CN109517831A

CN109517831A - It is a kind of from the chalcone enzyme gene of anoectochilus formosanus and its application

Info

Publication number: CN109517831A
Application number: CN201811456216.3A
Authority: CN
Inventors: 杨琳; 张君诚; 付凤玲; 李晚忱; 宋育红; 邢建宏
Original assignee: Sanming University
Current assignee: Sanming University
Priority date: 2018-11-30
Filing date: 2018-11-30
Publication date: 2019-03-26
Anticipated expiration: 2038-11-30
Also published as: CN109517831B

Abstract

The present invention provides a kind of chalcone synthase gene from anoectochilus formosanus and its application, is related to gene engineering technology field.A kind of chalcone synthase gene from anoectochilus formosanus has the nucleotide sequence as described in SEQ ID NO.1, the amino acid sequence as described in SEQ ID NO.2, the coding region sequence as shown in SEQ ID NO:3.The sequence is a kind of new chalcone synthase gene sequence, can be applied in rice, can be with the content of flavone compound in adjusting and controlling rice.It has great importance to the route of synthesis of research secondary metabolite.

Description

It is a kind of from the chalcone enzyme gene of anoectochilus formosanus and its application

Technical field

The present invention relates to genetic engineering fields, and in particular to a kind of chalcone synthase gene from anoectochilus formosanus And its application.

Background technique

Anoectochilus formosanus (Aoectochilus roxburghii) is herbaceos perennial, is grown in Taiwan, is belonged to Orchid family Anoectochilus Blume roxburgh anoectochilus terminal bud kind.The active substance of valuable pharmacological is main in anoectochilus formosanus are as follows: flavone compound, steroid Compound, triterpene compound, carbohydrate content, alkaloid, Cardiac glycosides, esters, taurine, a variety of amino acid, microelement And inorganic elements etc..The laudatory titles such as " king of medicine ", " gold grass ", " god grass ", " bird ginseng " are known as civil.Wherein, in anoectochilus formosanus Polysaccharide, flavone compound, steroidal etc. be considered being important active material, the synthesis of these substances directly affects Taiwan The medical value of roxburgh anoectochilus terminal bud.The route of synthesis of parsing secondary metabolite is the premise and base for carrying out related synthetic biology research Plinth.

Chalcone synthase (Chalcone synthase, CHS) is enzyme base crucial in Flavonoid substances metabolic pathway of synthesizing Cause.In flavone compound metabolic pathway of synthesizing, the enzyme being catalyzed at first is phenylalanine lyase (Phenylalanine Ammonialyase, PAL), it can be catalyzed L-phenylalanine (L-phenylalanine) production trans-cinnamic acid (Trans- cinnamic acid).The trans-cinnamic acid generated in upper step reaction, in oxygen and nicotinamide-adenine dinucleotide phosphate Under conditions of (Nicotinamide adenine dinucleotide phosphate, NADPH) is existed simultaneously, by cortex cinnamomi Sour 4- hydroxyl enzyme (Cinnamic acid 4-hydroxy enzyme, C4H) catalysis, can be generated 4- hydroxyl coumaric acid (4- hydroxycinnamate acid).Then 4- hydroxyl coumaric acid-CoA ligase (4-hydroxycinnamate acid- Coenzyme Aligase, 4CL) catalysis connection, 4- hydroxyl coumaric acyl-is converted by the 4- hydroxyl coumaric acid that previous step generates The thioesters such as CoA, this step need ATP to provide energy.The 4- hydroxyl coumaric acyl-CoA of generation can be with malonyl-CoA in chalcone It reacts, generates chalcone (Chalcone) under the action of synthase.Chalcone synthesizes 5 branch (different Huangs as flavone compound Ketone branch, aurones branch, flavones branch, anthocyanidin branch and flavonols branch) common substrate, in phenylpropyl alcohol alkane metabolic process It plays a role.Therefore, chalcone synthase is the key that vegetable flavonoid metabolic pathway of synthesizing rate-limiting enzyme, active and each The anabolism of kind flavone compound and accumulation are closely related.Research to chalcone synthase gene in anoectochilus formosanus is The basis for studying specific purpose secondary metabolite enrichment in anoectochilus formosanus, has great importance.

Summary of the invention

The purpose of the present invention is to provide a kind of chalcone synthase gene, this genes from anoectochilus formosanus to encode Amino acid, the application containing the recombinant plasmid of this gene and the chalcone synthase gene are Flavonoids from Plants Anabolism research provides basis.

The present invention solves its technical problem and adopts the following technical solutions to realize.

A kind of chalcone synthase gene derived from anoectochilus formosanus, the nucleotide sequence of the chalcone synthase gene is such as Shown in SEQ ID NO:1.

Optionally, the amino acid sequence of chalcone synthase gene coding is as shown in SEQ ID NO:2.

Optionally, the coding region sequence of chalcone synthase gene is as shown in SEQ ID NO:3.

A kind of recombinant plasmid containing above-mentioned chalcone synthase gene.

Optionally, the plasmid is pZZ00026-Ubi-CHS-T-nos.

A kind of application of above-mentioned chalcone synthase gene in adjusting and controlling rice flavones content.

Optionally, application as described above, comprising the following steps:

The ORF sequence of the chalcone synthase gene of anoectochilus formosanus is inserted into pZZ00026-Ubi-CHS-T-nos by S1 In plasmid, expression vector is obtained；

S2 converts the expression vector to Agrobacterium, infect to rice with the Agrobacterium after conversion and turns Change, obtains transgenic paddy rice.

Compared with prior art, the beneficial effects of the present invention are:

Provided by the present invention look into closes ketone synthase gene and clones obtain from anoectochilus formosanus for the first time.Flavonoids Compound is a kind of Secondary metabolites of great application value.Chalcone synthase gene is flavone compound synthesis generation Thank to the crucial rate-limiting enzyme in approach.The successful clone of the gene provides important evidence for roxburgh anoectochilus terminal bud breed breeding, is also flavonoids The transgenic plant line research of compound enrichment provides basis.The present invention obtains anoectochilus formosanus in the method that transcript profile is sequenced Unigene sequence expand the CDS complete sequence of chalcone synthase in the both ends design primer of Unigene sequence.It is this to pass through The method of transcript profile sequencing obtains the CDS sequence of target gene, and the method for amplification gene sequence is simple to operate.Utilize this hair It is bright genetic engineering transformation to be carried out to rice, paddy gene is regulated and controled by transgenosis, to enhance the flavones in rice Kind compound content.Plant variety identification, screening etc. are either utilized the gene for, the plant variety of high flavones content is obtained Deng providing basis for purpose secondary metabolite enrichment specific in roxburgh anoectochilus terminal bud.Cha Er is cloned in anoectochilus formosanus simultaneously Ketone synthase gene is the basis for studying Flavonoid substances anabolism access in anoectochilus formosanus, can be selected for roxburgh anoectochilus terminal bud kind It educates and important foundation is provided, also provide basis for the transgenic line research of flavone compound enrichment.

Detailed description of the invention

In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

Fig. 1 is the roxburgh anoectochilus terminal bud CHS gene C DS sequence electrophoresis detection result figure that the embodiment of the present invention 4 provides.

Fig. 2 is the albumen tertiary structure figure for the anoectochilus formosanus CHS prediction that the embodiment of the present invention 5 provides.

Fig. 3 is related for the anoectochilus formosanus CHS albumen that the embodiment of the present invention 5 provides and sets analysis.Red frame is Taiwan gold Line lotus present position.

