CN116837007A - Phyllostachys Pubescens flavonoid compound synthesis related gene PnF H and application thereof - Google Patents

Abstract

A purple bamboo flavonoid compound synthesis related gene PnF H and application thereof belong to the technical field of molecular biology. The invention provides a flavonoid compound synthesis related gene PnF H and a coded protein thereof, and provides application of the flavonoid compound synthesis related gene PnF H. The invention provides a gene resource which is important for purple bamboo and can regulate and control flavonoid synthesis with universality, and provides excellent candidate genes for cultivating plant varieties with bamboo stalk color change.

Description

Phyllostachys Pubescens flavonoid compound synthesis related gene PnF H and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a gene PnF H related to synthesis of a purple bamboo flavonoid compound and application thereof.

Background

Flavonoid (Flavonoids) compounds are a class of natural polyhydroxyphenols secondary metabolites found in plants, widely distributed in the plant kingdom, and have high biological activity. Flavonoids are important secondary metabolites synthesized naturally in plants and are closely related to the color development of leaves, stems and flowers of plants. Furthermore, flavonoids are now widely recognized as a large class of active ingredients in phytochemicals, having diverse functions in plant growth, development, reproduction and response to various stresses, and also play a non-negligible critical role in animal growth, medical development, human health, etc. The related studies have found that naturally occurring flavonoids have many beneficial physiological activities and functional effects including antibacterial, antioxidant, bacteriostatic, anticancer, etc.

Basic structure of flavonoids: the two benzene rings are connected by a pyran ring containing 3 carbon and a 15 carbon phenylpropane ring to form a core C skeleton structure. The backbone can be acylated, methoxylated, hydroxylated or O-glycosylated with different enzymes to form different classes, and can be specifically classified into six general classes, respectively Flavonols (Flavonols), anthocyanins (Anthiocyanins), flavones (Flavones), flavanones (Flavones), isoflavones (Isoflavones) and Flavanols (Flavonols).

The phenylalanine metabolic pathway is the main process of flavonoid biosynthesis, and generally starts from phenylalanine metabolism and enters the biosynthetic branch of flavonoid. Firstly, phenylalanine generates cinnamic acid through deamination of phenylalanine lyase (Phenylalanine ammonia-lyase, PAL), cinnamic acid 4-carboxylase (Cinnamate 4-hydroxyase, C4H) can introduce hydroxyl group on benzene ring of cinnamic acid to generate coumaric acid, and secondly, under the action of coumaroyl-CoA ligase, coumaroyl-CoA is catalyzed by coumaroyl-CoA. Coumaroyl-coa and malonyl-coa produce naringenin (an important precursor of flavonoid biosynthesis under the catalysis of chalcone synthase (Chalcone synthase, CHS) and chalcone isomerase (Chalcone isomerase, CHI). Naringenin then enters other flavonoid synthesis pathways as a main metabolite, and finally forms precursors of Flavone, isoflavone, flavonol and anthocyanin through catalysis of Flavanone-3-carboxylase (F3H), flavanone-3 '-hydroxylase (F3' H), flavonol synthase (Flavonol synthase, FLS) and other enzymes.

Flavanone-3-carboxylase (F3H) is a key enzyme gene on the plant anthocyanin biosynthesis pathway and is mainly responsible for catalyzing naringenin into flavanonol, and is ubiquitous in plants. However, the synthesis of the gene F3H related to the flavonoid compound of Phyllostachys Pubescens and the use thereof have not been reported in the prior art.

In conclusion, the research on F3H gene in flavonoid synthesis pathway involved in flavonoid synthesis is helpful for understanding the mechanism of flavonoid synthesis, and lays a foundation for molecular mechanism research for bamboo stalk color development.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention aims to design and provide a technical scheme for synthesizing related genes PnF H and application thereof by using the purple bamboo flavonoid compounds.

