔCT Calculating the relative expression level of the target gene; setting the expression inducing value of 0h as 1.

TABLE 2 FdFAR1 expression level detection primers

3 biological repeated tests are carried out, and the expression quantity of Ft FAR1 is detected on a BAI 7500 real-time fluorescent Quantitative PCR instrument by utilizing real-time fluorescent Quantitative PCR (Quantitative real-time PCR, qRT-PCR). RQ (relative expression amount) =2 was used in this experiment ^-ΔΔCT Calculating the relative expression level of the target gene; fig. 3 shows a schematic diagram of induction of the FdFAR1 gene in response to jasmonic acid stress in the embodiment of the present invention, in fig. 3, the horizontal axis represents jasmonic acid treatment time, the vertical axis represents relative expression, the expression value of induced expression 0h is set to be 1, the expression value of induced expression 1h is set to be close to 11, the expression value of induced expression 4h is set to be close to 6, and the expression value of induced expression 12h is set to be close to 5, and the result shows that the FdFAR1 gene significantly responds to jasmonic acid stress, and the expression of the FdFAR1 gene exhibits significant up-regulation. Among them, FAR1 showed a tendency of increasing and decreasing in expression level after induction with jasmonic acid, and reached the highest level in 1 hour. The data in the figure are the expression analysis of seedlings under 50mM methyl jasmonate induction, FtH3 as an internal reference gene, showing that the FdFAR1 gene is able to respond to the induction of SA, and each data set represents the mean. + -. SD (standard deviation) of three replicates.

The FdFAR1 gene can be operably constructed in an expression control sequence to form a plant expression vector containing the FdFAR1 gene, and the construction of a1302-FdFAR1 overexpression vector is included, wherein the 1302-FdFAR1 overexpression vector is constructed: designing a homologous recombination primer, constructing a primer by taking an FdFAR1-T vector as a template, 1302-FdFAR 1F/R as a primer and an FdFAR1 gene overexpression vector shown in table 3, amplifying a full-length sequence of the FdFAR1 by PCR, then carrying out enzyme digestion, recovery, connection and transformation, positively inserting the full-length sequence of the FdFAR1 into the downstream of a CaMV35S promoter of a pCAMBIA-FdFAR1 vector, and obtaining the overexpression vector pCAMBIA1302-FdFAR1 after complete sequencing.

TABLE 3 primers for construction of FdFAR1 Gene overexpression vector

Specifically, the plant expression vector containing 1302 gene obtained in the step (4) is transformed into agrobacterium rhizogenes to obtain agrobacterium rhizogenes strain containing FdFAR1 gene plant expression vector for transforming wild buckwheat rhizome, and the method is characterized in that: sequencing verification the correct pCAMBIA1302-FdFAR1 recombinant plasmid and pCAMBIA 1302-empty vector plasmid were transformed into Agrobacterium rhizogenes A4 competent cells by heat shock method, respectively. After colony PCR identification, pCAMBIA1302-FdFAR1 recombinant plasmid positive bacteria and pCAMBIA1302 empty vector positive bacteria are obtained.

The genetic transformation of the agrobacterium rhizogenes strain containing the FdFAR1 gene into the wild buckwheat rhizome tissue in the step (6) comprises the following steps: FIG. 4 is a schematic diagram showing the cultivation process of wild buckwheat rhizome hairy roots in the example of the present invention, which takes 40 to 60 days from the infection of the explant to the induction of the transgenic hairy roots. Cotyledons of wild buckwheat rhizome, a 4-week-old seedling, were induced by explant culture hairy roots (FIG. 4 a). The Agrobacterium was blotted dry using sterile filter paper, the explants were dried and co-cultured on MS solid medium for 48h at 25 ℃ in the dark. After co-cultivation, the explants were washed three times with MS liquid medium containing 100mg/mL cefotaxime and four times again with sterile water. Subsequently, the explants were cultured in a growth incubator on MS solid medium containing 100mg/mL cefotaxime to induce hairy roots (FIG. 4 b). After 7 days, the explants began to grow hairy roots (FIG. 4 c). Subsequently, hairy roots were excised from the explants and placed on detoxification media to maintain growth. Transgenic testing was performed using PCR to verify that the vector FdFAR1 gene contained had been transformed into the genome; after 14 days of culture, the hairy roots grew at high speed to fill the medium (FIG. 4 d). Finally, 2 cm of transgenic positive hairy root lines were excised from the detoxification medium and transferred to MS liquid medium for growth (FIG. 4e), eventually producing large numbers of hairy roots in the liquid medium (FIG. 4 f). On the other hand, the induction of wild-type hairy roots is the same as that of transgenic hairy roots.