Fig. 4 is the anoectochilus formosanus CHS gene different tissues expression pattern figure that the embodiment of the present invention 6 provides.

Fig. 5 be the anoectochilus formosanus CHS gene that provides of the embodiment of the present invention 6 at 4mg/L Phe for 24 hours in expression pattern Figure.

Fig. 6 be the anoectochilus formosanus CHS gene that provides of the embodiment of the present invention 6 at 100nM NaCl for 24 hours in expression mould Formula figure.Light pillar indicates that the expression quantity of anoectochilus formosanus CHS gene, dark pillar indicate the expression quantity of anoectochilus formosanus.

Fig. 7 be the anoectochilus formosanus CHS gene that provides of the embodiment of the present invention 6 under the ultraviolet stress of 253.7nm for 24 hours in table Expression patterns figure.

Fig. 8 is the anoectochilus formosanus CHS gene promoter Tail-PCR result that the embodiment of the present invention 7 provides.M is Marker, AD1-AD6 are the second wheel and third round segment of the segment of six degenerate primers and special primer amplification.

Fig. 9 is the anoectochilus formosanus CHS gene promoter cis-acting elements that the embodiment of the present invention 7 provides.

Figure 10 is the transient expression vector pC2300-35S-CHS-eGFP that the embodiment of the present invention 8 provides.

Figure 11 is the subcellular localization for the roxburgh anoectochilus terminal bud CHS albumen that the embodiment of the present invention 8 provides.

Figure 12 is prokaryotic expression carrier pET-28a (+)-CHS that the embodiment of the present invention 9 provides.

Figure 13 is the protokaryon inducing expression for the roxburgh anoectochilus terminal bud CHS albumen that the embodiment of the present invention 9 provides.

Figure 14 is the purifying for the roxburgh anoectochilus terminal bud CHS albumen that the embodiment of the present invention 9 provides.

Figure 15 is the roxburgh anoectochilus terminal bud CHS prolease activity measurement that the embodiment of the present invention 10 provides.

Figure 16 is the unifacial leaf over-express vector pZZ00026-Ubi-CHS-T-nos that the embodiment of the present invention 11 provides.

Figure 17 is the PCR detection figure for turning roxburgh anoectochilus terminal bud CHS trans-genetic hybrid rice that the embodiment of the present invention 11 provides.

Figure 18 is the general flavone enrichment condition for turning roxburgh anoectochilus terminal bud CHS trans-genetic hybrid rice that the embodiment of the present invention 11 provides.

Specific embodiment

It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

The embodiment of the present invention is specifically described below.

In each of the embodiments described below, the chalcone synthase gene of anoectochilus formosanus is referred to as AfCHS, by experimental example, Finally obtain the amino acid sequence of the chalcone sequence (as shown in SEQ ID NO.1) and its coding from anoectochilus formosanus (as shown in SEQ ID NO.2).

Embodiment 1

The present embodiment provides the Total RNAs extractions of anoectochilus formosanus, are extracted using the Trizol of the precious biological Cable companies in Dalian The extraction of kit progress roxburgh anoectochilus terminal bud blade total serum IgE, comprising the following steps:

(1) take the fresh blade of about 100mg, the grind into powder in liquid nitrogen is transferred to 1.5mL centrifuge tube, immediately after plus Enter 1mL RNAiso Plus, is mixed by inversion, obtains homogenate.

(2) homogenate mixed well is stored at room temperature 5min, is then centrifuged 5min under the conditions of 12000g, 4 DEG C.

(3) Aspirate supernatant 800ml is transferred in new centrifuge tube, is sure not to draw precipitating, supernatant volume is then added 200ml chloroform, and shake vigorously and mix well to homogenate emulsification and be creamy white, it is then stored at room temperature 5min, obtains mixed liquor.

(4) mixed liquor is centrifuged 15min under the conditions of 12000g, 4 DEG C, the homogenate after being centrifuged at this time is divided into three layers.From It is respectively up to lower: supernatant (containing RNA), intermediate white layer (most of is DNA) and the colored organic phase of lower layer.

(5) the supernatant 400ml in aspiration step (4) is transferred in new centrifuge tube, is sure not to touch middle layer.Then plus Enter the isopropanol of 400ml and be mixed by inversion, then stands 10min at room temperature.

(6) solution after standing in step (5) is centrifuged 10min under the conditions of 12000g, 4 DEG C, visible white is wadded a quilt with cotton at this time The RNA of shape.It carefully discards supernatant and 75% ethyl alcohol of 1mL is added, turn upside down and discard supernatant liquid after washing RNA.

(7) after opening centrifuge tube lid drying at room temperature RNA a few minutes, the RNase-free water that 30 μ l are added dissolves RNA.

(8) concentration that total serum IgE is calculated using the value of ultramicrospectrophotometer (Bio-Rad, the U.S.) measurement A260, is read The value of OD260/OD280 is taken to estimate total serum IgE purity and integrality.With the agarose gel electrophoresis of 1.2% concentration, 135V is quick Detect RNA mass.

Embodiment 2

The present embodiment provides the sequencings of the transcript profile of anoectochilus formosanus, comprising the following steps:

According to the method that embodiment 1 provides, the total serum IgE of anoectochilus formosanus blade is extracted.It detects RNA and extracts quality, meet Requirement for construction data base (RNA concentration > 250ng/ μ L, total amount > 20 μ g, OD₂₆₀/OD₂₈₀Between 1.8~2.2, integrality is good, and RIN > 6.5).Then, be broken into a section segment with enrichment with magnetic bead poly (A) mRNA, as template, successively synthesize the 1st article of cDNA chain and 2nd article of cDNA chain connects sequence measuring joints after purifying, elution, end repair, add poly (A).Select 200bp~700bp big Small segment carries out PCR amplification, establishes cDNA sequencing library, is sequenced with IIIumina HiSeq 2000.The part Experiment Mei Yin science service Cable companies are entrusted to complete.

De novo is using short reads composite software Trinity (v2.4.0) to assemble, and is obtained the Contig without N and is assembled After segment, de-redundancy is carried out using tgicl (v2.1), removes that quality in sequence is low, uncertain sequence, remains larger than 200bp Sequence carry out subsequent analysis.Prediction of gene structure is carried out to above-mentioned splicing result using transdecoder (v2.0.1), is obtained To the CDS sequence of AfCHS gene.

Embodiment 3

The present embodiment provides the first chains of anoectochilus formosanus cDNA, comprising the following steps:

Using the roxburgh anoectochilus terminal bud blade total serum IgE that embodiment 1 provides as template, with oligo (dT) 18 for reverse transcriptase primer, use PrimeScriptTM Reverse Transcriptase (TakaRa China) is according to SMART^TMPCR cDNASynthesis The synthesis of Kit (Clontech USA) operating instruction progress the first chain of cDNA.Reaction system total volume is 20 μ L.

(1) it is prepared in 0.2mL PE pipe according to the reagent in table 1 and obtains reverse transcription mixed liquor 1；

(2) it is prepared in another 0.2mL PE pipe according to the reagent in table 2 and obtains reverse transcription mixed liquor 2；

(3) the reverse transcription mixed liquor 1 in step (1) is kept the temperature to after 5min chilling 2min on ice rapidly at 65 DEG C, from Calculation second makes the mixed liquor of template ribonucleic acid, primer etc. be gathered in PE bottom of the tube；

(4) the reverse transcription mixed liquor 2 configured in step (2) is added to the reverse transcription mixed liquor 1 in step (3) after reaction In, 42 DEG C of reaction 90min after being gently mixed with liquid-transfering gun；

(5) reaction solution obtained in step (4) is kept the temperature at 80 DEG C cooled on ice after 5min, obtains cDNA solution.