The invention is realized by the following technical scheme:

the first aspect of the invention provides a related gene PnF H synthesized by the purple bamboo flavonoid compound, and the nucleotide sequence of the related gene is shown as SEQ ID NO. 1.

The second aspect of the invention provides a coding protein of a related gene PnF H synthesized by the purple bamboo flavonoid compound, and the amino acid sequence of the coding protein is shown as SEQ ID NO. 2.

In a third aspect, the present invention provides a vector comprising a gene PnF H related to the synthesis of a purple bamboo flavonoid as described above.

In a fourth aspect, the present invention provides the use of a gene PnF H related to the synthesis of a purple bamboo flavonoid compound or a vector as described above for controlling the synthesis of a flavonoid compound.

Further, the use may comprise the gene PnF H in plants or the gene PnF H may be over-expressed in plants.

In a fifth aspect, the present invention provides a method of modulating the synthesis of a flavonoid compound in a plant, the method comprising: plants were made to contain the gene PnF H or plants were made to overexpress the gene PnF H.

Further, the method specifically comprises the following steps:

1) Collecting annual purple bamboo leaves, extracting RNA, reversely transcribing into cDNA, cloning a CDS sequence of PnF H, connecting the CDS sequence with a pC-1300GFP vector for sequencing, and constructing a 35S PnF H overexpression vector after identification is correct;

2) And transferring the constructed 35S PnF H over-expression vector into plant callus to make the plant contain gene PnF H or make the plant over-express gene PnF H.

The invention has the following beneficial effects:

1. the PnF H gene and the coding protein are obtained from the purple bamboo for the first time, and the biological function of regulating and controlling the flavonoid synthesis is verified.

2. According to the invention, the purple bamboo PnF H gene is overexpressed in the calli of tobacco and Malaysia japonica for the first time, and the PCR method is used for proving that the gene is successfully integrated into the calli of tobacco genome and Malaysia japonica, namely a positive strain.

3. The invention carries out qRT-PCR analysis on transgenic tobacco leaves, wild tobacco leaves, common Mallotus japonicus callus and cDNA of the transgenic Mallotus japonicus callus as templates, and determines the flavonoid content in PnF H transgenic tobacco and Mallotus japonicus callus, and the result shows that in the transgenic strain of over-expressed PnF H, the expression level of most genes in the tobacco flavonoid synthesis pathway is up-regulated but not obvious, and it is presumed that PnF H can play a certain role in the synthesis of tobacco flavonoids. In transgenic calli of Mallotus japonicus over-expressing PnF H, the expression level of the key genes of flavonoid synthesis pathway in different strains of PnF H transgenic Mallotus japonicus was increased, and it was speculated that PnF H may play a role in the synthesis of bamboo flavonoids.

Drawings

FIG. 1 shows a thermal map of the expression level of the F3H-like gene of the key enzyme in the flavonoid synthesis pathway;

FIG. 2PnF H gene clone;

FIG. 3 shows that the expression level of PnF H gene of different strains of tobacco (A) and Malaysia japonica (B) is extremely remarkable (P.ltoreq.0.01);

FIG. 4 tobacco (A) and Malaysia tabacum (B) differ in the flavonoid content of the PnF H overexpressed strain species, representing significant (P.ltoreq.0.05);

FIG. 5PnF H shows the expression level of key genes of flavonoid synthesis pathway in different lines of transgenic tobacco, representing significant (P.ltoreq.0.05); * Represents extremely significant (p.ltoreq.0.01);

FIG. 6PnF H expression levels of key genes of flavonoid synthesis pathways in different strains of Mallotus japonicus, representing significant (P.ltoreq.0.05); * Represents extremely significant (p.ltoreq.0.01).

Detailed Description

The invention will now be described in further detail with reference to the following specific examples, which are given by way of illustration and not limitation.

Example 1: cloning of Phyllostachys Pubescens PnF H Gene

1. Material preparation

Annual purple bamboo leaves from purple bamboo forest of Zhejiang agriculture and forestry university in Linan area of Hangzhou, zhejiang province are collected and stored at-80 ℃ for subsequent experiments.

