CN109517812A - Azalea chalcone synthase RsCHS albumen and its encoding gene - Google Patents

Abstract

The invention discloses a rhododendron chalcone synthase RsCHS protein and its coding gene, including the amino acid sequence of the sequence table and its coding gene; the gene is expressed in leaves, stamens, pistils and petals, and the expression in leaves is remarkable It is higher than other tissues; in different developmental stages, the expression amount in the blooming stage is the largest; the present invention provides a theoretical basis for the use of genetic engineering technology to improve plant quality and obtain plants with different colors in the future, and makes up for the current related fields of rhododendron research. The blank has application value.

Description

Azalea chalcone synthase RsCHS albumen and its encoding gene

Technical field

The present invention relates to the key enzymes and its encoding gene in azalea anthocyanidin route of synthesis, and in particular to a kind of cuckoo Flower chalcone synthase (RsCHS) albumen and encoding gene, belong to field of biotechnology.

Background technique

Flavone compound is the secondary metabolite of majority of plants in plant, wherein flavonoids route of synthesis The anthocyanidin of middle synthesis is most noticeable one kind.Since the presence of anthocyanidin makes plant produce different colors, There are many more other functions for anthocyanidin simultaneously, but the general presence all in the form of anthocyanin in plant.Chalcone synthase It is the effect of first rate-limiting enzyme and the key enzyme in anthocyanin biosynthesis pathway, coumaric acyl CoA and malonyl CoA in CHS Lower formation tetrahydroxy chalcone.Tetrahydroxy chalcone provides basic framework for generated compound in this approach, deposits The reaction in approach is being enabled to go on smoothly.

The expression of CHS gene is reacted to have and is directly contacted with flavonoids route of synthesis in plant.Using same Source antisense CHS cDNA converts three kinds of Lisianthus kinds, and after conversion, the purple Lisianthus more than 50% occurs white Spot, informal voucher line even complete white flower

Cuckoo (Rhododendron simsii) is Ericaceae rhododendron.Its form is mainly shrub, only Yunnan Province of China a few species are arbor.Azalea pattern is in riotous profusion, is usually used in urban afforestation, landscape garden and indoor potted landscape. As such a resourceful flower section plant, the Utilization prospects as natural pigment are self-evident.And anthocyanin is not only It can be used for the processing coloring of food, and have and improve visual capacity, reduce the different physiology such as blood lipid level reduction artery sclerosis Sexual function.

Currently, having cloned chalcone synthase genes (CHS), such as arabidopsis, petunia, Portugal from many plants Grape, soybean etc..Clone, expression of the azalea as one of anthocyanidin content colored section plant abundant, for azalea CHS gene The relevant reports such as situation are not common.

Summary of the invention

It is an object of the invention to fill up the clone of azalea CHS family member, expression analysis and azalea CHS The blank of albumen and encoding gene.The present invention provides cDNA, gDNA of a kind of azalea CHS gene and amino acid sequences Column；Further the present invention also provides azalea CHS genes in different tissues and the expression of petal different developmental phases.

To solve the above-mentioned problems, the present invention provides one kind to have the active protein of azalea chalcone synthase, The protein includes:

(a) protein of the composition of the amino acid sequence as shown in SEQ ID NO:4；Or

(b) amino acid sequence of SEQ ID NO:4 shown in (a) is substituted, lacks and/or increases one or more Amino acid and have the active protein as derived from (a) of azalea chalcone synthase.

As a preferred option, it is above-mentioned with the active egg of azalea chalcone synthase that the present invention provides a kind of codings The gene of white matter.

As a preferred option, the sequence of the gene with the active protein of azalea chalcone synthase is such as Shown in SEQ ID NO:1.

As a preferred option, the present invention provides a kind of coding, this has the active albumen of azalea chalcone synthase The nucleic acid sequence of matter, the gDNA sequence of the gene is as shown in SEQ ID NO:2.

As a preferred option, the present invention provides a kind of coding, this has the active albumen of azalea chalcone synthase The nucleic acid sequence of matter, the cDNA sequence of the gene is as shown in SEQ ID NO:3.