1 reverse transcription mixed liquor 1 of table composition

2 reverse transcription mixed liquor 2 of table composition

Embodiment 4

The present embodiment provides the cloning process of AfCHS gene, comprising the following steps:

(1) design of primers

On the basis of obtaining the CDS sequence of AfCHS gene by transcript profile sequencing splicing in embodiment 2, use 5.0 software Design primers of Primer Premier, the specificity of designed primer analyte primer in Oligo6.0.

Upstream primer CHSF: the nucleotide sequence (5'-ATGCCGAGCCTCGAATCCA- as shown in SEQ ID NO:4 3')；

Downstream primer CHSR: as shown in SEQ ID NO:5 (5'-TTAAAGAGGAACGCTGCGAA-3').

(2) PCR reacts

Response procedures are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 1min are pressed later Expand 38 circulations.PCR reaction uses 50 μ L systems, as shown in table 3.

3 PCR reaction system of table

*: the first chain of cDNA provided for embodiment 3 dilutes 3-10 times.

Take gained amplified production 50ul through 1% non denatured Ago-Gel 180V electrophoresis 30min, after GoLdenView dyeing Ultraviolet inspection amplified fragments, as a result as shown in Figure 1.Figure 1A is the amplification of open reading class, and Figure 1B is gene coding region amplification, In, M indicates marker, and 1 indicates Anoectochilus roxburghii, and 2 indicate anoectochilus formosanus.

(3) recycling of target fragment

The specific band come will be amplified in step (2) in clean blade in the UV lamp fast and standard slave agarose It digs out, is placed in 1.5ml centrifuge tube, with gel reclaims kit (the E.Z.N.A.TM Gel Extraction of OMEGA company Kit the DNA fragmentation in glue) is recycled.

(4) connection reaction

It will be in the Pmd19-T carrier of the DNA fragmentation clone of recycling and TaKaRa company.Connection reaction is using connection kit (TaKaRa company), linked system total volume 10ul, 16 DEG C of connections are overnight.

(5) preparation of competent cell:

The E.coLiDH5a single colonie newly activated from picking on LB plate, is inoculated in 3-5ml LB liquid medium, 37 DEG C, 225r/min shaken cultivation 12h or so, until the logarithmic growth later period.By the bacterium solution suspension with the ratio of 1::10-1:50 It is inoculated in 100ml LB liquid medium, 37 DEG C of shaken cultivation 2-3h to OD600=0.35-0.5 or so.

Culture solution is transferred to centrifuge tube, places 10min on ice, then 3000r/min is centrifuged 10min. at 4 DEG C

It discards supernatant, with the CaCl2 solution 10ml of the 0.05mol/L of pre-cooling gently suspension cell, places 15- on ice After 30min, 3000r/min is centrifuged 10min at 4 DEG C.

Supernatant is abandoned, the CaCl2 solution of 0.05mol/L of the 4ml pre-cooling containing 15% glycerol is added, gently suspension cell, on ice Place a few minutes, competent cell suspension.

Competent cell is distributed into the aliquot of 100ul, is stored in -70 DEG C of storable half a year.

(6) conversion of Plasmid DNA

The competent escherichia coli cell DH5a in a pipe step (5) is taken, is placed on ice to melt.Then aseptically The connection reaction solution in 10ml step (4) is added, after gently concussion mixes, places 30min on ice.42 DEG C of water-bath thermal shock 90s, It not shake, be put into the cooling 2-3min of ice bath rapidly later.LB liquid medium of the 890ul without Amp is added, after mixing, 37 DEG C, The shaking culture of 150r/min shaking table, incubates 1h, the bacterium solution converted.

The bacterium solution for taking 100ul to convert is applied on the culture plate of a LB Amp, face-up places 30min, to bacterium After liquid is cultured base absorption completely, with ParafilmTM, culture dish is then reversed, 37 DEG C are protected from light culture 12-16h；Then screening Positive bacterium colony.

(7) identification and preservation of bacterium colony are recombinated

From the appearance point of view, the bacterium colony of general white and circle is the positive, further identified by bacterium solution PCR, step is such as Under:

Several white colonies are taken with the lancet choicest of sterilizing on the plate being incubated overnight, dibbling is in 0.1g/L Amp respectively LB liquid medium 1.5ml centrifuge tube in, 37 DEG C, 150r/min shaking culture 4-6h.

It takes 1uL bacteria suspension as template, carries out PCR amplification, pyrolysis time in PCR reaction condition with primer CHSF/CHSR 5min is extended to, detects amplified production with 1% non denatured agarose gel electrophoresis.If product is purpose band, this bacterium It falls as positive colony, is otherwise feminine gender.The negative control that bacteria suspension is not added is set simultaneously.

The bacterium colony suspension 750ul for taking identified positive colony, is added the sterile glycerol of 250ul, after mixing, uses liquid Nitrogen is quick-frozen, is placed in -70 DEG C of refrigerators and saves backup.

(8) it is sequenced

By the bacterium colony of the positive colony of identified preservation send handsome biotech company's sequencing, gained sequence in step (7) The BLastn for being listed in NCBI is compared, and verifying cloned sequence is correct.

Expanding obtained product is AfCHS gene layer cDNA intermediate segment, obtains nucleotide sequence SEQ ID NO.1 institute Show, shown in the amino sequence SEQ ID NO.2 of coding, coding region sequence is as shown in SEQ ID NO.3.

Embodiment 5

The present embodiment provides the protein biology bioinformatics analysis of AfCHS gene, comprising the following steps:

Using ProtParam (http://web.expasy.org/protparam/) to AfCHS gene physics and chemistry Property is analyzed.Anoectochilus formosanus CHS full length gene 1173bp, coding region sequence 1260bp, the 180th between 267bp It is inserted into the introne of 1 long 87bp, encodes 390 amino acid.13 activation sites of CHS protein specificity

(G¹³⁸-G¹⁶³-G¹⁶⁷-L²¹⁴-D²¹⁷-G²⁵³-P³⁰⁵-G³⁰⁶-G³⁰⁷-G³³⁶-G³⁸⁵-P³⁸⁶-G³⁸⁷), 4 catalytic residues (Cys-His-Asp+F) and 6 conserved residues (G³⁷⁹-V³⁸⁰-L³⁸¹-F³⁸²-G³⁸³-F³⁸⁴) identical, only in malonyl coenzyme A Binding motif

(V³¹⁴-E³¹⁵-E³¹⁶-R³¹⁷-V³¹⁸-G³¹⁹-L³²⁰-K³²¹-P³²²-E³²³-K³²⁴-L³²⁵-T³²⁶-T³²⁷-S³²⁸-R³³⁰) it is close The end C- has 2 residues different from iris.Moreover, it has been found that 1 classical nuclear localisation signal (Nuclear localization signal,NLS)R⁶⁶-K⁶⁷-R⁶⁸-H⁶⁹.The online work provided using ExPASy Proteomics Server Tool ProtParam predicts the physicochemical property of CHS gene coded protein, thus it is speculated that the albumen relative molecular mass is 42.9kDa, isoelectric point pI 6.04.

