CN116239661A - Tobacco flavonol and flavone substance expression regulatory factor NtMYB184 and application thereof - Google Patents

Abstract

The invention discloses a tobacco flavonol and flavone substance expression regulatory factor NtMYB184 and application thereof, wherein the nucleotide sequence of the regulatory factor is SEQ ID NO:2 is shown in the figure; is used for regulating and controlling the content of flavonols and flavones in tobacco. The obtained regulating factor can regulate and control the content of flavones and flavone substances in tobacco, inhibit the expression of NtMYB184 genes, inhibit the expression of structural genes of flavones and flavone synthesis pathways, and further realize the negative expression regulation and control of flavones and flavone substances.

Description

Tobacco flavonol and flavone substance expression regulatory factor NtMYB184 and application thereof

Technical Field

The invention relates to the technical field of synthesis regulation of tobacco substances, in particular to tobacco flavonol and flavone substance expression regulation factor NtMYB184 and application thereof.

Background

Flavonols are a class of phenolic secondary metabolites and play an important role in the adverse effect of plants on stress resistance. There has been increasing evidence in recent years that it has antioxidant capacity in plants, and is capable of scavenging Reactive Oxygen Species (ROS) produced by ultraviolet (UV-B) radiation or inhibiting ROS production. In Arabidopsis and tomato, ROS accumulation can be eliminated by inducing flavonol production through exogenous addition, mutant endogenous accumulation, ethylene treatment and other modes. In addition, tobacco flavonol substances, such as rutin and the like, are aroma substances closely related to the aroma quality of tobacco leaves, so that the tobacco flavonol regulating factors and regulating mechanisms are revealed to have important values for improving the aroma quality of tobacco.

Flavones (flavanes) belong to a subclass of flavonoids (flavanoids) and are secondary metabolites widely distributed in higher plants. The flavone is involved in physiological processes such as cell membrane protection, antioxidation, xanthine oxidase inhibition, lipid reduction and the like, and has important biological effects. In addition, the flavone has pharmacological functions of antivirus, antibacterial, antifungal, photo-protecting agent, cosmetic additive, etc. Among them, there are studies showing that 6 flavonoids such as apigenin, echinocandin, baicalein, luteolin, twinin and lignan have anticancer activity. At present, the research on structural genes of flavone synthesis in higher plants is clearer, but the research on regulatory factors and regulatory mechanisms is less.

Prior art CN 201911257021.0, "a preparation method of high flavonol tobacco and application thereof", discloses: constructing a plant expression vector of a tobacco gene NtMYB12, infecting wild tobacco through agrobacterium-mediated transformation, and obtaining a NtMYB12-Oe transgenic homozygous line which is the high flavonol tobacco through cultivation and screening. This document discloses that tobacco plants overexpressing NtMYB12 can up-regulate the expression levels of NtCHS, ntCHI, ntF H and NtFLS, resulting in a significant 12-fold increase in flavonol content in tobacco leaves compared to wild-type tobacco (WT), while allowing the tobacco plants to attain low phosphorus stress tolerance. However, this prior document does not disclose how to control the flavonol content in tobacco, which is a model plant, and at the same time, control the flavonol content.

Disclosure of Invention

Aiming at the technical problems, the invention provides a tobacco flavonol and flavone substance expression regulating factor NtMYB184 and application thereof, so as to realize simultaneous regulation and control of the content of the tobacco flavonol and flavone substances.

The invention provides a flavonol and flavone substance regulatory factor NtMYB184, the nucleotide sequence of which is SEQ ID NO:1 is shown in the specification; is used for regulating and controlling the content of flavonols and flavones in tobacco.

In another aspect of the present application, there is provided a method for regulating and controlling flavonols and flavones in tobacco, comprising: ligating a gene sequence of a regulatory factor NtMYB184 onto the vector; or transforming the expression vector in a host cell.