2. RNA extraction

The RNA extraction is carried out by referring to the RNA extraction kit instruction book of Hangzhou New JingZhou Simmen biological Limited company, and the specific steps are as follows:

(1) Grinding the leaves of Phyllostachys Pubescens into powder in a sterilized mortar, weighing 300mg sample in 1.5mL RNA-free centrifuge tube, adding 600 μl Buffer RCT with beta-mercaptoethanol, and vortex shaking to dissolve thoroughly.

(2) 600. Mu.L of BufferEX was added to the 1.5mL RNA-free centrifuge tube in step (1), and the mixture was thoroughly shaken and centrifuged at 12,000 rpm for 5min.

(3) 400 μl of the supernatant from step (2) was transferred into a new 1.5mL RNA-free centrifuge tube.

(4) Adding 400 mu L of Buffer K into the supernatant in the step (3) and uniformly mixing, transferring the whole mixed solution into a filter column, covering a tube cover, and centrifuging at 3500 rpm for 2min.

(5) The column from step (4) was discarded, 700. Mu.L of 70% ethanol was added to the filtrate and mixed directly with a pipette tip.

(6) The mixture in the step (5) was pipetted into a nucleic acid purification column at 700. Mu.L and centrifuged at 13,000 rpm for 1min.

(7) The filtrate in the 2mL centrifuge tube was discarded, and the nucleic acid purification column was returned to the 2mL RNA-free centrifuge tube, and the remaining mixture was aspirated and added to the nucleic acid purification column, and centrifuged at 13,000 rpm for 1min.

(8) The filtrate in the 2mL centrifuge tube WAs discarded, the nucleic acid purification column WAs returned to the 2mL RNA-free centrifuge tube, 500. Mu.L Buffer WA (absolute ethanol WAs added) WAs added to the nucleic acid purification column, the tube WAs capped, and the mixture WAs centrifuged at 13,000 rpm for 1min.

(9) The 2mL of the filtrate in the centrifuge tube was discarded, the nucleic acid purification column was returned to the 2mL RNA-free centrifuge tube, 600. Mu.L of Buffer WBR (absolute ethanol was added) was added to the nucleic acid purification column, the tube was capped, and the mixture was centrifuged at 13,000 rpm for 1min.

(10) The filtrate in the 2mL centrifuge tube was discarded, and the nucleic acid purification cartridge was returned to the 2mL centrifuge tube and centrifuged at 14,000 rpm for 1min.

(11) The 2mL centrifuge tube was discarded, the nucleic acid purification column was placed in a clean 1.5mL RNA-free centrifuge tube, 40. Mu.L RNase-free Water was added to the center of the membrane of the purification column, the tube cap was closed, and the mixture was allowed to stand at room temperature for 1min at 13,000 rpm for centrifugation for 1min.

3. RNA reverse transcription

The reactions were all performed on ice with reference to NovoScriptv Plus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNAPurge) kit instructions.

(1) And removing genome DNA. The following reagents were prepared on ice to prepare a reaction mixture, and reacted at 42℃for 5min after centrifugal mixing.

(2) RNA reverse transcription cDNA. The following reaction mixture is added into the PCR tube in the first step, and after thorough mixing and centrifugation, the mixture is reacted for 15min at 50 ℃ and 5min at 75 ℃ and cooled on ice.

(3) After 10-fold dilution of the reverse transcribed cDNA, the cDNA was stored at-20℃and used in subsequent experiments.