As a preferred option, the present invention provides a pair of for expanding drawing for the gDNA of the nucleic acid sequence of the gene Object, the sequence of the primer is as shown in SEQ ID NO:5 and SEQ ID NO:6.

As a preferred option, the present invention provides a pair of for expanding drawing for the cDNA of the nucleic acid sequence of the gene Object, the sequence of the primer is as shown in SEQ ID NO:5 and SEQ ID NO:6.

The technical solution has technical effect beneficial below:

Azalea is at home and abroad all popular as ornamental plant, and the anthocyanidin of azalea also considerably enriches, Uncommon for relevant reports such as clone, the expressions of azalea CHS gene at present, the present invention is looked into for current azalea The also weaker status of the research of your ketone synzyme CHS albumen, provides a kind of clonal fashion of azalea CHS gene, And the analysis of different tissues again and different times performance situation is carried out, for cloning resulting CHS gene to shut out from now on The correlative study of cuckoo flower provides theoretical foundation, to have changed plant quality using technique for gene engineering from now on, having obtained different patterns Plant provide theoretical foundation, application value with higher.

Detailed description of the invention

Fig. 1 is azalea amino acid and cowberry (BAO58433.1), camellia (BAI66465.1), Malus toringoides Hughes (ADV29810.1), the CHS amino acid series comparison diagram in sand pear (AFQ92052.1) and Chinese gooseberry (PSR98361.1), In figure, black surround indicates that conserved sequence " RLMMYQQGCFAGGTVL " and " GFGPG ", "●", " ◆ ", " # " and " ▲ " are respectively indicated Malonyl coenzyme A binding site, active site, polypeptide binding site and product binding site.

Fig. 2 is that expression of the azalea CHS albumen in different tissues organ shows figure；

Fig. 3 is expression of the azalea CHS albumen in the different developmental phases of petal；

Specific embodiment

Below in conjunction with specific embodiment, the present invention is described further.

Embodiment provided below is not intended to limit the invention covered range, and described step is also not use Sequence is executed to limit its.Those skilled in the art combine existing common knowledge to do conspicuous improvement to the present invention, also fall Enter the present invention claims protection scope within.

The clone of embodiment 1, azalea RsCHS gene

1. the acquisition of vegetable material

This experiment vegetable material used is the high-quality germ plasm resource belgian azalea of azalea.Belgian azalea phenotype is red Flower.Experimental material is cultivated in the greenhouse in Ningbo City, Chai Qiaowan scape cuckoo breeding garden, Beilun District.Nursery under field conditions (factors), growth And it blooms.The petal of the phase in full bloom of azalea is acquired for extracting RNA and DNA.

The extraction of 2.RNA and DNA

Extracting RNA using RNAprep Pure polysaccharide polyphenol plant total RNA extraction reagent box, (kit is biochemical purchased from Tiangeng Scientific and technological (Beijing) Co., Ltd).The integrality for identifying RNA after denaturation with 1% agarose gel electrophoresis, in spectrophotometer The purity and concentration of RNA are measured on (Thermo Scientific NanoDrop2000).

Total DNA (OMEGA Bio-tek (U.S.) company) is extracted using HP Plant DNA Kit kit

3. the full-length clone of gene

Conserved sequence based on CHS gene in Genebank designs pair of primers, and the RNA of extraction is carried out reverse transcription (SuperRTcDNA Synthesis Kit: health is century Biotechnology Co., Ltd), using the first chain cDNA as template, utilizes Primer

F1:ATGGTCACCGTCGAGGATGTCA(SEQ ID NO:5)

R1:TCAAGTGCACAAACTGTGCAGCA(SEQ ID NO:6)

Carry out PCR, amplification recycles after obtaining expected length and is connected to pMD18-T (precious day doctor biotechnology (Beijing) has Limit company) on carrier, bacillus coli DH 5 alpha is converted, picking white single colonie passes through bacterium solution PCR screening positive clone, send to raw work Bioengineering (Shanghai) limited liability company is sequenced, and cDNA sequence is obtained.