Preferably, the vector used is an RNAi vector; more preferably: a pK7GWIW2 vector; more preferably, the regulatable flavonol substance is selected from at least one of quercetin, kaempferol, derivatives of isorhamnetin; the adjustable flavone substance is selected from at least one of apigenin, chrysin, luteolin, eriodictyol, chrysin derivatives. More preferably, the negative regulation results are: the content of the derivatives of quercetin, kaempferol and isorhamnetin is reduced by 20-80 percent; the content of derivatives of apigenin, chrysoeriol, luteolin, eriodictyol and chrysin is reduced by 37-100%.

Preferably, the host cell is selected from any of tobacco, E.coli or Agrobacterium; plant cells or microbial cells may be selected.

In another aspect, the application also provides a protein for regulating and controlling the content of flavonols and flavones in tobacco, the amino acid sequence of the protein is shown as SEQ ID NO. 2, or the protein is coded as the amino acid sequence shown as SEQ ID NO. 2 according to the NtMYB184 gene.

Specifically, in the present invention, SEQ ID NO:1 is:

>NtMYB184_cds

atgggaagag caccttgttg tgagaaagtg ggtctcaaaa gaggcagatg gactgcagaa 60

gaggatgaaa ttctcactaa atatattcaa actaacggcg aaggctcttg gagatcatta 120

cccaaaaatg ctgggttact tagatgtgga aagagttgcc gactgagatg gattaattac 180

ttgaggtctg atttgaggag aggtaacata acttctgaag aggaagacat aatcatcaag 240

ttacatgcaa ctttgggtaa cagatggtct ctaatagcgg gacatttacc aggtagaaca 300

gacaatgaga ttaaaaacta ctggaactct catctaagca gaaaagttga aagcttaaga 360

attccaagcg acgaaaagct gcctcaagct gtagttgatt tggctaataa agggactttg 420

aaccctatca aatgtagagt tggcaaaaca agccgaccca cagtaaagaa aaacagaaca 480

tttaaaaagt ctagttcaag tttgccagag cctaagcaac ctaaagaaag tagtgaacct 540

ttaaattcaa cagttcctat gccctcaact ccaaacatgg aaaaagaggc cttatctagc 600

accattaatg ccatggattc catgcaagaa gaggtagcaa acgtagccgc cgctccaaat 660

ccttggccag ggtctagaga ggcccagagc agccttagct cagatagtgg tatggaatgg 720

cttgacgata ttatgccaat ggtcatggac gatcaaaata tggatccaaa tgaattcatt 780

ttgacttgtt tagacaacgg gcaaggagaa agtccagaaa aagtcagcaa cgacgcagaa 840

aataactgcg ggaccacgaa cagagtcaat gaacataaca tcaaagatca cgataataaa 900

atggtaccaa gtgaagatac acaactggag agtagtccag agagtgaggc tgtatcaata 960

aatattctga aagatgtaca agagaatagc aacgaaacaa gattattaat agaggatggt 1020

acagttgaat gggattggca agagatagct gaagacagaa gagaagtatg gtcatgggaa 1080

gaaacagggc aagagaacat gtcaattaat cacagctggc cgctatggga taatactgac 1140

accgagttac ttcaaaattg cacgaatgaa atcacggtgg aaatggattc cgtgctgcac 1200

agtgaaaacc aaaaccatag ttcccttgtc gcttggcttt tgtcttag 1248

in the invention, SEQ ID NO:2 is:

>NtMYB184 amino acids

MGRAPCCEKV GLKRGRWTAE EDEILTKYIQ TNGEGSWRSL PKNAGLLRCG KSCRLRWINY 60

LRSDLRRGNI TSEEEDIIIK LHATLGNRWS LIAGHLPGRT DNEIKNYWNS HLSRKVESLR 120

IPSDEKLPQA VVDLANKGTL NPIKCRVGKT SRPTVKKNRT FKKSSSSLPE PKQPKESSEP 180

LNSTVPMPST PNMEKEALSS TINAMDSMQE EVANVAAAPN PWPGSREAQS SLSSDSGMEW 240

LDDIMPMVMD DQNMDPNEFI LTCLDNGQGE SPEKVSNDAE NNCGTTNRVN EHNIKDHDNK 300

MVPSEDTQLE SSPESEAVSI NILKDVQENS NETRLLIEDG TVEWDWQEIA EDRREVWSWE 360

ETGQENMSIN HSWPLWDNTD TELLQNCTNE ITVEMDSVLH SENQNHSSLV AWLLS 415

the SEQ ID NO:2 amino acid sequence, wherein R2 repeat, R3 repeat, SG7 domain [ K/R ] [ R/x ] [ R/K ] xGRT [ S/x ] [ R/G ] xx [ M/x ] K, are useful for modulating flavonol, flavone content. The amino acid sequence of the regulatory factor NtMYB184 comprises R2 repetitive sequence, R3 repetitive sequence, SG7 structural domain [ K/R ] [ R/x ] [ R/K ] xGRT [ S/x ] [ R/G ] xx [ M/x ] K from amino terminal to carboxyl terminal; the 12 th-62 th amino acid of the amino acid sequence of the regulatory factor NtMYB184 is R2 repetitive sequence; the 64 th-115 th amino acid of the amino acid sequence of the regulatory factor NtMYB184 is R3 repetitive sequence; the 144 th-156 th amino acid of the amino acid sequence of the regulatory factor NtMYB184 is SG7 structure [ K/R ] [ R/x ] [ R/K ] xGRT [ S/x ] [ R/G ] xx [ M/x ] K.

In another aspect, the present application further provides a method for synthesizing a substance for modulating a flavonol or flavonol in tobacco, comprising at least: any one of a to c:

a) Inhibiting expression of the regulatory factor NtMYB184 gene of claim 1 in tobacco;

b) Transforming an RNAi vector linked to the regulatory factor NtMYB184 gene of claim 1;

c) A transformant obtained by ligating the RNAi vector of the regulatory factor NtMYB184 gene of claim 1 infects tobacco.

Reagents used in the synthesis method are all commonly used in the art to achieve synthesis of the substance.

Preferably, the obtaining of the expression vector comprises the steps of: PCR amplification of the gene sequence of the regulatory factor NtMYB184 to obtain PCR amplification products, and connecting the PCR amplification products with a vector to obtain an expression vector.

The gene sequence can be subjected to PCR amplification, and the gene sequence of the flavonol and flavone regulatory factor NtMYB184 is subjected to PCR amplification to obtain a PCR amplification product; the PCR amplification system comprises: 4. 4 ng/. Mu.L of plant cDNA, 0.2. Mu.L/mu.L of 5 XPhusion HF reaction buffer, 0.2mM dNTP,0.04U/. Mu.L of Phusion High-Fidelity DNA Polymerase, 0.2. Mu.M forward and reverse primers, and the balance of water.

More preferably, the amplification primers comprise:

forward primer S194_F_BamHI 5'GGATCCTCAAACTAACGGCGAAGG-3’，

Reverse primer s194_r_xhoi:5' -CTCGAGCTGGTAAATGTCCCGCTA-3’。

Preferably, the PCR amplification procedure comprises: 98 ℃ for 30 seconds; at 98℃for 7 seconds; 62 ℃,30 seconds; 72 ℃,45 seconds is 1 cycle, and total 35 cycles are adopted; extending at 72 ℃ for 7 minutes;

preferably, the PCR amplification product of the gene is ligated to an RNAi vector to obtain an expression vector.

Preferably, the connection means that the vector pENTR ™ NtMYB184 connected with the gene sequence of the flavonol and flavone substance regulating factor NtMYB184 is further connected through LR reaction to obtain RNAi vector pK7GW-NtMYB184 capable of regulating and controlling the synthesis of the flavone substance. The system of LR reaction includes: 6.25 ng/. Mu.L-18.75 ng/. Mu.L pENTR ™ NtMYB184, 0.0625. Mu.L/. Mu.L ligation vector, the remainder being TE Buffer.