Do you use GOR IV (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl? page= Npsa_gor4.html) secondary structure of CHS gene is predicted.Secondary structure on-line prediction the result shows that, alpha-helix (Alpha helix), accounts for the 32.82% of total amino acid, extended chain (Extended strand), Zhan Suoyou amino acid 20.51%, random coil (Random coil) accounts for the 46.67% of total amino acid.

By SWISS-MODEL (https: //swissmodel.expasy.org/) to the FPS gene of anoectochilus formosanus The prediction for carrying out albumen tertiary structure, is mapped, threedimensional model is as shown in Figure 2 by PyMOL Viewer.

The CHS amino acid sequence for finally first finding the higher plant of homology carries out amino acid sequence using ClustalW method It is as shown in Figure 3 to construct Neighbor-joining systematic evolution tree by MEGA7.0 for the comparison of column.Wherein, box mark in Fig. 3 It is anoectochilus formosanus present position at knowledge.

Embodiment 6

The present embodiment provides the endogenous detection of expression of AfCHS gene, comprising the following steps:

(1) material processing

The processing of 1.1 ultraviolet lights:

By the anoectochilus formosanus tissue culture transplantation of seedlings of culture 4 months to the flowerpot that Nutrition Soil (Nutrition Soil: vermiculite=3:1) is housed In, in 200 μm of 25 DEG C of (daytime)/20 DEG C (night) of temperature, relative humidity 60~70% and illumination ol/m²S (14h)/dark (10h) Under conditions of cultivate.It adapting to 3~5 days, the uniform untreated seedling of growing way takes root, stem, leaf sample, and -70 DEG C of liquid nitrogen flash freezer It saves.The uniform seedling of remaining growing way carries out ultraviolet light processing.

Treatment conditions are as follows: the roxburgh anoectochilus terminal bud of potting is placed in ultraviolet incubator, ultraviolet radiation wave-lengths 253.7nm, respectively Handle 0h, 0.5h, 1h, 2h, 4h, 8h, 12h and for 24 hours.

Each each processing is repeated 3 times, and throughout reason time point samples -70 DEG C of liquid nitrogen flash freezer preservations immediately.

The processing of 1.2 red light irradiations:

By the anoectochilus formosanus tissue culture transplantation of seedlings of culture 4 months to the flowerpot that Nutrition Soil (Nutrition Soil: vermiculite=3:1) is housed In, in 200 μm of 25 DEG C of (daytime)/20 DEG C (night) of temperature, relative humidity 60~70% and illumination ol/m²S (14h)/dark (10h) Under conditions of cultivate.It adapting to 3~5 days, the uniform untreated seedling of growing way takes root, stem, leaf sample, and -70 DEG C of liquid nitrogen flash freezer It saves.The uniform seedling of remaining growing way carries out Infrared irradiation processing.

Treatment conditions are as follows: the roxburgh anoectochilus terminal bud of potting is placed in feux rouges incubator, red light wavelength 650nm, respectively handle 0h, 0.5h, 1h, 2h, 4h, 8h and 12h.

The processing of 1.3 salt stress:

The anoectochilus formosanus tissue-cultured seedling cultivated 4 months is moved on plastic foam plate with holes, with Hoagland nutrient solution 3~5d is cultivated, the uniform seedling of growing way is selected to carry out salt stress processing.Salt stress is handled i.e.: Hoagland is added in NaCl Nutrient solution handles 0h, 0.5h, 1h, 2h, 4h, 8h, 12h and for 24 hours to final concentration 100mmol/L respectively.Each each processing repeats 3 Secondary, throughout reason time point samples -70 DEG C of liquid nitrogen flash freezer preservations immediately.

The processing of 1.4 phenylalanines:

The anoectochilus formosanus tissue-cultured seedling cultivated 4 months is moved on plastic foam plate with holes, with Hoagland nutrient solution 3~5d is cultivated, the uniform seedling of growing way is selected to carry out phenylalanine processing.Phenylalanine is handled i.e.: phenylalanine is added Hoagland nutrient solution handles 0h, 0.5h, 1h, 2h, 4h, 8h, 12h and for 24 hours to final concentration 4mg/L respectively.Each each processing weight 3 times multiple, throughout reason time point samples -70 DEG C of liquid nitrogen flash freezer preservations immediately.

(2) RNA is extracted

The blade of above-mentioned 4 kinds of Stress treatments and control group seedling is taken, liquid nitrogen flash freezer grinding, is tried using Total RNAs extraction respectively Agent box Trizol (TaKaRa, Dalian) simultaneously extracts total serum IgE.Step such as embodiment 1.

(3) the first chain of cDNA synthesizes

It using the RNA of extraction as template, is loaded according to 4 system of table, 42 DEG C of reaction 2min remove possible genomic DNA (gDNA), it with PrimeScriptTM RT reagent Kit with gDNA Eraser kit (TaKaRa, Dalian), presses 5 system of table sample-adding, 37 DEG C of warm bath 30min, 85 DEG C of holding 5s terminate reaction and synthesize cDNA with reverse transcription, and standby in -20 DEG C of preservations With.

4 gDNA of table removes reaction system

5 cDNA synthetic system of table

(4) specific detection of qRT-PCR primer

The template reacted after 3 times of cDNA sample dilutions as qRT-PCR is compareed with 0h, using 50~65 DEG C of annealing temperature Gradient carries out qRT-PCR reaction to determine optimal annealing temperature, using 20 μ L reaction systems as shown in table 6.QRT-PCR draws Object sequence is as shown in table 7.

6 qRT-PCR reaction system of table

7 qRT-PCR primer sequence of table

Reaction tube uses the 0.2mL PCR plate after hundred happy silication.QRT-PCR is in iQ^TM5thermal cycler(Bio-Rad USA it) carries out.Response procedures are as follows: 95 DEG C of initial denaturation 10s；Then by 95 DEG C of denaturation 10s, 50~65 DEG C of annealing 20s, 72 DEG C extend 20s, and in this temperature collection fluorescence, the program of read plate (Plate read) carries out 46 circulations.Later since 50 DEG C, with every The temperature gradient of 0.5 DEG C of step is increased to 95 DEG C, and every step temperature keeps 5s.

(5) qRT-PCR reacts

It is used as qRT-PCR after the cDNA sample of the reverse transcription of samples different in the present embodiment step (3) is diluted again to expand Template, reaction tube use silication after 0.2mL PCR plate, using 20 μ L reaction systems (table 6), and set up with ddH₂O is mould The negative control of plate.The cDNA sample of single reverse transcription is set to be repeated three times.

QRT-PCR response procedures are as follows: then 95 DEG C of initial denaturation 10s press 95 DEG C of denaturation 10s, most suitable annealing temperature annealing 20s, 72 DEG C of extension 20s, and in this temperature collection fluorescence, the program of read plate (Plate read) carries out 46 circulations.Later most Afterwards since 50 DEG C, 95 DEG C are increased to 0.5 DEG C of speed of every step, each temperature keeps 5s, draws melting curve.

(6) qRT-PCR data are analyzed

This experiment is carried out using expression of double calibration curve methods to the key gene in Flavone metabolism route of synthesis Relative quantification.The relative expression quantity of gene is calculated using △ △ CT (Normalized Gene Expression) method.

The differential expression SPSS between differential expression and each processing between 0h control and processing (version10.0Inc.Chicago, IL) statistical analysis software is analyzed.Set up two differences of P=0.05 and P=0.01 Significance.Anoectochilus formosanus CHS gene different tissues expression pattern figure is as shown in Figure 4.

