CN106498009A - Verbascoside biosynthesis pathway and its synzyme related gene - Google Patents

Verbascoside biosynthesis pathway and its synzyme related gene Download PDF

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CN106498009A
CN106498009A CN201610849755.8A CN201610849755A CN106498009A CN 106498009 A CN106498009 A CN 106498009A CN 201610849755 A CN201610849755 A CN 201610849755A CN 106498009 A CN106498009 A CN 106498009A
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verbascoside
seq
kegg
biosynthesis pathway
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周延清
王向楠
段红英
张亮
张丹丹
杨柯
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Henan Normal University
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Abstract

The invention discloses verbascoside biosynthesis pathway and its synzyme related gene, tuber with 6 different developmental phases of Radix Rehmanniae is as material, extract and detect its total serum IgE, transcript profile sequencing is combined to which with Illumina HiSeq2500 platforms, 149.8 million reads are obtained, from the beginning assembling obtains 96961 Unigenes sequences；Unigenes sequences are compared with KEGG data bases carries out metabolic pathway analysis, obtains 280 KEGG approach, and metabolic pathway is compared with the verbascoside route of synthesis for speculating, 5 candidate's KEGG approach are filtered out；Compare the verbascoside route of synthesis of supposition and filter out 5 candidate's KEGG approach, setting up has the verbascoside biosynthesis pathway of 19 gene code enzyme catalysiss, Unigenes sequences are carried out BlastX with data base to compare, additional ESTscan software functions, obtain the verbascoside biosynthetic enzyme related gene of 13 candidates again.The present invention lays the foundation for illustrating the complete verbascoside biosynthesis pathway of Radix Rehmanniae and producing verbascoside using Secondary metabolic engineering or synthetic biology.

Description

Verbascoside biosynthesis pathway and its synzyme related gene
Technical field
The invention belongs to verbascoside biosynthesis technology field, and in particular to a kind of verbascoside biosynthesiss way Footpath and its synzyme related gene.
Background technology
Verbascoside is phenethyl alcohol glycosides compound, is distributed in more than the 200 of 23 sections such as Herba Verbasci Thapsi, Radix Rehmanniae and Herba Cistanches The root of kind of plant, stem, leaf and spend, adjust with antioxidation, antiinflammatory, antitumor, healing wounds, hepatoprotective, neuroprotective and immunity The effect such as section.Related researcher has been based on cold labeling precursor feeding trial and deduces verbascoside biology Route of synthesis（As shown in Figure 1）, and chemosynthesis verbascoside accordingly（Synthesis of verbascoside:a dihydroxyphenylethyl glycoside with diverse bioactivity. Eur J Org Chem 1999, 10: 2623–2632）.However, real verbascoside biosynthesis pathway is unknown, its enzyme and its corresponding gene is participated in also Know little about it.Therefore, it is badly in need of understanding its biosynthetic enzyme and its corresponding gene.Transcript profile sequencing technologies are to solve this problem One of effective technology（Verbascoside-A review of its occurrence, (bio) synthesis and Pharmacological significance. Biotechnology Advances, 2014,32：1065–1076）.
Transcript profile sequencing have flux height, low cost, sensitivity height, low-abundance expressing gene can be obtained, be not limited to Known Genomic sequence information, the species for being applied to unknown gene group sequence, cloning process, simple to operate and can be complete is not needed The advantages of medicinal plants genomic information is understood in face, is that medicinal plants functional gene excavates maximally effective instrument.Medicinal plants are lived Property the parsing of ingredients Biogenic route of synthesis and the excavation of key gene for the research of medicinal plants transcript profile main target.With transcription Salvia miltiorrhiza Bge root secondary metabolitess synthesis related gene has been excavated in group sequencing（Pellet based on high-flux sequence 454GSFLX Ginseng transcription group research, Acta Pharmaceutica Sinica, 2010,45（4）：524-529）, glycyrrhizin biosynthesis pathway enzyme gene, polygoni cuspidati,radix The gene for participating in shikimic acid Biosynthetic pathway and may be relevant with glucosides class secondary metabolites biosynthesiss（Chinese medicinal plant Rhizoma Polygoni Cuspidati The high flux transcript profile sequencing of root and transcript profile specificity analysises, Chinese science：Life sciences, 2012,42（5）：398-412）, Chinese holly Qi and Phenylalanine biosynthesis related gene（Identification of phenylpropanoid biosynthetic genes and phenylpropanoid accumulation by transcriptome analysis ofLycium chinense. BMC Genomics, 2013,14 (1): 802）, gentiana straminea iridoid and flavone route of synthesis gene（De novosequencing transcriptome of endemicGentiana straminea(Gentianaceae) to Identify genes involved in the biosynthesis of active ingredients, Gene, 2016, 575:160–170）Magnolia obovata mevalonate pathway related gene（The identification of Cortex Magnoliae Officinalis MVA approach related gene and bio information credit Analysis, CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2015,40（11）：2077-2083）Deng.