According to same primers, using DNA as template, the gDNA of CHS is expanded, amplification is connected to after obtaining product recycling On pMD18-T (precious day cures biotechnology (Beijing) Co., Ltd) carrier, bacillus coli DH 5 alpha, picking white single colonie warp are converted Bacterium solution PCR screening positive clone is crossed, send to Sangon Biotech (Shanghai) Co., Ltd. and is sequenced, obtains gDNA sequence Column.

Pair of primers is designed according to obtained cDNA sequence

By the RNA of extraction carry out reverse transcription (5 '/3 ' Kit: precious day cures biotechnology (Beijing) limited public affairs Department) 3 ' and 5 ' cDNA of synthesis using the cDNA of synthesis as template utilize primer

3 ' RACE:TCTGCGGCCCAACCATTCTACCTG (SEQ ID NO:7)

5 ' RACE:GGATTTGTCGCACATGCGCTGGAA C (SEQ ID NO:8)

Carry out PCR, amplification recycles after obtaining expected length and is connected to pMD18-T (precious day doctor biotechnology (Beijing) has Limit company) on carrier, bacillus coli DH 5 alpha is converted, picking white single colonie passes through bacterium solution PCR screening positive clone, send to raw work Bioengineering (Shanghai) limited liability company is sequenced, and 3 ' and 5 ' sequences are obtained.Obtained sequence is spliced, base is obtained Because of full length sequence.

The sequence information and homology analysis of example 2, azalea RsCHS gene

Obtained CHS gene predicts that 1037-2017bp is ORF sequence, length 981bp in sequence by ORF, thus it is speculated that Encode 326 amino acid.CHS molecular weight of the encoded protein is 41.6kDa, and theoretical isoelectric point pI is 6.13.The genome of CHS Contain 2 exons and 1 introne.

The open reading frame sequence of azalea CHS and its amino acid sequence for encoding albumen is online in NCBI, EMBL etc. Nucleotide and protein homology search are carried out using blast program in analysis.

The performance situation of the differential expression and petal different developmental phases of example 3, azalea CHS gene in different tissues

1. the acquisition of vegetable material

The petal for acquiring different developmental phases, acquires leaf, petal and hero (female) stamen in florescence respectively, sample is distinguished It is wrapped with valve bag, after liquid nitrogen flash freezer, is stored in -80 DEG C of ultra low temperature freezers.

The extraction of 2.RNA

Extracting RNA using RNA prep Pure polysaccharide polyphenol plant total RNA extraction reagent box, (kit is raw purchased from Tiangeng Change scientific and technological (Beijing) Co., Ltd).With 1% agarose gel electrophoresis (1TAE after deformation；120V, 20min) identify that RNA's is complete Whole property measures the purity and concentration of RNA on spectrophotometer (Thermo Scientific NanoDrop2000).

The acquisition of 3.cDNA

Using the total serum IgE of 500ng as template, according to Nuo Weizan companyⅡQ RT SuperMix for qPCR (+gDNA wiper) kit operating instruction carries out reverse transcription and prepares cDNA.

4. designing specific primer carries out real-time fluorescence quantitative PCR analysis gene in different tissues and different developmental phases Expression quantity.According to the azalea CHS gene order obtained, Real-time is designed for using primer-design software The specific primer of PCR.

CHS-F:CAGCGAGCACAAGGCAGAGC (SEQ ID NO:9)

CHS-R:AACTTCCACCACAACCATGTCCTG (SEQ ID NO:10)

Reference gene is 18s, and primer is 18S-F:AACCATAAACGATGCCGACCAGG (SEQ ID NO:11)

18S-R:TTCAGCCTTGCGACCATACTCCC (SEQ ID NO:12)

5. the Real time PCR of target gene in sample to be tested.Using the cDNA of synthesis as template, purpose is used respectively The primer amplified of gene and reference gene carries out quantitative fluorescence analysis, and Real-time PCR reaction is real-time in CFX-96 It is carried out on quantitative instrument, reaction system is 20 μ L.Using three-step approach, 95 DEG C of denaturation 1min；95 DEG C of 10s, 60 DEG C of 30s, 40 circulations； 60 DEG C of 60s, 95 DEG C of 15s acquire melt curve analysis.