Fig. 5 is that (dark pillar represents anoectochilus formosanus CHS gene expression pattern figure interior for 24 hours under 4mg/L phenylalanine Anoectochilus formosanus CHS gene), 4h and 8h expression quantity compared with 0h, difference is extremely significant.Fig. 6 is anoectochilus formosanus CHS gene The expression pattern figure interior for 24 hours at 100mmol/L NaCl, for expression quantity in 8h, 12h and for 24 hours compared with 0h, difference is extremely significant. Fig. 7 be anoectochilus formosanus CHS gene under the ultraviolet stress of 253.7nm for 24 hours in expression pattern figure, 0.5h, 1h, 2h, 4h, Compared with 0h, difference is extremely significant for 8h, 12h and expression quantity for 24 hours.

Embodiment 7

The present embodiment provides the amplification methods of anoectochilus formosanus AfCHS gene promoter sequence, comprising the following steps:

(1) according to AfCHS gene order, it is special to synthesize nido as shown in table 8 for design within transcription initiation site 300bp Different primer SP1, SP2 and SP3 synthesize hot asymmetric interlaced PCR (Thermal asymmetric as shown in table 9 Interlaced PCR, TAIL-PCR) degenerate primer.

The nested specific primers of 8 TAIL-PCR of table amplification

The degenerate primer of 9 TAIL-PCR of table amplification

Note: S=G/C, W=A/T, N=A/T/C/G.

(2) to do template after the anoectochilus formosanus genomic DNA dilution of extraction, with every kind of degenerate primer and special primer SPC1, SPC2 and SPC3 are successively matched, and are loaded by reaction system shown in table 10, by temperature cycling program shown in table 14, into The 3 wheel amplifications of row TAIL-PCR.

10 TAIL-PCR reaction system of table

The temperature cycles of 11 TAIL-PCR of table amplification

(3) third round amplified production is separated with 2% non denatured agarose gel electrophoresis, as a result as shown in Figure 8.In Fig. 8, M is marker, and AD1-AD6 is the second wheel and third round segment of the segment of six degenerate primers and special primer amplification.Recycling Purify the specific fragment of degenerate primer AD3 amplification.

(4) analysis recycling specific fragment base sequence, reuse Plant CARE software (http: // Bioinformatics.psb.ugent.be/webtools/plantcare/html/ the anoectochilus formosanus material amplified) is analyzed The promoter sequence of material, and predict cis-acting elements therein, as a result as shown in Figure 9.

Embodiment 8

The present embodiment provides the subcellular localization of anoectochilus formosanus AfCHS albumen the following steps are included:

(1) building of transient expression vector:

1.1 according to anoectochilus formosanus CHS gene ORF sequence, needed for its 5 ' and 3 ' end addition transient expression vector building Restriction enzyme site and protection base, as shown in Figure 10, with 5.0 software design of Premier without containing terminator codon Special primer [5 '-TCCCGGG (Sma I)

ATGCCGAGCCTCGAATCCA-3′/5′-CGCGGATCC(BamH I)

AAGAGGAACGCTGCGAA-3′]。

1.2, using the pDM19-T plasmid for being inserted into CHS gene ORF as template, are loaded by 4 reaction system of table, carry out PCR amplification. The setting of PCR temperature cycles system program are as follows: 94 DEG C, 3min；98 DEG C of 10s, 62 DEG C of 30s, 72 DEG C of 60s, amplification cycles 38 times；Finally 72 DEG C of extension 5min.

1.3 amplified productions are separated with 1% non denatured agarose gel electrophoresis,ChemiDocTM XRS type gel Imaging system (Bio-Rad, the U.S.) imaging, with gel reclaims kit (Tiangeng, Beijing) recovery purifying target fragment.

1.4 by 12 reaction system of table be loaded, using fast enzyme cutting under the conditions of 30 DEG C digestion 0.5h.Digestion products are with 1.2% Ago-Gel be separated by electrophoresis, with gel reclaims kit (Tiangeng, Beijing) recovery purifying.

12 target fragment Sma I/BamHI double enzyme digestion reaction system of table

1.5 are loaded according to 13 reaction system of table, with double enzyme products of T4DNA ligase connection recovery purifying, 16 DEG C of connections 8h。

13 coupled reaction system of table

Connection product is converted with heat-shock transformed method and carries out PCR identification to recombination bacterium colony by 1.6.

1.7 extract the Plasmid DNA in above-mentioned steps, after the fast enzyme cutting Sma I and BamH I double digestion identification of correspondence, take A small amount of plasmid send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced, and checking carrier building is correct.

(2) transient expression vector is detected in onion bulb stem epidermal cell.

2.1 take the 5th layer of scale of fresh onion bulb stem, are cut into 2cm × 2cm square, and endepidermis is taken to be laid on 1/2MS plate, and 25 DEG C culture 4h.

2.2, which weigh 60mg bronze (60 μm of diameter), is placed in 1.5mL centrifuge tube, adds into the centrifuge tube equipped with bronze 70% ethyl alcohol of 1mL, acutely shakes 15min in vortex oscillation, with the revolving speed of 1300g, is centrifuged 1min at room temperature, and abandoning is taken to be centrifuged Supernatant fluid in pipe repeats the step three times.It is eventually adding sterile water, -20 DEG C is put into and saves backup.

2.3 take 5 μ L bronze suspension in the import centrifuge tube of 1.5mL, and 1 μ L transient expression vector DNA (1.0 μ g/ are added μ L), the autoclaved 2.5mol/L CaCl of 8 μ L₂, 4 μ L filter sterilizing 0.1mol/L spermidine, after mixing, in vortex oscillation 3min is acutely shaken on device, and stands 15min in ice bath.

2.4 with the revolving speed of 1200g, and under room temperature, moment is centrifuged 10s, takes abandoning supernatant fluid, and it is pure that 100 μ L analysis is added The dehydrated alcohol of concentration, bronze is resuspended in concussion on turbula shaker.

2.5 with the revolving speed of 12000g, and under room temperature, moment is centrifuged 10s, and except supernatant is abandoned, it is pure dense that 15 μ L analysis is added The dehydrated alcohol of degree shakes in turbula shaker and is resuspended, keeps liquid uniform.

2.6 particle guns use PDS-1000/He type particle gun (Bio-Rad, the U.S.), condition are arranged are as follows: sample vacuum chamber 26in Hg is spent, can split film pressure is 1100psi, at range 6cm.10 μ L suspension equalization points are taken to be coated in conversion slide glass Region is entreated, under the above conditions, the bronze that conversion slide glass middle section is wrapped in transient expression vector is uniformly squeezed into and is cultivated Onion bulb stem endepidermis cell.

2.7 are placed with the culture medium of the onion endepidermis after conversion with black plastic bag package, cultivate at 25 DEG C.

2.8 cultures 16 take the onion epidermis film-making on culture medium to after for 24 hours, BX63 type fluorescence microscope (Olimpas, Japan) under observe and take pictures.As a result as shown in figure 11, subcellular localization is in nucleus.Wherein, Figure 11 A is pC2300-35S- The subcellular localization of eGFP, Figure 11 B are the subcellular localization of pC2300-35S-CHS-eGFP.