Radix Rehmanniae iridoid biosynthesis related genes have been excavated by transcript profile sequencing technologies（Transcriptome analysis reveals putative genes involved in iridoid biosynthesis inRehmannia glutinosa,Int. J. Mol. Sci.2012,13, 13748-13763）, the main crop and continuous cropping Radix Rehmanniae tuber miRNAs Target gene（Differential miRNA expression inRehmannia glutinosaplants subjected To continuous cropping. BMC Plant Biology, 2011,11:53）, continuous cropping obstacle related gene （Transcriptome-wide identification of the genes responding to replanting disease inRehmannia glutinosaL. roots, Mol Biol Rep, 2015,42:881–892）With hormone phase Correlation gene（http://www.xzbu. com/1/view-6200742.htm）.But, have no that Radix Rehmanniae verbascoside is given birth to so far The research report of thing synthetic molecules mechanism.In addition, plant verbascoside biosynthesis gene has few report, such as Radix RehmanniaePAL Gene overexpression improves the verbascoside content of Radix Rehmanniae（Turn the side that RgPAL1 genes improve verbascoside content in Radix Rehmanniae Method, CN201410628062.7）Synthesize verbascoside in Flos Caryophylli suspension cell with TyrDC genes（Production of hydroxy phenylethanol glycosides in suspension cultures ofSyringa vulgaris. Phytochemistry 1983; 22:1941–1943）.But, more unknown participation biosynthetic genes of verbascoside Require study.
Content of the invention
In order to make up the deficiency for lacking verbascoside biosynthetic enzyme genes in the even plant gene research of existing Radix Rehmanniae, The invention provides a kind of verbascoside biosynthesis pathway and its synzyme related gene, the route of synthesis is more using Radix Rehmanniae The combination tuber transcript profile sequencing of stage of development and data processing, obtain transcript（Unigenes）, by functional annotation, obtain KEGG approach；By the verbascose for comparing these KEGG approach and speculate based on cold labeling precursor feeding trial Glycosides biosynthesis pathway（Fig. 1）, candidate's verbascoside biosynthesis pathway is found, after comprehensive analysis, verbascoside is set up Biosynthesis pathway, excavates and verifies the synthase gene needed for some of them reaction with RT-PCR technology.
The present invention adopts the following technical scheme that for solving above-mentioned technical problem verbascoside biosynthesis pathway, its are special Levy and be that detailed process is：Tuber with 6 different developmental phases of Radix Rehmanniae extracts and detects its total serum IgE as material, uses Illumina HiSeq2500 platforms combine transcript profile sequencing to which, obtain 149.8 million reads, and from the beginning assembling obtains 96961 Individual Unigenes sequences；Above-mentioned Unigenes sequences are compared with KEGG data bases carries out metabolic pathway analysis, obtains 280 KEGG approach, and then the verbascoside speculated by KEGG metabolic pathways and based on cold labeling precursor feeding trial Route of synthesis is compared, and filters out 5 candidate's KEGG approach；Compare the verbascoside route of synthesis of supposition and filter out 5 times KEGG approach is selected, setting up has the verbascoside biosynthesis pathway of 19 gene code enzyme catalysiss.
Further preferably, described verbascoside biosynthesis pathway, it is characterised in that comprise the following steps：
Step（1）, with the generation in the verbascoside route of synthesis that speculated based on cold labeling precursor feeding trial Step and product are thanked as reference；
Step（2）, transcript profile sequencing, take 6 different developmental phases mixing roots of a Radix Rehmanniae kind, extract total serum IgE, determining After its quality and purity, reverse transcription builds sequencing library, carries out high-flux sequence with Illumina HiSeq2500 into cDNAs；
Step（3）, sequence analysis and transcript profile assembling, carry software using Illumina and above-mentioned sequencing gained view data passed through Cross Basecalling and be converted into corresponding nucleotide sequence data, remove low quality sequence and uncertain sequence in sequencing procedure, Obtain clear reads sequences, the from the beginning assembling for carrying out transcript profile using composite software, obtain contigs sequences or Unigenes sequences；
Step（4）, transcript functional annotation and classification, by above-mentioned Unigenes sequences and Nr, Swiss-Prott, KEGG and COG Protein Data Bank is compared, and obtains GO, COG functional annotation and classified statistic and EGG metabolic pathways；
Step（5）, verbascoside biosynthesis pathway foundation, by the above-mentioned KEGG approach of comparison and be based on stable isotope The verbascoside biosynthesis pathway that labelled precursor thing feeding trial speculates, finds and includes latter reaction's step and reaction product Synzyme needed for the candidate KEGG approach and its corresponding approach of thing, sets up verbascoside biosynthesis pathway.