Preferably, the LR reaction step comprises: mixing the connection reaction system, ice-bathing for 2min, and mixing; adding 0.2 mu L/mu L LR Clonease II enzyme Mix, mixing well, centrifuging, and water-bathing at 25 ℃ 1 h; then adding 0.9 mu L/mu L protease K, mixing well, and carrying out water bath at 37 ℃ for 10 min; the mixing mode is preferably flicking.

Transformant acquisition can be performed using existing methods, for example, comprising the steps of: RNAi vector pK7GWIW2 for regulating and controlling synthesis of flavonol and flavone substanceNtMYB184Transforming into competent cells to obtain transformant capable of regulating and controlling synthesis of flavonol and flavone substancesThe method comprises the steps of carrying out a first treatment on the surface of the And infecting plant cells with the transformant capable of regulating and controlling the synthesis of flavonol and flavone substances. Competent cells are agrobacterium competent cells; the transformant capable of regulating and controlling the synthesis of the flavonol and the flavonol substances refers to RNAi vector pK7GW containing the sameNtMYB184Is a clone of Agrobacterium.

In another aspect of the present application, there is provided a kit comprising at least: such as regulatory factor NtMYB184; and/or the expression vector described above; and/or transformants as described above.

Preferably, the kit comprises: the gene sequence amplification primer, PCR reagent, connection conversion reagent and transgene reagent.

The above reagents may be selected according to a kit commonly used in the art, for example, PCR reagents used include: DNA polymerase, dNTP, PCR buffer solution and double distilled water; the DNA polymerase used is preferably Phusion-Fidelity DNA Polymerase. The PCR buffer used is preferably 5 XPhusion HF reaction buffer.

The vector used is RNAi vector pK7GWIW2 vector, then the ligation transforming reagent used comprises: the corresponding restriction enzyme is selected fromBamHI、XhoI, a step of I; the restriction enzyme is Clonase ^TM II enzyme Mix; the connection Buffer solution is TE Buffer; the protease is Proteinase K. When the kit is used for regulating the expression level of flavonols, the amplification primers are as described above.

Competent cells in the ligation transformation reagent were selected from: coli competent cells, agrobacterium competent cells; the culture medium used was LB medium.

The invention has the beneficial effects that:

1) The regulating factor NtMYB184 provided by the invention can regulate and control the expression of flavonols and flavones in tobacco, and realize the negative expression regulation and control of flavonols and flavones.

2) The flavonol and flavone substance regulating factor NtMYB184 provided by the invention can reduce the content of derivatives of flavonols such as quercetin, kaempferol, isorhamnetin and the like by 20% -80% by inhibiting the expression of the regulating factor in plants; celeryThe content of flavonoid derivatives such as element, chrysoeriol, luteolin, eriodictyol, chrysin is reduced by 37% -100%. Indicating inhibition ofNtMYB184The gene can reduce the content of flavonols and flavones in tobacco leaves, namely, the NtMYB184 regulates and controls the synthesis of the flavonols and flavones in the tobacco leaves.

Drawings

FIG. 1 shows experimental example 1 of the present inventionNtMYB184Agarose gel electrophoresis diagram of gene PCR amplified product and TOPO cloning vector. A:NtMYB184amplification product, M: DL2,000 DNA markers; b:BamHI+Xhoi double cleavage detection of pTOPO-NtMYB184Plasmid, M: DL2, 000 DNA markers, 1-2: pTOPO-NtMYB184Plasmid cleavage products.

FIG. 2 shows RNAi vector pK7GW-NtMYB184Is a PCR detection electropherogram of (C). A: primer IntronF2/P35SR2 plasmid PCR detection of pK7GW-NtMYB184，M：DL2，000，1～3: pK7GW-NtMYB184A plasmid PCR product; primer T35SF1/P35SR1 plasmid PCR detection pK7GW-NtMYB184，M：DL2，000，1～3: pK7GW-NtMYB184Plasmid PCR products.

FIG. 3 shows the gene expression level test of transgenic plants in Experimental example 4 of the present invention. KD3, KD4: RNAi transgenic lines; y87: wild type plants.