Embodiment 9

The present embodiment provides the prokaryotic expression methods of CHS albumen, comprising the following steps:

(1) Prokaryotic expression vector construction

1.1 according to CHS gene ORF sequence, in its 5 ' and 3 ' end addition prokaryotic expression carrier pET28a (+)-CHS (Figure 12) Restriction enzyme site needed for building and protection base, with Premier5.0 software design special primer, [5 '-CGGGATCC (BamHI)ATGCCGAGCCTCGAATCCA-3’/5’-CCAAGCTT(HindШ)TTAAAGAGGAACGCTGCGAA-3’]。

1.2 using the pDM19-T plasmid of above-mentioned insertion CHS gene ORF as template, is loaded by 4 reaction system of table, carries out PCR Amplification.Temperature cycling program are as follows: 94 DEG C of 3min；98 DEG C of 10s, 62 DEG C of 30s, 72 DEG C of 90s are recycled 36 times；72℃5min.

1.3 amplified productions are separated with 1% non denatured Ago-Gel,ChemiDocTM XRS type gel at As system (Bio-Rad, the U.S.) imaging, with gel reclaims kit (Tiangeng, Beijing) recovery purifying target fragment.

1.5 are loaded by 13 reaction system of table, with double enzyme products of T4DNA ligase connection recovery purifying, under the conditions of 16 DEG C, It is at least connected with 8h.

1.7 extract the Plasmid DNA in above-mentioned steps, after the fast enzyme cutting BamH I and Hind Ш double digestion identification of correspondence, Take a small amount of plasmid that Sangon Biotech (Shanghai) Co., Ltd. is sent to be sequenced, checking carrier building is correct.

(2) prokaryotic expression:

Prokaryotic expression carrier pET28a (+)-CHS built is converted BL21 competent cell with heat-shock transformed method by 2.1, And it is inoculated in LB plate (kanamycins containing 100mg/L), the monoclonal in picking LB solid medium tablets does bacterium solution PCR detection After the positive, it is inoculated in LB liquid medium (kanamycins containing 100mg/L), with 150g revolving speed, under the conditions of 37 DEG C, shaking culture Overnight.

2.2 take culture solution by the dilution proportion of 1:100, are inoculated in LB liquid medium (kanamycins containing 100mg/L), With 150g revolving speed, under the conditions of 37 DEG C, culture to OD value about 0.6.

2.3 take 20mL culture solution, and 1mmol/L isopropylthiogalactoside (Isopropyl β-D- is added Thiogalactoside, IPTG) 20 μ L, under 38 DEG C of inductive conditions, inducing expression 2h.

2.4 with the revolving speed of 8000g, and under room temperature, centrifugation 10min collects thallus, with the combination buffer of 1/25 volume [25mmol/L Tris-HCl (pH 8.0), 0.5mol/L NaCl, 5mmol/L imidazoles] is resuspended, by the centrifuge tube of resuspension fluids It is steadily put into and turns to have in the conical flask of mixture of ice and water, be crushed 40min using ultrasonic Syrup-homogenizing instrument, it is mixed that every 20min changes an ice water Close object.Part bacterium solution is taken, with the revolving speed of 14000g, under room temperature, 2min is centrifuged, takes abandoning supernatant fluid, precipitating is collected, to lure The thallus of leading is control, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sodium dodecyl Sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) separation, and contaminated using Coomassie brilliant blue Color, as a result such as Figure 13.In Figure 13, anoectochilus formosanus and Anoectochilus roxburghii CHS albumen can be induced to express.M represents marker, PET-28a (+)-CHS of 1 expression anoectochilus formosanus CHS is not induced, and 2 indicate pET-28a (+)-CHS of Anoectochilus roxburghii CHS not Induction, 3 indicate pET-28a (+)-CHS induction of anoectochilus formosanus CHS, and 4 indicate the pET-28a (+)-of Anoectochilus roxburghii CHS CHS induction.

2.5 protein purifications: Ni-NTA prepacked column is balanced using combination buffer, the liquid in step 1.4 is taken, is used after loading Washing buffer [(25mmol/L Tris-HCl (pH 8.0), 0.5mol/L NaCl, 20mmol/L imidazoles] washing, it reuses Elution buffer containing 25mmol/L Tris-HCl (pH 8.0), 0.5mol/L NaCl, 600mmol/L imidazoles, which elutes, to be collected Destination protein, 12%SDS-PAGE separation, coomassie brilliant blue staining, as a result as shown in figure 14.In Figure 14, M represents marker, and 1 Anoectochilus formosanus CHS purifying protein is represented, 2 represent 3 times of concentration anoectochilus formosanus CHS purifying proteins, and 3 represent Anoectochilus roxburghii CHS purifying protein, 4 represent 3 times of concentration Anoectochilus roxburghii CHS purifying proteins.Anoectochilus formosanus and Anoectochilus roxburghii CHS albumen are equal It is purified.

2.6 rinse PD-10 column with displacement buffer (phosphate buffer containing 10% glycerol).

Embodiment 10

The present embodiment provides the detection methods of ultrasonic disruption bacterium solution and the CHS enzymatic activity of purifying protein:

Using plant CHS Enzyme assay kit (ABRaxis, USA) and spectrophotometer detection, specific step is as follows:

(1) spectrophotometer (UV752) is opened, using the cuvette of 100 μ L, wavelength 412nm was read every 5 minutes, altogether Read 7 times (totally 30 minutes), and zero setting.

(2) 205 μ L buffers are taken, 25 μ L substrate solutions mix in centrifuge tube；Under the conditions of 30 DEG C, it is added after warm bath 3min 20 μ L sample to be tested (200 μ g) albumen topple over mixing, and as blank control, (30 minutes degrees are to 0 point for spectrophotometer detection Clock).

(3) 185 μ L buffers, 20 μ L reaction solutions and 25 μ L substrate solutions is taken to mix in centrifuge tube；In 30 DEG C of temperature conditions Under, 20 μ L sample to be tested (200 μ g) albumen are added after warm bath 3min, topple over mixing, spectrophotometer is detected as sample readout (30 Minute, degree was by 0 minute).

(4) the obligate liquid of 20 μ L and 20 μ L sample to be tested (200 μ g) albumen, 30 DEG C of incubation 15min are taken.

(5) 165 μ L buffers, 20 μ L reaction solutions and 25 μ L substrate solutions mix in centrifuge tube；Under the conditions of 30 DEG C of temperature, 40 μ L liquid in step (3) are added after warm bath 3min, topple over mixing, spectrophotometer is detected as sample readout (30 minutes degree It counts to 0 minute).

(6) relative value of sample room enzymatic activity is calculated, as shown in figure 15.Figure 15 A indicates that internal enzyme activity, Figure 15 B indicate External enzyme activity.Although anoectochilus formosanus and anoectochilus formosanus CHS translation albumen have identical functional domain, internal enzyme activity and External enzyme activity is different.

Embodiment 11

The present embodiment provides the heterogenous expression of CHS gene in rice, include the following steps:

(1) according to CHS gene ORF sequence, with 5.0 software design special primer of Premier, and it is single in its 5 '-end addition Restriction enzyme site and guarantor needed for cotyledon plant Overexpression vector pZZ00026-Ubi-CHS-T-nos (Figure 16) building Protect base [5 '-TCCCGGG (Sma I)-CGCGGATCC of ATGCCGAGCCTCGAATCCA-3 '/5 ' (Bam HI) AAGAGGAACGCTGCGAA-3′]。

(2) it using the pDM19-T plasmid of above-mentioned insertion CHS gene ORF as template, is loaded by 4 reaction system of table, carries out PCR Amplification.Temperature cycling program are as follows: 94 DEG C of initial denaturation 3min；98 DEG C of 10s, 62 DEG C of 30s, 72 DEG C of 60s, this three temperature cycles 38 It is secondary；72 DEG C of extension 5min.