The identification of the transgenic wild buckwheat rhizome hairy root is specifically that the infected wild buckwheat rhizome hypocotyl and cotyledon are placed on an MS solid culture medium, after the amount of the hairy root is enough, a proper amount of the infected wild buckwheat rhizome hypocotyl and cotyledon are placed in an MS liquid culture medium, and the mixed culture medium is shaken at room temperature (120 r/min) (meanwhile, the transgenic wild buckwheat rhizome hairy root pCAMBIA1302-FdFAR1 is used as a positive control, and the transgenic wild buckwheat rhizome 1302-empty vector hairy root is used as a negative control). Detecting gene expression by using FdFAR1-F/R, extracting DNA from hairy roots obtained after infection and arabidopsis thaliana, and performing PCR verification, wherein an upstream primer: 1302-FdFAR 1-F: 5'-acgggggactcttgaccatggATGGAAAATCAACCCGAAATAGAC-3',

A downstream primer: 1302-FdFAR 1-R: 5'-aagttcttctcctttactagtTCAATGTTTCAGTATCAAGTGAACGT-3', FIG. 5 shows the hairy root positive identification chart of the overexpression FdFAR1 gene in the invention example, and

hairy root lines

3, 4, 5, 6, 7 and 8 in FIG. 5 are all positive to transgenes.

Analysis of FdFAR1 gene expression level of overexpressed hairy roots, FIG. 6 shows an analysis chart of FdFAR1 gene expression level of overexpressed hairy roots in the examples of the present invention, FIG. 6 shows strains on the horizontal axis and relative expression level on the vertical axis, and FdFAR1-OE4 (strains), OE7, OE8, which are well grown, and FdFAR1 gene in empty vectors A4 and 1302 are analyzed for expression level, and Table 4 shows transgenic hairy root detection primers. The result shows that the expression level of FdFAR1 gene of wild buckwheat over-expressing hairy roots is obviously up-regulated.

TABLE 4 transgenic hairy root detection primers

In the present example, HPLC analysis of flavone content was performed on transgenic hairy roots, and the flavonoids produced in different hairy roots were analyzed by high performance liquid chromatography using an Ultimate 3000 HPLC system (Thermo Fisher Scientific), a C18 column (2.1 mm. times.75 mm, 2.7 μm) with a detection temperature of 30 ℃. The mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.3 mL/min. The related substances are identified by the retention time of rutin and kaempferol-3-O-rutinoside standard substances. And finally, quantifying the compounds in the wild buckwheat rhizome according to the peak areas and the standard curves of the standard substances with different concentrations, and setting three biological repetitions.

In the embodiment of the invention, the contents of rutin and kaempferol-3-O-rutinoside in the transgenic hairy root are measured by high performance liquid chromatography, fig. 7 shows a schematic diagram of the content of rutin in the transgenic hairy root in the embodiment of the invention, in fig. 7, the horizontal axis represents a strain, and the vertical axis represents the concentration of rutin; FIG. 8 shows a schematic representation of kaempferol-3-O-rutinoside content in transgenic hairy roots in an example of the invention, in FIG. 8, the horizontal axis represents the strain and the vertical axis represents the kaempferol-3-O-rutinoside concentration, and the results show that: the concentrations of rutin and kaempferol-3-O-rutinoside in the hairy root of the over-expressed FdFAR1 are higher than those of the wild-type hairy root, which proves that the FdFAR1 gene influences the generation of specific flavonoids in the transgenic hairy root.

In the above examples, it was shown that the FdFAR1 gene can significantly increase the content of kaempferol-3-O-rutinoside and rutin in the hairy root, and in the examples of the present invention, the expression level of the flavone pathway gene in the transgenic hairy root was quantitatively analyzed, fig. 9 shows a schematic diagram of the expression level of the brass gene in the FAR1 hairy root in the examples of the present invention, fig. 9 shows the strain on the horizontal axis and the relative expression level on the vertical axis, fls (Flavonol synthases) shows the relative expression level of the Flavonol synthase gene, F3H (flavonone-3-hydroxyase) shows the relative expression level of the Flavanone-3-hydroxylase gene, F3' H (Flavonol 3' -hydroxyase) shows the relative expression level of the flavonoid-3 ' -hydroxylase gene, F3'5' H (flavonone-3 ',5' -hydroxyase) shows the relative expression level of the flavonoid-3 ',5' -hydroxydase gene, quantitative analysis on the expression level of part of flavone pathway genes shows that the expression level of FLS and F3'5' H genes is remarkably increased in the transgenic hairy roots influenced by FdFAR1 genes, while the expression level of F3H and F3' H genes is remarkably reduced in the transgenic hairy roots influenced by FdFAR1 genes, which shows that the FdFAR1 genes play an important role in synthesizing rutin and kaempferol-3-O-rutinoside pathways.