4. Gene cloning

(1) PnF3H specific PCR primers were designed using Oligo7.0 software and were synthesized by Hangzhou Kangshengmbh with the following sequences:

PnF3H-cds-F: ATGGCGGACCAGCTCATCTCCAC (shown as SEQ ID NO. 3)

PnF3H-cds-R: CTAGGTTCTGAAGAACTCCAAACAGTGCTC (shown as SEQ ID NO. 4)

(2) PCR amplification

Taking the DNA of the purple bamboo leaves as a template, and carrying out PCR amplification by using corresponding primers, wherein the reaction procedures are as follows: 95 ℃ for 5min;95 ℃ for 5 seconds, 60 ℃ for 30 seconds, 72 ℃ for 60 seconds, 32cycles; and at 72℃for 10min. The amplification instrument is CFX96 ^TM The Real-time PCR instrument and the amplification system are as follows:

(3) Preparing 1.2% agarose gel, performing agarose gel electrophoresis experiment, setting the voltage to 125V, setting the time to 20min, detecting whether the fragment size of the target gene meets the expectations, and performing tapping recovery and purification on the target fragment meeting the expectations.

(4) Gel recovery and purification gel recovery product purification was performed using the san prep column DNA gel recovery kit from Shanghai biosciences.

(5) Ligation reaction

Reference to Takara company pMD ^TM 18-T Vector Cloning Kit instruction, the connection time is 3-10h, and the connection system is as follows:

(6) Ligation product transformation of E.coli competent DH 5. Alpha

Referring to the specification of the transformation of competent DH5 alpha of Escherichia coli of Shanghai Weidi Biotechnology Co., ltd, the specific steps are as follows:

(1) the purchased escherichia coli competent DH5 alpha is placed on ice for 5min for freeze thawing, 10 mu L of connecting product is added, the mixture is uniformly mixed at the bottom of a flick tube, and the mixture is kept stand on ice for 30min.

(2) And standing on ice for 2min at 42 ℃ for 45 s.

(3) 300. Mu.L of the antibiotic-free LB medium was added thereto, and the mixture was subjected to shaking culture at 220rpm and 37℃for 1 hour.

(4) About 100. Mu.L of the culture solution was uniformly spread on Luria-Bertani (LB) medium (ampicillin-containing) and cultured at 37℃for 12 hours.

(5) And selecting a monoclonal colony, culturing the monoclonal colony in a 1mL liquid culture medium centrifuge tube containing corresponding antibiotics at 37 ℃ under shaking at 220rpm for 6 hours until bacterial liquid is turbid, and carrying out bacterial liquid PCR identification.

(6) And (3) delivering the positive bacterial liquid identified in the step (5) to Shanghai biological company for sequencing, wherein more than 3 monoclonal antibodies are selected for each sequence, so that the influence of mismatch on gene sequence confirmation in the PCR process is reduced.

The nucleotide sequence of the finally cloned flavonoid compound synthesis related gene PnF H is shown as SEQ ID NO.1, and the amino acid sequence of the flavonoid compound synthesis related gene PnF H encoded protein is shown as SEQ ID NO. 2.

A thermal chart of the expression level of the key enzyme gene F3H-like in the flavonoid compound synthesis pathway is shown in figure 1, and the color depth represents the expression level in figure 1. PnF3H gene clone is shown in FIG. 2, where M: DL2000Marker, the band from top to bottom represents 2000, 1000, 750, 500, 250, 100bp.

Example 2: genetic transformation of tobacco and Malayan

1. Genetic transformation of tobacco

(1) Pre-culture

Sterilizing tobacco seeds with 75% alcohol for 30s, cleaning with sterile water for 1min, sterilizing with 84 disinfectant for 3-5min, and cleaning with sterile water for 3 times and 1 min/time. Sowing the sterilized tobacco seeds on germination culture medium, culturing at 23deg.C for 16h/8h (light/dark) for 4-5 weeks, cutting sterilized tobacco leaves into small pieces with a scalpel, and inoculating on culture medium.