FIG. 4 shows the flavonol content of transgenic plants in Experimental example 5 of the present invention. KD3, KD4: RNAi transgenic lines; y87: wild type plants.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.

The technical means which are not described in detail and are not used for solving the technical problems of the invention are all arranged according to common general knowledge in the field, and various common general knowledge arrangement modes can be realized.

Examples

The reagents and apparatus used in the examples below are commercially available unless otherwise specified.

Experimental example 1 cloning of amplification product of regulatory factor NtMYB184

1. Carrying out PCR reaction by adopting the following primers to obtain a target product;

forward primer S194_F_BamHI 5'GGATCCTCAAACTAACGGCGAAGG-3’，

Reverse primer s194_r_xhoi:5' -CTCGAGCTGGTAAATGTCCCGCTA-3’。

To construct RNAi vectors, primer cleavage sites (underlined) are included in the above primersBamHI andXhoI；

2. RNA from tobacco leaves was extracted using Trizol kit (Invitrogen) and operated according to the instructions provided by the kit.

3. The first strand cDNA obtained by reverse transcription is used as a template, and primer S194_F_BamHI/S194_R_XhoI is used for PCR amplification, and a Phusion high-fidelity amplification enzyme reaction system is selected, wherein the total volume of the system is 50 mu L, and the PCR amplification method comprises the following steps: 200 10 mu L of 5 XPhusion HF reaction buffer, 1 mu L of 10 mM dNTP, 2U Phusion of High-Fidelity DNA Polymerase,1 mu L of 10 mu M forward and reverse primers, and water to 50 mu L. The PCR reaction is carried out on a Mastercycler pro-amplicon instrument, and the reaction procedure is as follows: 98 ℃ for 30 seconds; 98℃for 7 seconds; 62 ℃,30 seconds; 72 ℃,45 seconds; 35 cycles; extension was performed at 72℃for 7 minutes. The PCR product was recovered and purified. The electrophoretogram of the PCR amplification product is shown in FIG. 1.

Experimental example 2 RNAi vector construction

1. CloningNtMYB184Ligation of TOPO vectors

a. TOPO cloning is carried out on the recovered amplified product fragment, the amplified product fragment is connected to a PCR-Blunt II-TOPO (3.5 kb) vector, then escherichia coli DH5 alpha competent cells are transformed, plasmids are extracted for PCR detection, plasmids with amplified product size of about 0.2kb are selected for DNA extraction, and the constructed vector is named pTOPO-NtMYB184；

b. Since the forward and backward primers of the gene are respectively provided withBamHI、XhoI recognition site, therefore, the two enzymes were selected to perform double digestion of the plasmid DNA sample, resulting in two fragments, about 3.5 kb and 0.2kb, respectively, indicating that the fragment of interest has been inserted into the TOPO vector (FIG. 1).

2. Construction of plant RNAi vector

a. Entrance cloning pENTR ™ B-NtMYB184Construction of (3)

（1）BamHI/XhoI cleavage of pTOPO-NtMYB184And pENTR ™ B to obtain the target gene fragmentNtMYB184And vector pENTR ™ B linearization fragment, and connecting and transforming competent cells DH5 alpha after glue recovery;

(2) Selecting clone after DH5 alpha transformation, extracting plasmid DNA,BamHI/Xhoi cleavage, the vector fragment of 3.8 kb and the fragment of about 0.2kb were the correct clones, designated pENTR ™ B-NtMYB184；

b. Plant RNAi vector obtained through LR reaction

(1) Entrance cloning pENTR ™ B-NtMYB184Transforming E.coli DH5 alpha competent cells after reacting with RNAi vector pK7GW1WG2 LR to obtain plant RNAi vector pK7GW-NtMYB184. With pK7GW-NtMYB184As templates, PCR was performed with two pairs of primers (T35 SF1/P35SR1, intronF2/P35SR 2), respectively, to check the correctness of the recombinant plasmid. In theory, the size of the fragment amplified by the T35SF1/P35SR1 primer is 1.7 kb, and the size of the fragment amplified by the IntronF2/P35SR2 primer is 750 bp. The PCR amplification results were consistent with the expectations, indicating that the expression vector pK7GW was constructedNtMYB184(FIG. 2).