(3) amplified production is separated with 1% non denatured agarose gel electrophoresis,ChemiDocTM XRS type is solidifying Glue imaging system (Bio-Rad, the U.S.) imaging, with gel reclaims kit (Tiangeng, Beijing) recovery purifying target fragment.

(4) by 12 reaction system of table be loaded, using fast enzyme cutting under the conditions of 30 DEG C digestion 0.5h.Digestion products are with 1.2% Ago-Gel be separated by electrophoresis, with gel reclaims kit (Tiangeng, Beijing) recovery purifying.

(5) it is loaded by 13 reaction system of table, with double enzyme products of T4DNA ligase connection recovery purifying, under the conditions of 16 DEG C, It is at least connected with 8h.

(6) connection product is converted with heat-shock transformed method and PCR identification is carried out to recombination bacterium colony.

(7) Plasmid DNA in above-mentioned steps is extracted, after the fast enzyme cutting BamH I and Hind Ш double digestion identification of correspondence, Take a small amount of plasmid that Sangon Biotech (Shanghai) Co., Ltd. is sent to be sequenced, checking carrier building is correct.

(8) by anoectochilus formosanus CHS gene ORF monocotyledon Overexpression vector pZZ00026-Ubi-CHS-T- Nos, heat shock method convert Agrobacterium (Agrobacterium tumefaciens) EHA105 bacterial strain.

(9) improvement N6 culture medium, evoked callus are inoculated into after spending 11 rice paddy seeds that grain husk is gone to sterilize by.

(10) after subculture, embryo callus is selected, is transferred to pre-culture medium dark culture 3d or so.

(11) callus is transferred to and is co-cultured on base, the agrobacterium suspension 100mL after adding activation, 19 DEG C of dark cultures After 3d, with sterile ddH₂O is cleaned 3 times, and the sterile purified water of cephalothin containing 400mg/mL (Cephalothin) is added, with The revolving speed of 200g rocks culture 30min.

(12) callus after co-cultivation is transferred to screening and culturing medium, under the conditions of 28 DEG C, dark culture 20d or so.

(13) transfer screening and culturing medium on callus in differential medium, under the conditions of 28 DEG C, illumination cultivation to divide Dissolve seedling.

(14) the differentiation callus transferred on root media is sufficiently sent out under the conditions of 28 DEG C of illumination cultivations to root system After educating, hardening 7d or so intermediate house or crop field.

Embodiment 11

The present embodiment provides the PCR verification methods for turning CHS trans-genetic hybrid rice of embodiment 10, comprising the following steps:

(1) regrowth foot leaf is taken, genomic DNA is extracted.

(2) special with Primerblast software (https: //blast.ncbi.nlm.nih.gov/Blast.cgi) design Different the primer (- AAGAGGAACGCTGCGAAGAA-3 ' of 5 '-GCTCACCCTGTTGTTTGGTG-3 '/5 '), and expand across promoter 1205bp segment.

(3) 1.2% non denatured agarose gel electrophoresis separation,ChemiDocTM XRS type gel imaging system System (Bio-Rad, the U.S.) imaging, screens positive transgenic plant from regeneration plant.As a result as shown in figure 17.In Figure 17, F table To show and turns anoectochilus formosanus CHS gene, F expression turns anoectochilus formosanus CHS gene, and R expression turns Anoectochilus roxburghii CHS gene ,+indicate Positive plasmid ,-indicate non-transgenic line.Anoectochilus formosanus and Anoectochilus roxburghii CHS gene are transferred in rice genome.

Embodiment 12

The present embodiment provides the detections of transgenic paddy rice general flavone content, the specific steps are as follows:

(1) rice leaf for turning anoectochilus formosanus CHS gene is weighed, drying grinding pulverized specimen 1.000g is in 20mL in cone In shape bottle, it is repeated 3 times.

(2) 95% ethyl alcohol 10mL is added, in KQ-100DV type ultrasonoscope (Kunshan Ultrasonic Instruments Co., Ltd.), 30 DEG C 30min is extracted, is filtered with 3 μm of filter paper, collect filtrate.

(3) residue adds same concentration ethanol, and by step (2), filtrate is collected in ultrasonic wave extraction, 3 μm of filter paper filterings again, with Step (2) filtrate merges.

(4) each 2mL of above-mentioned filtrate (ethyl alcohol of blank control addition 2mL 95%) is taken, 100g/L Al (NO3) 3 is added 1mL, 9.8g/L potassium acetate 1mL, stand 30min after shaking up.

(5) 1cm cuvette is used, in UV-1800 type ultraviolet-visible spectrophotometer (Shimadzu, Japan), measures 415nm Light absorption value.As a result as shown in figure 18.After anoectochilus formosanus CHS gene is transferred to rice, water is transferred to anoectochilus formosanus CHS gene Rice, it is different to the change of Rice flavone content.In Figure 18, F expression turns anoectochilus formosanus CHS gene, and R expression turns Anoectochilus roxburghii CHS gene (R-1 and R-2 are respectively represented and turned two independent transformation events that Anoectochilus roxburghii CHS gene obtains), * indicate poor Different significant, * * indicates that difference is extremely significant.

Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts Every other embodiment, shall fall within the protection scope of the present invention.

SEQUENCE LISTING

<110>Sanming College

<120>a kind of from the chalcone synthase gene sequence of anoectochilus formosanus and its application