Further preferably, described verbascoside biosynthetic enzyme related gene, it is characterised in that：Will be above-mentioned Unigenes sequences carry out BlastX with data base and compare, then additional ESTscan software functions, obtain the Herba Verbasci Thapsi of 13 candidates The base sequence of glucosides biosynthetic enzyme genes ORFs, base sequence such as SEQ ID NO.1, SEQ of synthase gene ORFs ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID Shown in NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13.
The invention has the advantages that：Verbascoside biosynthesis pathway disclosed by the invention be illustrate complete Verbascoside biosynthesis pathway lays the foundation, and is to produce Herba Verbasci Thapsi by Secondary metabolic engineering or synthetic biology technology Glucosides lays the foundation, and the utilization for wherein functional gene is provided as improved Radix Rehmanniae verbascoside content by technique for gene engineering Theory support.
Description of the drawings
Fig. 1 is the verbascoside biosynthesis pathway figure speculated based on cold labeling precursor feeding trial；
Fig. 2 is the biosynthetic Technology Roadmap of verbascoside of the invention；
Fig. 3 is phenylalanine, L-Tyrosine and tryptophan biosynthesis approach figure；
Fig. 4 is phenylalanine metabolic pathway figure；
Fig. 5 is L-Tyrosine metabolic pathway figure；
Fig. 6 is phenylpropanol biosynthesis pathway figure；
Fig. 7 is isoquinoline alkaloid biosynthesis pathway figure；
Fig. 8 is verbascoside biosynthesis pathway figure, 1. chorismate mutase CM：chorismate mutase[EC: 5.4.99.5]Prephenate dehydratase ADT:arogenate/prephenate dehydratase[EC:4.2.1.91]In advance Benzoic acid dehydratase PD：prephenate dehydrogenase [EC:1.3.1.12]PAL enzyme PAT： aspartate prephenate aminotransferase[EC:2.6.1.79]Aspartate transaminase ASP5: aspartate aminotransferase, chloroplastic [EC:2.6.1.1]Aspartate transaminase GOT2： aspartate aminotransferase, mitochondrial [EC:2.6.1.1]PAL PAL： phenylalanine ammonialyase[EC:4.3.1.24]4- coumarate CoA-ligase 4CL:4- coumarate-CoA ligase[EC:6.2.1.12]Cinnamic acid hydroxylase C4H: Cinnamate-4- hydroxylase [EC:1.14.13.11] 10. quininic acid hydroxy cinnamate acyltransferase HCT:shikimate O-hydroxycinnamoyl transferase [EC:2.3.1.133] coumaric acid 3- hydroxylase C3'H: coumaroylquinate (coumaroylshikimate) 3'-monooxygenase[EC:1.14.136] caffeoyl shikimic acid esterase CSE: caffeoylshikimate esterase [EC:3.1.1.-] cyclohexadienyl dehydratase Phe:cyclohexadienyl dehydratase[EC:4.2.1.51] prephenate dehydratase TYRAAT:arogenate dehydrogenase [EC: 1.3.1.78] tryrosinase TYR：tyrosinase[EC:1.14.18.1] tyrosine decarboxylase TyrTDC： tyrosine decarboxylase[EC:4.1.1.25] dopamine β-monooxygenase DBH: dopamine beta- monooxygenase[EC:1.14.17.1] primary amine oxidase AOC3: primary-amine oxidase[EC: 1.4.3.21] aryl-alcohol dehydrogenase AAD: aryl-alcohol dehydrogenase [EC:1.1.1.90], ┇ ┈ tables Show that enzyme is unknown；
Fig. 9 be 8 participation verbascoside biosynthesis pathway genes RT-PCR proof diagrams, swimming lane M：Trans2K® DNAMarker, swimming lane 1-8：ASP5、4CL、AOC3、C4H、ADT、C3’H、HCTWithGOT2, swimming lane 9：Negative control.