The wild buckwheat rhizome transcription factor FdFAR1 gene and the application thereof, which are disclosed by the invention, adopt a transgenic engineering strategy of the transgenic wild buckwheat rhizome FdFAR1 gene to obtain a transgenic hairy root system, fill up the blank of cloning the FAR transcription factor gene in the wild buckwheat rhizome of a medicinal plant and regulating and controlling the synthesis of flavonoid compounds, lay a molecular foundation for further researching the biological metabolism engineering of the flavonoid as a component of the wild buckwheat rhizome of a Chinese medicinal herb, and have important significance for the genetic engineering research and the innovative utilization of the active ingredients of the wild buckwheat rhizome of the medicinal plant.

SEQ ID NO.1 (FdFAR 1 gene coding region sequence (CDS)):

ATGGAAAATCAACCCGAAATAGAGGAACTAGATTATTCGGATGTGTTTGGAGTCGAACAATTTTTTCCGGATAAAGATTCTCTAGTTGATTGGGTTCGTGAGGAAGGTAGAAAATATTATATGGTGATGATCATATTAAAATCATACAAGCCGAGCGATCATCAATATGGGAAGGTTGTATTGGTATGTGAGAAATTTGGGAGGCGGAAGAGTGTGAAGATGGATGATATTGATCCTTCGAATAGGCGTAGATCAAAGTCGAAGAAAAGAGATTGTAAATTTAAGATAGTGGGGGATGAAGTACAACGGAGCAATGGAGTGGTACCGACTAAGTGGAAGATTACCGTGGTTCATGGTAGTCACAACCACGATATTCTGCCATCACTAGAAGGCCATTCATTTGCGGGTCGGCTTACGAGCGAACAAATGCAGAAAGTGAGAACTTGTTCCGCTTCCGGCGTTAAACCGGTGAAAATTTTGGATATGCTTCGAAAGGAATATCCGTCAAATGTCACATCTACACAACAAATCTACAACGCTCGATATAAATTGAAGGTCGAAGATAGAAGCGGGCGGACGATAACACAACAAGCATTACATTTTTTGGGTGAGCGCAATTATTATACGGATTATAGGACTCTTCCCGGGACAAACATCATTAGTGATCTTATCTTGAGTCACCATATGTCACGACATTGGTTGAGCATGTTTCCGTATGTCCTCGTGATGGACACAACGTACAAGACAAACCGGTACGAAATGCCGTTATTGGAGGTGGTCGGATTCACTAGCACGGAGAAGACCTTTGCCGTCGCATTTGTATTCATGTCGAGGGAAACTACGGATCACTATGAGTGGGCATTGGAGCGTGTTAAGATAATATGCGGGGATCGGCTACCAGACGTGATTACGACGGACCGGGAACTTGCCCTTATCAACGCCATCGGAAAGGTCTTCCCCAATGCGAAACATATAATTTGCAACGTTCACTTGATACTGAAACATTGA

SEQ ID NO. 2 (FdFAR 1 gene encoding amino acid results):

MENQPEIEELDYSDVFGVEQFFPDKDSLVDWVREEGRKYYMVMIILKSYKPSDHQYGKVVLVCEKFGRRKSVKMDDIDPSNRRRSKSKKRDCKFKIVGDEVQRSNGVVPTKWKITVVHGSHNHDILPSLEGHSFAGRLTSEQMQKVRTCSASGVKPVKILDMLRKEYPSNVTSTQQIYNARYKLKVEDRSGRTITQQALHFLGERNYYTDYRTLPGTNIISDLILSHHMSRHWLSMFPYVLVMDTTYKTNRYEMPLLEVVGFTSTEKTFAVAFVFMSRETTDHYEWALERVKIICGDRLPDVITTDRELALINAIGKVFPNAKHIICNVHLILKH*

in the above amino acid sequence, a represents a stop codon.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Sequence listing

<110> Chinese medicine Co Ltd, and Chinese medicine speciation Co Ltd

<120> wild buckwheat rhizome transcription factor FdFAR1 gene and application thereof