(2) Agrobacterium infection, co-cultivation, induction

And (3) taking agrobacterium liquid stored at the temperature of minus 80 ℃, carrying out a large amount of shaking, collecting colonies in tobacco invasion liquid, preparing agrobacterium heavy suspension with OD600 = 0.2, inoculating tobacco leaves subjected to pre-culture for 2-3d into the agrobacterium suspension, infecting for 10-15min, inoculating the infected tobacco leaves onto filter paper, airing, inoculating the tobacco leaves onto a co-culture medium, and culturing in the dark for 48-72h. And transferring the leaves subjected to co-culture for 2d into an induction culture medium to induce callus for about 10d, and growing callus.

(3) Screening for resistant callus

Selecting callus meeting the standard, inoculating on a screening culture medium with corresponding resistance, and culturing for 15-30d at the temperature: 21-25 ℃.

(4) Differentiation and rooting

The resistant calli with vigorous growth of the second sieve are inoculated on a differentiation medium, 4-5 calli/dish are cultured for 15-30d at 23 ℃ for 16h/8h (light/dark). In the differentiation process, the callus to be healed has seedlings formed, and the callus is inoculated to a strong seedling culture medium to grow for 7-10d.

2. Genetic transformation of Malaysia japonica

Selecting a plurality of embryogenic calli of Malaysia malaica with consistent state, dividing the calli into calli with the size of about 0.5-1cm, infecting the calli by using EHA105 agrobacterium tumefaciens bacteria solution added with 100 mu L acetosyringone, completely immersing the calli in the bacteria solution during infection, slightly shaking the bacteria solution for 15min, removing redundant bacteria solution after infection is finished, placing the calli on an infected calli co-culture medium, placing the calli in a culture box at 25 ℃, and culturing in darkness for 2-3d. After the dark culture is finished, aseptic water (with the timentin added) is used for degerming the infected calli, and the calli are placed on a screening culture medium for screening of resistant calli after degerming (dark culture).

Example 3:35S PnF H over-expression vector construction

The annual purple bamboo leaves from Zhejiang agriculture and forestry university planted in Linan area of Hangzhou, zhejiang province in example 1 are taken, RNA extraction is carried out, cDNA is reversely transcribed, a CDS sequence of PnF H is cloned, then the CDS sequence is connected with pC-1300GFP vector for sequencing, and an over-expression vector 35S is constructed after identification is correct, wherein the expression vector is PnF H.

Example 4: application of PnF H gene heterologous transformation tobacco and phyllostachys malabarica callus to improvement of flavonoid synthesis amount

1. Screening and determination of transgenic tobacco and Malaysia japonica transformed callus positive plants

(1) Positive plant DNA and RNA extraction and PCR identification of transgenic material

(1) Liquid nitrogen is added into 300-500 mg of young leaves of positive plants, and the young leaves are quickly ground into powder. A1.5 mL centrifuge tube was pre-cooled with liquid nitrogen, and 100mg of the sample was weighed and ground into powder.

(2) Adding 500 μL of Buffer PL preheated at 65 ℃ into the ground sample, mixing uniformly by vortex oscillation for 30s, carrying out water bath at 65 ℃ for 10min, and shaking the sample every 3min to enable the sample to be fully cracked.

(4) Add 350. Mu.L Buffer K, cover the tube and shake with force for 15s and vortex for 30s.13 Centrifuge at 000rpm for 5min.

(4) Pouring the supernatant obtained by centrifugation in the step 3 into a nucleic acid purification column, placing the nucleic acid purification column into a 2mL centrifuge tube, covering a tube cover, and centrifuging at 12000rpm for 30s.

(5) The 2mL of the filtrate in the centrifuge tube WAs discarded, the nucleic acid purification column WAs returned to the 2mL centrifuge tube, 500Buffer WA (absolute ethanol had been added) WAs added to the nucleic acid purification column, the tube WAs capped, and the mixture WAs centrifuged at 12,000 rpm for 30s. The 2mL of the filtrate in the centrifuge tube was discarded, the nucleic acid purification cartridge was returned to the 2mL centrifuge tube, 600. Mu.L of Buffer WB (absolute ethanol had been added) was added to the nucleic acid purification cartridge, and the mixture was centrifuged at 12 rpm for 30s.