6. obtaining target gene to the expression of reference gene, often using using 2- [Ct (target gene)-Ct (18s)] Kind sample all sets 3 repetitions.CHS gene has expression in the leaf, hero (female) stamen and petal of azalea as the result is shown, wherein Expression highest in leaf, followed by petal, stamen and gynoecium are then minimum, illustrate that the expression of CHS gene has apparent space parallax It is anisotropic.And in the different stages of development, downward trend after first rising is presented in the expression of CHS gene, reaches in the expression of full-bloom stage To highest.

The alignment of embodiment 4, azalea RsCHS albumen and other plant CHS albumen

As shown in FIG. 1, FIG. 1 is the present invention clone resulting azalea CHS albumen sequence and cowberry (BAO58433.1), Camellia (BAI66465.1), Malus toringoides Hughes (ADV29810.1), sand pear (AFQ92052.1) and Chinese gooseberry (PSR98361.1) comparison diagram, the sequence for further illustrating that clone obtains belongs to chalcone synthase family gene, with other Known chalcone synthase sequence has identical malonyl coenzyme A binding site, active site, polypeptide binding site and product Binding site, the albumen may play similar function in azalea pattern route of synthesis.

Chalcone synthase is directly affected as first rate-limiting enzyme in flavonoid biosynthesis pathway, expression activity The progress entirely reacted.Domestic pattern breeding now is substantially also in crossbreeding is relied on, for utilizing genetic engineering means Molecular breeding research it is less.Therefore we can use genetic engineering means and operate to chalcone synthase, can be with It is directionally altered the character of cuckoo, the new varieties of completely new pattern are brought for cuckoo flowers, shorten breeding cycle and time.

Specific embodiments of the present invention are described above.It is to be understood that the invention is not limited to above-mentioned spies Determine embodiment, those skilled in the art can make various denaturation or modification within the scope of the claims, this has no effect on Substantive content of the invention.

Sequence table

<110>Wanli College, Zhejiang

<120>azalea chalcone synthase CHS albumen and its encoding gene

<130> 2018-12-11

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2236

<212> DNA

<213>azalea (Rhododendron simsii)