<160> 12

<170> SIPOSequenceListing 1.0

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<211> 1008

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggaaaatc aacccgaaat agaggaacta gattattcgg atgtgtttgg agtcgaacaa 60

ttttttccgg ataaagattc tctagttgat tgggttcgtg aggaaggtag aaaatattat 120

atggtgatga tcatattaaa atcatacaag ccgagcgatc atcaatatgg gaaggttgta 180

ttggtatgtg agaaatttgg gaggcggaag agtgtgaaga tggatgatat tgatccttcg 240

aataggcgta gatcaaagtc gaagaaaaga gattgtaaat ttaagatagt gggggatgaa 300

gtacaacgga gcaatggagt ggtaccgact aagtggaaga ttaccgtggt tcatggtagt 360

cacaaccacg atattctgcc atcactagaa ggccattcat ttgcgggtcg gcttacgagc 420

gaacaaatgc agaaagtgag aacttgttcc gcttccggcg ttaaaccggt gaaaattttg 480

gatatgcttc gaaaggaata tccgtcaaat gtcacatcta cacaacaaat ctacaacgct 540

cgatataaat tgaaggtcga agatagaagc gggcggacga taacacaaca agcattacat 600

tttttgggtg agcgcaatta ttatacggat tataggactc ttcccgggac aaacatcatt 660

agtgatctta tcttgagtca ccatatgtca cgacattggt tgagcatgtt tccgtatgtc 720

ctcgtgatgg acacaacgta caagacaaac cggtacgaaa tgccgttatt ggaggtggtc 780

ggattcacta gcacggagaa gacctttgcc gtcgcatttg tattcatgtc gagggaaact 840

acggatcact atgagtgggc attggagcgt gttaagataa tatgcgggga tcggctacca 900

gacgtgatta cgacggaccg ggaacttgcc cttatcaacg ccatcggaaa ggtcttcccc 960

aatgcgaaac atataatttg caacgttcac ttgatactga aacattga 1008

<210> 2

<211> 335

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Glu Asn Gln Pro Glu Ile Glu Glu Leu Asp Tyr Ser Asp Val Phe

1 5 10 15

Gly Val Glu Gln Phe Phe Pro Asp Lys Asp Ser Leu Val Asp Trp Val

20 25 30

Arg Glu Glu Gly Arg Lys Tyr Tyr Met Val Met Ile Ile Leu Lys Ser

35 40 45

Tyr Lys Pro Ser Asp His Gln Tyr Gly Lys Val Val Leu Val Cys Glu

50 55 60

Lys Phe Gly Arg Arg Lys Ser Val Lys Met Asp Asp Ile Asp Pro Ser

65 70 75 80

Asn Arg Arg Arg Ser Lys Ser Lys Lys Arg Asp Cys Lys Phe Lys Ile

85 90 95

Val Gly Asp Glu Val Gln Arg Ser Asn Gly Val Val Pro Thr Lys Trp

100 105 110

Lys Ile Thr Val Val His Gly Ser His Asn His Asp Ile Leu Pro Ser

115 120 125

Leu Glu Gly His Ser Phe Ala Gly Arg Leu Thr Ser Glu Gln Met Gln

130 135 140

Lys Val Arg Thr Cys Ser Ala Ser Gly Val Lys Pro Val Lys Ile Leu

145 150 155 160

Asp Met Leu Arg Lys Glu Tyr Pro Ser Asn Val Thr Ser Thr Gln Gln

165 170 175

Ile Tyr Asn Ala Arg Tyr Lys Leu Lys Val Glu Asp Arg Ser Gly Arg

180 185 190

Thr Ile Thr Gln Gln Ala Leu His Phe Leu Gly Glu Arg Asn Tyr Tyr

195 200 205

Thr Asp Tyr Arg Thr Leu Pro Gly Thr Asn Ile Ile Ser Asp Leu Ile

210 215 220

Leu Ser His His Met Ser Arg His Trp Leu Ser Met Phe Pro Tyr Val

225 230 235 240

Leu Val Met Asp Thr Thr Tyr Lys Thr Asn Arg Tyr Glu Met Pro Leu

245 250 255

Leu Glu Val Val Gly Phe Thr Ser Thr Glu Lys Thr Phe Ala Val Ala

260 265 270

Phe Val Phe Met Ser Arg Glu Thr Thr Asp His Tyr Glu Trp Ala Leu

275 280 285

Glu Arg Val Lys Ile Ile Cys Gly Asp Arg Leu Pro Asp Val Ile Thr

290 295 300

Thr Asp Arg Glu Leu Ala Leu Ile Asn Ala Ile Gly Lys Val Phe Pro

305 310 315 320

Asn Ala Lys His Ile Ile Cys Asn Val His Leu Ile Leu Lys His

325 330 335

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggaaaatc aacccgaaat agac 24

<210> 4

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcaatgtttc agtatcaagt gaacgt 26