(6) The 2mL centrifuge tube was discarded, the nucleic acid purification column was returned to the 2mL centrifuge tube, and the tube lid was closed. 13 Centrifuge at 500rpm for 2min.

(7) The 2mL centrifuge tube was discarded, the nucleic acid purification column was placed in a 1.5mL centrifuge tube, 20. Mu.L of Buffer TE preheated at 65℃was added to the purification column, the tube was covered with a cap, and the mixture was allowed to stand at room temperature for 2min at 12,000 rpm for 30s. The eluted DNA was stored at-20℃for subsequent experiments.

(8) Extraction of positive plant RNA was performed with reference to the RNA extraction method in example 1.

(9) Using PnF H CDS primer to identify DNA and RNA level of the selected transgenic plant seedling, the PCR reaction program is: 95℃for 5s,60℃for 30s,72℃for 60s,26cycles.

(2) Overexpression PnF H tobacco and Malaysia japonica callus gene expression level analysis

In order to explore the function of PnF H genes, 35S is constructed by transferring PnF H overexpression vectors into wild tobacco (K326) and Malaysia japonica callus, respectively extracting transgenic plant leaf DNA and RNA, and DNA and RNA of the Malaysia japonica callus, and then carrying out PCR amplification sequencing of target fragments to determine a transgenic positive strain. qRT-PCR analysis is carried out by taking cDNA of transgenic tobacco leaves, wild tobacco leaves, common Mallotus japonicus callus and transgenic Mallotus japonicus callus as templates, and PnF H gene expression is shown in figure 3. As shown in fig. 3 (a), pnF H over-expressed tobacco lines were designated OE1, OE2, OE3, OE4, OE5, respectively. As shown in FIG. 3 (B), the PnF H overexpressing Mallotus japonicus strain was designated as D.asper-OE1, D.asper-OE2, D.asper-OE3, D.asper-OE4, and D.asper-OE5, respectively.

(3) Quantitative determination of PnF H transgenic tobacco and Malayan bamboo flavonoid synthesis pathway related genes

(1) Reference is made to the relevant literature reports. And selecting flavonoid synthesis pathway related genes in tobacco to detect the expression quantity. The genes selected mainly are as follows: C4L, CHS, CHI, C4H, F3H, F3' H, FLS, ANR, DFR and UFGT.

(2) The expression level was analyzed by reverse transcription using transgenic tobacco and Mallotus japonicus leaf RNA as templates, and real-time fluorescence quantitative analysis was performed by referring to SYBR Premix (Novoprotein) kit instructions, and the reaction system was as follows. The fluorescence quantitative reaction procedure is 95 ℃ for 30s;95℃for 5s,60℃for 30s,39 cycles, 65℃for 5s,95℃for 5s, the dissolution profile was measured from 60℃to 95 ℃. Each reaction has three or more biological replicates.

(4) Determination of flavonoid content of transgenic plants

Referring to the kit instruction book of the plant flavonoid content detection kit of Beijing Solarbio biological company, the flavonoid content of the transgenic plant is determined by the following specific steps:

(1) sample processing: the sample is dried to constant weight, crushed by a crusher, sieved by a 30-50 mesh sieve, weighed about 0.1g, added with 1mL of 60% ethanol, extracted by an ultrasonic extraction method, the ultrasonic power is 300W, the crushing is carried out for 5s, the gap is 8s, the temperature is set to 60 ℃, and the extraction is carried out for 30min. Then, the mixture was centrifuged (12000 rpm,25 ℃ C., 10 min) with a centrifuge, and the supernatant was taken and was fixed to 1mL with 60% ethanol solution to be measured.