<400> 1

attatttttt ccggcgaaaa gatggtcacc gtcgaggatg tcaggagggc gcaacgggct 60

gaaggcccag caacagtcat ggcaatcgga accgcgaccc catcgaactg cgtcgatcag 120

agcacgtacc cggatttcta cttccggatt actaacagcg agcacaaggc agagctcaaa 180

gagaagttcc agcgcatgtg taagtgaata tcaccatctt tccttctgta cgtaattttg 240

gtaatccatt ttcaagtttc taatcagggc caggtgtttg ggagccgact ttttgaatta 300

tggatcgtgc atatattctg attacggggg tttggaaacc aagtagatat gcaaaatgat 360

ctgaaaaaca ccgaattcat gaatcgtcgt cggatgagcc ttttaaattc aattatagta 420

cacttccgta cacaaaacaa aaaaatttgt tctgctcctt tcatccaagt agttttacaa 480

attcttatcg taattttaac atctatgatt aacaatccaa aagctaaatc taaaaaatta 540

ttggcttcca aacaccccat tagtgtttcc ggcgaccgga atttatggtt ttacacacat 600

gcatgtgact tgctggatga tgtatatatg tagattaaat aggtacatgt acttcttatg 660

ggaatggaaa aggatttgga aattctatac tgaattaaca aatctagggt gacggttact 720

gacttatgtt tgtatttttc tcttctattt atttcctttt cttactaatt ggggaatttc 780

tatttaaaat tgtgcatgcg aatagatgca ttgtattttg gtagtgtcca ttgttagttt 840

tgttgttaat gtggtgaaat taaatggaca tcaccatcgg cagtagccca gtacttcttg 900

gcaccccccg ccgccccctc cctctctctc tctctctctt ctctctctca gaagagaatg 960

agtggattat tgatttttaa atctacaatt actatgatga actaatagga agattctgaa 1020

ttattagttt cttcaacaat ttttttgctg cttccatcaa tttttcaagg cgacaaatcc 1080

atgatcaaga agcgttatat gtacttaacc gaagaaatct tgaaggaaaa tcccagtgtg 1140

tgtgagtaca tggcgccttc tctggacgct aggcaggaca tggttgtggt ggaagttccc 1200

aaattgggca aagaggctgc caccaaggcc ataaaggaat ggggccagcc caaatccaaa 1260

atcacccact tggtcttttg caccaccagt ggtgtcgaca tgccaggcgc cgactaccag 1320

ctcaccaagc tcctcggcct ccgcccatcc gtcaagcgcc tcatgatgta ccaacaaggt 1380

tgcttcgctg gtggtacggt cctccgcctg gccaaggact tggcagagaa taacaaaggc 1440

gcccgggttt tagtcgtgtg ttctgagatc actgctgtca ctttccgtgg gccaagtgac 1500

acccaccttg acagtctggt tggccaagcc ctctttggag atggcgcagc tgctattatt 1560

gttggtgcgg acccggtacc cgaagttgag aagccattgt ttgagctggt ctctgcggcc 1620

caaaccattc tacctgatag cgatggggcc atcgatggac atctccgcga agtgggcctt 1680

actttccact tgctcaaaga tgttcctggg ctcatctcca agaacataga aaaggccttg 1740

accgaggctt ttcagccctt gggcatctcc gattggaatt ccattttctg gattgcgcac 1800

cccggcgggc ctgctattct agatcaggtg gaattgaagt tgagccttaa gcccgagaag 1860

ctacgggcca cgaggcacgt gctgagcgaa tacgggaaca tgtcgagcgc gtgcgtgctg 1920

ttcattttgg atgagatgag gaggaagtcg gccgaggaag ggctcaagac cactggtgag 1980

gggctcgagt ggggtgtgct cttcgggttc gggccagggc tcactgttga gactgtggtg 2040

ctgcacagtt tgtgcacttg aatgtgctac ccctcattag catcgtttct ggattctctt 2100

taattaattg ctccttgtgt gccaaaaaaa aaaaaaaaaa aaaaaaaaag tactctgcgt 2160

tgataccact gcttgcccta tagtgagtcg tattagaatc gtcgacctgc aggcatgcaa 2220

gctgcactgc gaggcc 2236

<210> 2

<211> 870

<212> DNA

<213>azalea (Rhododendron simsii)

<400> 2

aagtgaatat caccatcttt ccttctgtac gtaattttgg taatccattt tcaagtttct 60

aatcagggcc aggtgtttgg gagccgactt tttgaattat ggatcgtgca tatattctga 120

ttacgggggt ttggaaacca agtagatatg caaaatgatc tgaaaaacac cgaattcatg 180

aatcgtcgtc ggatgagcct tttaaattca attatagtac acttccgtac acaaaacaaa 240

aaaatttgtt ctgctccttt catccaagta gttttacaaa ttcttatcgt aattttaaca 300

tctatgatta acaatccaaa agctaaatct aaaaaattat tggcttccaa acaccccatt 360

agtgtttccg gcgaccggaa tttatggttt tacacacatg catgtgactt gctggatgat 420

gtatatatgt agattaaata ggtacatgta cttcttatgg gaatggaaaa ggatttggaa 480

attctatact gaattaacaa atctagggtg acggttactg acttatgttt gtatttttct 540

cttctattta tttccttttc ttactaattg gggaatttct atttaaaatt gtgcatgcga 600

atagatgcat tgtattttgg tagtgtccat tgttagtttt gttgttaatg tggtgaaatt 660

aaatggacat caccatcggc agtagcccag tacttcttgg caccccccgc cgccccctcc 720

ctctctctct ctctctcttc tctctctcag aagagaatga gtggattatt gatttttaaa 780

tctacaatta ctatgatgaa ctaataggaa gattctgaat tattagtttc ttcaacaatt 840

tttttgctgc ttccatcaat ttttcaaggc 870

<210> 3

<211> 1167

<212> DNA

<213>azalea (Rhododendron simsii)