<130> 1182628

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1173

<212> DNA

<213>anoectochilus formosanus

<400> 1

atgccgagcc tcgaatccat ccggaaggcg cccagagccg acggcttggc ctccatattg 60

gccatcggca gggccaatcc tgaaaacttc atggaacaaa gcaacttccc tgatttcttc 120

ttccgcgtca ccggcagcga ccacttggtc gacctcaaga agaaattcca acgcatatgc 180

gataggacgg caatcaggaa acgccatttt gtctggaacg aagagttcat caaagccaac 240

ccttgcttca gtaccttcat ggacaactct ttaaacgtca gacaagaggt agcgataagg 300

gagataccga agttgggtgc agaggcagcc accaaggcca tcaaggagtg ggggcagccc 360

aagtcaagca tcacccatct catcttttgc accaccagcg gcatggattt gcctggtgct 420

gactaccagc tcactcgaat tctaggcctc aacccgaacg tcgagagggt gatgctctac 480

cagcaaggat gttttgctgg cggaaccacg ctccggctag ccaagtgtct tgcagagagc 540

cgcaaaggcg cacgtgttct tatcgtttgt gcagagacca ccacagtgct cttccgcgca 600

ccctctgagg agcaccaaga ggaccttgtc actcaagctc tgtttgcgga tggcgcctcg 660

gcagttatag taggtgccga cccgaatgag gatgccaatg aacgtgccag ctttgtcatc 720

ttctccacat cacaggtatt attgccagac tctgaaggtg caattggcgg gcatgtatca 780

gagggaggcc tcttagccac gctccacagg gacgtcccac aacttgtttc caagaatgtc 840

gaaaaatgct tagaagaggc gttcactcct ttaggcattt cggactggaa ctccatcttt 900

tgggtgccgc acccaggtgg gcgtgccatt ctagaccaaa tcgaggagag ggtggggctg 960

aagccggaga aactaaccac ttccaggcac gtgcttgctg agtacggtaa catgtcaagc 1020

gtgtgtgtgc actttgttct ggatgagatg cgcaagaagt cttcaaaaga aggtaaagct 1080

acgacaggtg agggcctcga gtggggagtg ctcttcggat tcgggcctgg cctgactgtc 1140

gaaactgtag ttcttcgcag cgttcctctt taa 1173

<210> 2

<211> 390

<212> PRT

<213>anoectochilus formosanus

<400> 2

Met Pro Ser Leu Glu Ser Ile Arg Lys Ala Pro Arg Ala Asp Gly Leu

1 5 10 15

Ala Ser Ile Leu Ala Ile Gly Arg Ala Asn Pro Glu Asn Phe Met Glu

20 25 30

Gln Ser Asn Phe Pro Asp Phe Phe Phe Arg Val Thr Gly Ser Asp His

35 40 45

Leu Val Asp Leu Lys Lys Lys Phe Gln Arg Ile Cys Asp Arg Thr Ala

50 55 60

Ile Arg Lys Arg His Phe Val Trp Asn Glu Glu Phe Ile Lys Ala Asn

65 70 75 80

Pro Cys Phe Ser Thr Phe Met Asp Asn Ser Leu Asn Val Arg Gln Glu

85 90 95

Val Ala Ile Arg Glu Ile Pro Lys Leu Gly Ala Glu Ala Ala Thr Lys

100 105 110

Ala Ile Lys Glu Trp Gly Gln Pro Lys Ser Ser Ile Thr His Leu Ile

115 120 125

Phe Cys Thr Thr Ser Gly Met Asp Leu Pro Gly Ala Asp Tyr Gln Leu

130 135 140

Thr Arg Ile Leu Gly Leu Asn Pro Asn Val Glu Arg Val Met Leu Tyr

145 150 155 160

Gln Gln Gly Cys Phe Ala Gly Gly Thr Thr Leu Arg Leu Ala Lys Cys

165 170 175

Leu Ala Glu Ser Arg Lys Gly Ala Arg Val Leu Ile Val Cys Ala Glu

180 185 190

Thr Thr Thr Val Leu Phe Arg Ala Pro Ser Glu Glu His Gln Glu Asp

195 200 205

Leu Val Thr Gln Ala Leu Phe Ala Asp Gly Ala Ser Ala Val Ile Val

210 215 220

Gly Ala Asp Pro Asn Glu Asp Ala Asn Glu Arg Ala Ser Phe Val Ile

225 230 235 240

Phe Ser Thr Ser Gln Val Leu Leu Pro Asp Ser Glu Gly Ala Ile Gly

245 250 255

Gly His Val Ser Glu Gly Gly Leu Leu Ala Thr Leu His Arg Asp Val

260 265 270

Pro Gln Leu Val Ser Lys Asn Val Glu Lys Cys Leu Glu Glu Ala Phe

275 280 285

Thr Pro Leu Gly Ile Ser Asp Trp Asn Ser Ile Phe Trp Val Pro His

290 295 300

Pro Gly Gly Arg Ala Ile Leu Asp Gln Ile Glu Glu Arg Val Gly Leu

305 310 315 320

Lys Pro Glu Lys Leu Thr Thr Ser Arg His Val Leu Ala Glu Tyr Gly

325 330 335

Asn Met Ser Ser Val Cys Val His Phe Val Leu Asp Glu Met Arg Lys

340 345 350

Lys Ser Ser Lys Glu Gly Lys Ala Thr Thr Gly Glu Gly Leu Glu Trp

355 360 365

Gly Val Leu Phe Gly Phe Gly Pro Gly Leu Thr Val Glu Thr Val Val

370 375 380

Leu Arg Ser Val Pro Leu

385 390

<210> 3

<211> 1256

<212> DNA

<213>anoectochilus formosanus

<400> 3

atgccgagcc tcgaatccat ccggaaggcg cccagagccg acggcttggc ctccatattg 60

gccatcggca gggccaatcc tgaaaacttc atggaacaaa gcaacttccc tgatttcttc 120

ttccgcgtca ccggcagcga ccacttggtc gacctcaaga agaaattcca acgcatatgc 180

aagccgccct cgctctctcc ctgtactctc tcccttttag caaagctatg aacaaagatt 240

ggaattgata tataatcatc tgcgatagga cggcaatcag gaaacgccat tttgtctgga 300

acgaagagtt catcaaagcc aacccttgct tcagtacctt catggacaac tctttaaacg 360

tcagacaaga ggtagcgata agggagatac cgaagttggg tgcagaggca gccaccaagg 420

ccatcaagga gtgggggcag cccaagtcaa gcatcaccca tctcatcttt tgcaccacca 480

gcggcatgga tttgcctggt gctgactacc agctcactcg aattctaggc ctcaacccga 540

acgtcgagag ggtgatgctc taccagcaag gatgttttgc tggcggaacc acgctccggc 600

tagccaagtg tcttgcagag agccgcaaag gcgcacgtgt tcttatcgtt tgtgcagaga 660

ccaccacagt gctcttccgc gcaccctctg aggagcacca agaggacctt gtcactcaag 720

ctctgtttgc ggatggcgcc tcggcagtta tagtaggtgc cgacccgaat gaggatgcca 780

atgaacgtgc cagctttgtc atcttctcca catcacaggt attattgcca gactctgaag 840

gtgcaattgg cgggcatgta tcagagggag gcctcttagc cacgctccac agggacgtcc 900

cacaacttgt ttccaagaat gtcgaaaaat gcttagaaga ggcgttcact cctttaggca 960

tttcggactg gaactccatc ttttgggtgc cgcacccagg tgggcgtgcc attctagacc 1020

aaatcgagga gagggtgggg ctgaagccgg agaaactaac cacttccagg cacgtgcttg 1080

ctgagtacgg taacatgtca agcgtgtgtg tgcactttgt tctggatgag atgcgcaaga 1140

agtcttcaaa agaaggtaaa gctacgacag gtgagggcct cgagtgggga gtgctcttcg 1200

gattcgggcc tggcctgact gtcgaaactg tagttcttcg cagcgttcct ctttaa 1256

<210> 4

<211> 19

<212> DNA

<213>artificial sequence

<400> 4

atgccgagcc tcgaatcca 19

<210> 5

<211> 19

<212> DNA

<213>artificial sequence

<400> 5

ttaaaagggaacgctgcgaa 19

Claims

1. a kind of chalcone synthase gene from anoectochilus formosanus, which is characterized in that the chalcone synthase genes Nucleotide sequence is as shown in SEQ ID NO:1.

2. chalcone synthase gene as described in claim 1, which is characterized in that its amino acid sequence such as SEQ ID NO:2 institute Show.

3. chalcone synthase gene as described in claim 1, which is characterized in that its coding region sequence such as SEQ ID NO:3 institute Show.

4. containing the recombinant plasmid of chalcone synthase gene described in claim 1.

5. recombinant plasmid body as claimed in claim 4, which is characterized in that the plasmid is pZZ00026-Ubi-CHS-T- nos。

6. a kind of application of chalcone synthase gene as described in claim 1 in adjusting and controlling rice flavones content.

7. application according to claim 6, which comprises the following steps:

The ORF sequence of the chalcone synthase gene of anoectochilus formosanus is inserted into pZZ00026-Ubi-CHS-T-nos list by S1 In leaf over-express vector, expression vector is obtained；

S2 converts the expression vector to Agrobacterium, carries out infecting conversion to rice with the Agrobacterium after conversion, obtain To transgenic paddy rice.