(2) A flavonoid content determination step:

A. the visible light photometer is preheated for more than 30min, the wavelength is adjusted to 470nm, and zero is set.

B. Preparation of standard solution: and diluting the 10mg/mL rutin standard solution to 1.5,1.25,0.625,0.3125,0.15625,0.078,0.039 and 0.02mg/mL with the standard substance for later use.

C. The operation table is as follows:

(3) flavonoid content calculation:

a. drawing a standard curve: drawing a standard curve y=kx+b by taking rutin concentration as an abscissa and DeltaA' as an ordinate, and bringing DeltaA into a resolvable x (mg/mL);

b. flavonoid content (mg/g) =x×v/W (V: extract volume, W: sample mass).

2. Functional verification

(1) Determination of flavonoid content in PnF H transgenic tobacco and Mallotus japonicus callus: the results of the determination of flavonoid content in five overexpressed PnF H transgenic tobacco and three overexpressed PnF H transgenic Mallotus japonicus calli are shown in FIG. 4. By analyzing the changes in the content of flavonoids in transgenic tobacco versus wild type tobacco, we found that the synthesis of flavonoids in 5 tobacco over-expressed PnF H was significantly improved. As can be seen from FIG. 4 (B), in the selected three calli overexpressed PnF H by Malaysia japonica, the flavonoid content in the overexpressed calli was increased, and the flavonoid synthesis in line D.asper-OE3 was significantly increased.

(2) Analysis of expression level of flavonoid Synthesis pathway related genes in PnF H transgenic tobacco and Mallotus japonicus: the F3H enzyme gene is a key enzyme gene of flavonoid synthesis pathway, in order to verify the specific effect of PnF H in the flavonoid anabolism pathway of tobacco, respectively extracting RNA of over-expressed and wild tobacco, RNA of common Mallotus japonicus callus, over-expressing RNA of Mallotus japonicus callus, and detecting the expression level of related genes in the flavonoid synthesis pathway by RT-qPCR means, and the results are shown in figure 5 and figure 6. As can be seen from fig. 5, we found that the expression level of most genes in the tobacco flavonoid synthesis pathway was up-regulated but not significantly in the transgenic line overexpressing PnF H, wherein DFR, which is a downstream gene of the F3H enzyme gene, was significantly increased in the three lines overexpressing PnF H, the gene expression level of CHI was significantly increased in all the overexpressing lines, and it was assumed that PnF H may play a role in tobacco flavonoid synthesis, and as can be seen from fig. 5, F3' H and UFGT gene expression levels were increased in the transgenic calli of malassezia overexpressing PnF H, CHS were significantly increased, and that PnF H may play a role in bamboo flavonoid synthesis.

Claims (7)

1. A gene PnF H related to the synthesis of a purple bamboo flavonoid compound, which is characterized in that: the nucleotide sequence is shown as SEQ ID NO. 1.

2. The protein encoded by the gene PnF H related to the synthesis of the purple bamboo flavonoid compound according to claim 1, which is characterized in that: the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. A vector comprising a gene PnF H associated with the synthesis of a phyllostachys pubescens flavonoid compound according to claim 1.

4. Use of a gene PnF H related to the synthesis of a phyllostachys praecox flavonoid compound according to claim 1 or a vector according to claim 3 for modulating the synthesis of a flavonoid compound.

5. A use according to claim 3, wherein: plants were made to contain the gene PnF H or plants were made to overexpress the gene PnF H.

6. A method for regulating the synthesis of flavonoids in plants, which is characterized by: plants were made to contain the gene PnF H or plants were made to overexpress the gene PnF H.

7. The method of claim 6, wherein: the method specifically comprises the following steps:

1) Collecting annual purple bamboo leaves, extracting RNA, reversely transcribing into cDNA, cloning a CDS sequence of PnF H, connecting the CDS sequence with a pC-1300GFP vector for sequencing, and constructing a 35S PnF H overexpression vector after identification is correct;

2) And transferring the constructed 35S PnF H over-expression vector into plant callus to make the plant contain gene PnF H or make the plant over-express gene PnF H.