<400> 3

atggtcaccg tcgaggatgt caggagggcg caacgggctg aaggcccagc aacagtcatg 60

gcaatcggaa ccgcgacccc atcgaactgc gtcgatcaga gcacgtaccc ggatttctac 120

ttccggatta ctaacagcga gcacaaggca gagctcaaag agaagttcca gcgcatgtgt 180

gacaaatcca tgatcaagaa gcgttatatg tacttaaccg aagaaatctt gaaggaaaat 240

cccagtgtgt gtgagtacat ggcgccttct ctggacgcta ggcaggacat ggttgtggtg 300

gaagttccca aattgggcaa agaggctgcc accaaggcca taaaggaatg gggccagccc 360

aaatccaaaa tcacccactt ggtcttttgc accaccagtg gtgtcgacat gccaggcgcc 420

gactaccagc tcaccaagct cctcggcctc cgcccatccg tcaagcgcct catgatgtac 480

caacaaggtt gcttcgctgg tggtacggtc ctccgcctgg ccaaggactt ggcagagaat 540

aacaaaggcg cccgggtttt agtcgtgtgt tctgagatca ctgctgtcac tttccgtggg 600

ccaagtgaca cccaccttga cagtctggtt ggccaagccc tctttggaga tggcgcagct 660

gctattattg ttggtgcgga cccggtaccc gaagttgaga agccattgtt tgagctggtc 720

tctgcggccc aaaccattct acctgatagc gatggggcca tcgatggaca tctccgcgaa 780

gtgggcctta ctttccactt gctcaaagat gttcctgggc tcatctccaa gaacatagaa 840

aaggccttga ccgaggcttt tcagcccttg ggcatctccg attggaattc cattttctgg 900

attgcgcacc ccggcgggcc tgctattcta gatcaggtgg aattgaagtt gagccttaag 960

cccgagaagc tacgggccac gaggcacgtg ctgagcgaat acgggaacat gtcgagcgcg 1020

tgcgtgctgt tcattttgga tgagatgagg aggaagtcgg ccgaggaagg gctcaagacc 1080

actggtgagg ggctcgagtg gggtgtgctc ttcgggttcg ggccagggct cactgttgag 1140

actgtggtgc tgcacagttt gtgcact 1167

<210> 4

<211> 379

<212> PRT

<213>azalea (Rhododendron simsii)