Primer information is as follows:

T35SF1：5’-AGGTCACTGGATTTTGGTTTTA-3’ （SEQ ID NO.3）

P35SR1：5’-CTATCGTTCAAGATGCCTCTGC-3’ （SEQ ID NO.4）

IntronF2：5’-ATTGGTGGCTCAAATCATAGAA-3’ （SEQ ID NO.5）

P35SR2：5’-AGGACAGTAGAAAAGGAAGGTG-3’ （SEQ ID NO.6）

the LR reaction system described above: successfully constructed entry vector pENTR ™ -NtMYB184(50-150 ng) 1-7. Mu.L, 0.5. Mu. L Destination Vector (i.e., pK7GW1WG2 vector), TE Buffer to a total volume of 8. Mu.L; mixing, ice-bathing for 2min, flicking for 2 times; adding 2 μl of LR Clonease II enzyme Mix, flicking, mixing, centrifuging, and water-bathing at 25deg.C 1 h; then 1. Mu.L of Proteinase K is added for flicking, mixed well and water-bath at 37 ℃ for 10 min.

Experimental example 3 genetic transformation of tobacco

1. RNAi vector transformation of Agrobacterium

The agrobacteria competent cells were taken out from the refrigerator at-80℃and were dissolved on ice followed by addition of 4. Mu.L of recombinant RNAi vector pK7GW-NtMYB184The method comprises the steps of carrying out a first treatment on the surface of the Quick-freezing with liquid nitrogen for 1 minute, transferring into 37 ℃ water bath for 5 minutes, further ice-bathing for 2 minutes, adding 1mL of LB liquid medium into the mixture, and culturing for 3-4 hours at 28 ℃ and 220 rpm; the culture is coated on LB solid medium containing spectinomycin 100 mg/L and rifampicin 25 mg/L, and is cultured for 2-3 days at 28 ℃ in an inverted way, and agrobacterium clone containing the target vector is visible;

2. tobacco conversion

a. Selecting agrobacterium clone containing target vector, streaking on LB plate containing spectinomycin and rifampicin, culturing at 28 deg.C for 2-3 days; scraping streak bacterial plaque to connect bacteria in MS culture medium containing spectinomycin and rifampicin, shaking and culturing at 28 ℃ and 220 rpm, centrifuging at 6,000 rpm for 5 minutes when the bacterial liquid concentration reaches OD=0.5-0.8 to enrich the bacterial body, discarding the supernatant, and re-suspending the bacterial body by using 20 mL liquid MS culture medium to obtain agrobacterium suspension bacterial liquid containing target carrier;

b. putting tobacco leaves of flue-cured tobacco cloud 87 into a 500 mL wide-mouth bottle, adding a proper amount of 75% ethanol, and rinsing for 1min; discarding ethanol, adding 0.1% HgCl ₂ Placing the solution on a shaking table, and oscillating for 15-30 minutes at room temperature; discard HgCl ₂ The solution is washed 6 times by sterile water;

c. taking out tobacco leaves, sucking surface liquid with sterile absorbent paper, cutting the sterile leaves into small pieces of about 1 cm ×1 cm with scissors, placing the cut tobacco leaves into sterile MS liquid culture medium suspension containing target carrier, and standing for 15-20 min; taking out tobacco leaves, sucking excessive bacterial liquid by using sterile filter paper, and dark culturing in MS culture medium containing 6-BA (0.02 mg/L) and NAA (2 mg/L) at 25deg.C for two days; subsequently, transferring the tobacco leaves into a differentiation medium, contacting the incision with the medium, and performing differentiation culture under the greenhouse condition; the differentiation medium is MS medium containing 6-BA (0.5 mg/L), NAA (0.1 mg/L), kanamycin (100 mg/L) and cephalosporin (500 mg/L), and is cultured for 1 time every 2-3 weeks, callus grows gradually at the incision, and finally buds are differentiated;

d. cutting off buds growing to 3-5cm, transferring into an MS culture medium to induce rooting, taking out the rooted transgenic plants, washing the culture medium with tap water, and transplanting the transgenic plants into sterilized nutrient soil;

e. transgenic plants are subjected to NPTII gene specific primers:

NPTII-F: 5’-TCGGCTATGACTGGGCACAACAGA-3’ （SEQ ID NO.7）

NPTII-R: 5’-AAGAAGGCGATAGAAGGCGATGCG-3’ （SEQ ID NO.8）

and (5) PCR verification amplification and identification of transgenic positive plants.