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaaattcgca agtaccagaa gag 23

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccaacaaggt atgcctcagc 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tggtcggatt cactagcacg 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aagacctttc cgatggcgtt 20

<210> 9

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

acgggggact cttgaccatg gatggaaaat caacccgaaa tagac 45

<210> 10

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aagttcttct cctttactag ttcaatgttt cagtatcaag tgaacgt 47

<210> 11

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

acgggggact cttgaccatg gatggaaaat caacccgaaa tagac 45

<210> 12

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aagttcttct cctttactag ttcaatgttt cagtatcaag tgaacgt 47

Claims

1. A wild buckwheat rhizome transcription factor FdFAR1 gene is characterized in that the nucleotide sequence of the transcription factor FdFAR1 is shown as SEQ ID NO. 1.

2. A recombinant expression vector is characterized in that the recombinant expression vector comprises a nucleotide sequence shown as SEQ ID NO. 1.

3. An application of wild buckwheat rhizome transcription factor FdFAR1 gene in regulating and controlling the synthesis of flavonoid compounds is characterized in that the nucleotide sequence of the transcription factor FdFAR1 is shown in SEQ ID NO. 1.

4. The application of the wild buckwheat rhizome transcription factor FdFAR1 gene in regulating and controlling the synthesis of flavonoids compounds according to claim 3 is characterized by comprising the following steps:

5. The application of the wild buckwheat rhizome transcription factor FdFAR1 gene in regulating and controlling the synthesis of flavonoids compounds according to claim 4, wherein the transcription factor FdFAR1 gene obtained by cloning from wild buckwheat rhizome by a gene cloning method comprises the following steps:

6. The application of the wild buckwheat rhizome transcription factor FdFAR1 gene in regulating and controlling the synthesis of flavonoids compounds according to claim 4, wherein the quantitative PCR determination and analysis of the expression level of the FdFAR1 gene under the induction of jasmonic acid are further included after the transcription factor FdFAR1 gene is obtained.

7. The application of the wild buckwheat rhizome transcription factor FdFAR1 gene in regulating and controlling the synthesis of flavonoids compounds according to claim 6, wherein the quantitative PCR determination and analysis of the expression level of the FdFAR1 gene under the induction of jasmonic acid comprise:

8. The use of the wild buckwheat rhizome transcription factor FdFAR1 gene in regulating the synthesis of flavonoids as claimed in claim 4, wherein the FdFAR1 gene is operably constructed in an expression regulation sequence to form a plant expression vector containing the FdFAR1 gene, which comprises the construction of 1302-FdFAR1 over-expression vector.

9. The use of the wild buckwheat rhizome transcription factor FdFAR1 gene in regulating the synthesis of flavonoids as claimed in claim 8, wherein the construction of 1302-FdFAR1 overexpression vector comprises:

after enzyme digestion, recovery and connection transformation, the full-length sequence of FdFAR1 is inserted into the downstream of CaMV35S promoter of pCAMBIA-FdFAR1 vector, and the over-expression vector pCAMBIA1302-FdFAR1 is obtained after complete sequencing.

10. The application of the wild buckwheat rhizome transcription factor FdFAR1 gene in regulating and controlling the synthesis of flavonoids compounds according to claim 9, wherein the step of transforming the plant expression vector containing the FdFAR1 gene into agrobacterium rhizogenes to obtain the wild buckwheat rhizome agrobacterium rhizogenes strain containing the FdFAR1 gene for transforming the wild buckwheat rhizome comprises the following steps:

11. The application of the wild buckwheat rhizome transcription factor FdFAR1 gene in regulating and controlling the synthesis of flavonoids according to claim 9,

genetic transformation of the pCAMBIA1302-FdFAR1 recombinant plasmid positive bacteria and the pCAMBIA 1302-empty vector positive bacteria into wild buckwheat rhizome tissues;

12. The application of the wild buckwheat rhizome transcription factor FdFAR1 gene in regulating and controlling the synthesis of flavonoids according to claim 4,

analyzing flavonoids compounds produced in different hairy roots by high performance liquid chromatography, wherein the chromatographic conditions are as follows: the chromatographic column was a C18 column, the detection temperature was 30 ℃ and the mobile phase was 0.1% formic acid in water and 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL/min.