<400> 4

Arg Ser Asn Gln Gly Leu Arg Pro Ala Gln Ser Trp Gln Ser Gly Thr

1 5 10 15

Ala Thr Pro Pro Asn Cys Val Asp Gln Ser Thr Tyr Pro Asp Phe Tyr

20 25 30

Phe Arg Ile Thr Asn Ser Glu His Lys Ala Glu Leu Lys Glu Lys Phe

35 40 45

Gln Arg Met Asp Lys Ser Met Ile Lys Lys Arg Tyr Met Tyr Leu Thr

50 55 60

Glu Glu Ile Leu Lys Glu Asn Pro Ser Val Cys Glu Tyr Met Ala Pro

65 70 75 80

Ser Leu Asp Ala Arg Gln Asp Met Val Val Val Glu Val Pro Lys Leu

85 90 95

Gly Lys Glu Ala Ala Thr Lys Ala Ile Lys Glu Trp Gly Gln Pro Lys

100 105 110

Ser Lys Ile Thr His Leu Val Phe Cys Thr Thr Ser Gly Val Asp Met

115 120 125

Pro Gly Ala Asp Tyr Gln Leu Thr Lys Leu Leu Gly Leu Arg Pro Ser

130 135 140

Val Lys Arg Leu Met Met Tyr Gln Gln Gly Cys Phe Ala Gly Gly Thr

145 150 155 160

Val Leu Arg Leu Ala Lys Asp Leu Ala Glu Asn Asn Lys Gly Ala Arg

165 170 175

Val Leu Val Val Cys Ser Glu Ile Thr Ala Val Thr Phe Arg Gly Pro

180 185 190

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

195 200 205

Gly Ala Ala Ala Ile Ile Val Gly Ala Asp Pro Val Pro Glu Val Glu

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Ala Leu Thr Glu Ala Phe Gln Pro Leu Gly Ile Ser Asp Trp Asn Ser

275 280 285

Ile Phe Trp Ile Ala His Pro Gly Gly Pro Ala Ile Leu Asp Gln Val

290 295 300

Glu Leu Lys Leu Ser Leu Lys Pro Glu Lys Leu Arg Ala Thr Arg His

305 310 315 320

Val Leu Ser Glu Tyr Gly Asn Met Ser Ser Ala Cys Val Leu Phe Ile

325 330 335

Leu Asp Glu Met Arg Arg Lys Ser Ala Glu Glu Gly Leu Lys Thr Thr

340 345 350

Gly Glu Gly Leu Glu Trp Gly Val Leu Phe Gly Phe Gly Pro Gly Leu

355 360 365

Thr Val Glu Thr Val Val Leu His Ser Leu Cys

370 375

<210> 5

<211> 22

<212> DNA

<213>azalea (Rhododendron simsii)

<400> 5

atggtcaccg tcgaggatgt ca 22

<210> 6

<211> 23

<212> DNA

<213>azalea (Rhododendron simsii)

<400> 6

tcaagtgcac aaactgtgca gca 23

<210> 7

<211> 24

<212> DNA

<213>azalea (Rhododendron simsii)

<400> 7

tctgcggccc aaccattcta cctg 24

<210> 8

<211> 25

<212> DNA

<213>azalea (Rhododendron simsii)

<400> 8

ggatttgtcg cacatgcgct ggaac 25

<210> 9

<211> 20

<212> DNA

<213>azalea (Rhododendron simsii)

<400> 9

cagcgagcac aaggcagagc 20

<210> 10

<211> 24

<212> DNA

<213>azalea (Rhododendron simsii)

<400> 10

aacttccacc acaaccatgt cctg 24

<210> 11

<211> 23

<212> DNA

<213>azalea (Rhododendron simsii)

<400> 11

aaccataaac gatgccgacc agg 23

<210> 12

<211> 23

<212> DNA

<213>azalea (Rhododendron simsii)

<400> 12

ttcagccttg cgaccatact ccc 23

Claims (7)

1. one kind has the active protein of azalea chalcone synthase characterized by comprising

(a) protein of the composition of the amino acid sequence as shown in SEQ ID NO:4；Or

(b) amino acid sequence of SEQ ID NO:4 shown in (a) is substituted, lacks and/or increases one or more amino Acid and have the active protein as derived from (a) of azalea chalcone synthase.

2. encoding the gene described in claim 1 with the active protein of azalea chalcone synthase.

3. gene according to claim 2, which is characterized in that described that there is the active albumen of azalea chalcone synthase The gene order of matter is as shown in SEQ ID NO:1.

4. a kind of coding sequence as claimed in claim 2, which is characterized in that the gDNA sequence of the gene such as SEQ ID NO:2 It is shown.

5. a kind of coding sequence as claimed in claim 2, which is characterized in that the cDNA sequence of the gene such as SEQ ID NO:3 It is shown.

6. a pair for expand sequence described in claim 4 gDNA primer, the sequence of the primer such as SEQ ID NO:5 and Shown in SEQ ID NO:6.

7. a pair for expand sequence described in claim 5 cDNA primer, the sequence of the primer such as SEQ ID NO:5 and Shown in SEQ ID NO:6.