Analysis of Gene expression level of transgenic plants obtained in Experimental example 4, example 3

Total RNA of leaves of the T2 generation transgenic strain is extracted, and cDNA synthesized by using a TaKaRa PrimeScriptTM RT reagent Kit reverse transcription kit is used as a template for real-time fluorescence quantitative PCR analysis. Transgenic plants (KD 3/KD 4)NtMYB184Gene expression levels were significantly lower than wild-type plants (Y87) (FIG. 3), indicating inhibitionNtMYB184Genetic manipulation of the gene was successful. In the transgenic plant (KD 3/KD 4), the structural gene related to the flavone alcohol and flavone synthesis pathNtCHS、NtCHIExpression levels significantly lower than wild-type plants (Y87), indicating interferenceNtMYB184Expression of structural groups capable of inhibiting synthesis of flavonols and flavonesAs a result of expression (fig. 3).

Experimental example 5, detection of flavonol and flavone content of transgenic plant

1. Sample preparation

Fresh tobacco leaves were ground into powder in liquid nitrogen, freeze-dried, and then 20 mg dry samples were weighed, ground with 3 ml of 75% methanol, sonicated for 30 min, centrifuged at 10000 rpm for 10 min, 1ml of supernatant was transferred to 10 ml tubes, and then chlorophyll was removed with 1ml water and 2 ml chloroform. The mixture was spun for 1min, centrifuged at 10000 rpm for 10 min, and the supernatant was collected for analysis.

2. Detection of flavonols and flavones

The flavonoids content in the leaves and flowers of tobacco was analyzed using Waters ACQUITY UPLC system (Waters) in combination with AB Sciex Triple Quad 5500 mass spectrometer, the column was set to 30 ℃ using Waters BEH C18column (150×2.1 mm i.d., 1.7 um particle sizes, waters Corporation), the sample injection amounts were 1 μl, solvents a and B were water and acetonitrile, formic acid (0.1%, v/v) and 0.2mmol/L ammonium acetate were added to the solvents a and B, respectively, to facilitate the chromatographic separation, the eluent gradient was set to 10% solvent B1 min,10-90% solvent B8 min,90-100% solvent B2 min. The total run time was 13min, including a 2min equilibration time. The liquid chromatography eluate was directly introduced into the ESI interface at a flow rate of 0.2 ml/min and analyzed for flavonoids in combination with positive and negative ionization patterns. The collision energy of 20,30, 40, 50, V is used for MS2 fragmentation. Air curtain 40 psi; collision gas 6 psi; ion spray voltage ± 4000V; the temperature is 700 ℃; ion source gas 1, 60 psi; ion source gas 2, 50 psi. Helium was used as the collision gas in the fragmentation experiments.

3. Transgenic plant flavone content analysis

In transgenic plant leaves (KD 3/KD 4), the content of derivatives of flavonols such as quercetin, kaempferol, isorhamnetin and the like is reduced by 20-80%; the content of flavonoids derivatives such as apigenin, chrysoeriol, luteolin, eriodictyol, chrysin is reduced by 37% -100% (figure 4). This indicates inhibition ofNtMYB184The gene can reduce the content of flavonols and flavones in tobacco leaves, namely, the NtMYB184 regulates and controls the synthesis of the flavonols and flavones in the tobacco leaves.

From the above, the regulatory factor NtMYB184 provided by the invention can realize effective regulation of the flavonol and flavone contents of tobacco plants in tobacco, and can realize effective regulation of the contents of the two substances in tobacco.

Although the present invention has been described with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements and changes may be made without departing from the spirit and principles of the present invention.

Claims (9)

1. Flavonol, flavones substance regulating factor NtMYB184, characterized in that its nucleotide sequence is SEQ ID NO:1 is shown in the specification; is used for regulating and controlling the content of flavonols and flavones in tobacco.

2. A method for regulating and controlling flavonols and flavones of tobacco plants, which is characterized by comprising the following steps: ligating the gene sequence of the regulatory factor NtMYB184 of claim 1 onto a vector; or transforming an expression vector comprising the gene sequence of the regulatory factor NtMYB184 of claim 1 in a host cell.

3. The method according to claim 2, wherein the carrier used for regulating the flavonol and flavone substance content of the tobacco plant is RNAi carrier;

more preferably: a pK7GWIW2 vector;

more preferably, the NtMYB184 gene is used for regulating and controlling the flavonol content in tobacco to be negative;

more preferably, the regulatable flavonol substance is selected from at least one of quercetin, kaempferol, derivatives of isorhamnetin; the adjustable flavone substance is at least one selected from apigenin, chrysin, luteolin, eriodictyol and chrysin derivatives;

more preferably, the negative regulation results are: the content of the derivatives of quercetin, kaempferol and isorhamnetin is reduced by 20-80 percent; the content of derivatives of apigenin, chrysoeriol, luteolin, eriodictyol and chrysin is reduced by 37-100%.

4. The protein for regulating and controlling the content of flavonols and flavones in tobacco is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO. 2 or the protein is coded according to the NtMYB184 gene as shown in the SEQ ID NO. 2.

5. A method for regulating the synthesis of flavonoids and flavonols in tobacco, comprising at least: any one of a to c:

a) Inhibiting expression of the regulatory factor NtMYB184 gene of claim 1 in tobacco;

b) Transforming an RNAi vector linked to the regulatory factor NtMYB184 gene of claim 1;

c) A transformant obtained by ligating the RNAi vector of the regulatory factor NtMYB184 gene of claim 1 infects tobacco.

6. The method of claim 5, wherein the obtaining of the RNAi vector comprises the steps of: PCR amplification of the gene sequence of the regulatory factor NtMYB184 to obtain a PCR amplification product, and connecting the PCR amplification product with a carrier to obtain an RNAi carrier;

more preferably, the PCR amplification primers:

forward primer S194_F_BamHI 5'GGATCCTCAAACTAACGGCGAAGG-3’，

Reverse primer s194_r_xhoi:5' -CTCGAGCTGGTAAATGTCCCGCTA-3’。

7. The method of claim 5, wherein the transformant obtaining comprises the steps of: transforming RNAi vector into competent cells to obtain transformant; infecting plant cells with the resulting transformant;

more preferably, the competent cells are agrobacterium competent cells;

more preferably, the transformant refers to RNAi vectors containing a regulatable flavone, flavonol synthesisBody pK7GWNtMYB184Is a clone of Agrobacterium.

8. A kit, comprising at least: the gene sequence of regulatory factor NtMYB184 of claim 1; and/or an expression vector obtained by the method of claim 6; and/or a transformant synthesized by the method according to claim 7.

9. The kit of claim 8, wherein the kit comprises: a regulatory factor gene sequence amplification primer, a PCR reagent, a ligation conversion reagent and a transgenic reagent;

more preferably, the PCR reagents used include: DNA polymerase, dNTP, PCR buffer solution and double distilled water;

more preferably, the DNA polymerase is Phusion High-Fidelity DNA Polymerase; the PCR buffer used is preferably a 5 XPhusion HF reaction buffer;

more preferably, the vector used is the RNAi vector pK7GWIW2 vector, and the ligation transforming reagent used comprises: the corresponding restriction enzyme is selected fromBamHI、Xho I, a step of I; the restriction enzyme is Clonase ^TM II enzyme Mix; the connection Buffer solution is TE Buffer; the protease is Proteinase K.

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