Specific embodiment
By the following examples the above of the present invention is described in further details, but this should not be interpreted as this The scope for inventing above-mentioned theme is only limitted to below example, and all technology that is realized based on the above of the present invention belong to this Bright scope.
Embodiment 1
（1）The verbascoside biosynthesis pathway that search forefathers speculate
In website http://isiknowledge.com retrieves pertinent literature, has scientist to be based on stable isotope before finding Labelled precursor thing feeding trial, thus it is speculated that go out verbascoside biosynthesis pathway（Fig. 1）, and have researcher according to this way Footpath chemosynthesis verbascoside（Synthesis of verbascoside:a dihydroxyphenylethyl glycoside with diverse bioactivity. Eur J Org Chem 1999,10: 2623–2632）.But, Real verbascoside enzymatic living beings route of synthesis is unknown, and the enzyme and its corresponding gene for participating in approach is known little about it；
（2）Transcript profile is sequenced：6 different developmental phases mixing roots of appropriate Radix Rehmanniae temperature 85-5 plant are taken, plant Total RNAs extraction is used Test kit extracts total serum IgE, detects through 2100 Bioanalyzer of Alilent, meets transcript profile RNA examination criterias（RNA is total >=6 μ g of amount, OD260/280 is in 1.8-2.1 scopes, rRNA Ratio（28S:18S）≥1.5:1, RIN >=7（RNA integrity number））, and the integrity of total serum IgE is detected by denaturing formaldehyde glue.With with Oligo（dT）Magnetic bead from MRNA is enriched with above-mentioned total serum IgE, is added fragmentation buffer that mRNA is broken into short-movie section, with mRNA as template, is used hexabasic base Random primer synthesizes first cDNA chain, is subsequently adding buffer, dNTPs, RNaseH and archaeal dna polymerase synthesis Article 2 cDNA Chain, through QiaQuick PCR kits purification and does end after adding EB buffer solution elutions and repairs, plus poly（A）And connect survey Sequence joint, then clip size selection is carried out with agarose gel electrophoresiies, finally, Radix Rehmanniae sequencing library is built with PCR amplifications, used Illumina HiSeq2500 carry out high-flux sequence, obtain 1.498 hundred million reads；
（3）Sequence analysis are assembled with transcript profile：Software is carried using Illumina to pass through above-mentioned sequencing gained view data Basecalling is converted into corresponding nucleotide sequence data, is stored with fastq forms, to produced original series file Quality evaluation and Analysis on confidence is carried out, low quality sequence and uncertain sequence in sequencing procedure is removed, is obtained clear reads Sequence.Then, the from the beginning assembling of transcript profile is carried out using composite software, obtains contigs sequences.Believe in conjunction with Paired-end Breath, carries out scaffold to having obtained contigs sequences.In splicing with assembling process, part read pairs are filled up with N Between gaps, until obtaining containing the sequence that N is minimum, two ends can not re-extend, i.e. Unigenes.As a result from 1.498 hundred million In reads, 96961 UniGenes are from the beginning assembled；
（4）Transcript functional annotation and classification：By eggs such as above-mentioned Unigenes sequences and Nr, Swiss- Prott, KEGG and COG White matter data base carries out BLASTX, and locally batch is compared, and obtains optimum protein functional annotation information（E-value ＜ le-5）, take ratio The albumen best to result determines the sequence direction of Unigenes.If the comparison result between different storehouses is contradictory, just according to The priority of Nr, Swiss-Prott and COG determines its direction；For the Unigenes all not compared with above-mentioned data base, profit With its coding region of ESTScan software predictions, its sequence direction is determined.Finally, respectively using Blast2GO（http:// www.blast2go.de）Software and WEGO Web（http://wego.genomics.org.cn/ cgi-bin/wego/ index. pl）Instrument carries out GO functional annotations and classified statistic to Unigenes sequences.96, in 961 UniGenes, have 11,848 Unigenes annotations obtain the gene and ORFs of catalysis wherein reaction enzymes to 280KEGG approach；
（5）The foundation of verbascoside biosynthesis pathway：By the above-mentioned 280 KEGG approach of comparison and based on stable isotope The verbascoside biosynthesis pathway that labelled precursor thing feeding trial speculates（Fig. 1）, find and include latter portion reaction step 5 rapid KEGG approach and its enzyme needed for corresponding approach（Fig. 3-7）, 5 KEGG approach and its enzyme needed for corresponding approach with push away The verbascoside biosynthesis pathway of survey is integrated, and establishes 19 enzymatic verbascoside biosynthesis pathwaies（Fig. 8）；
（6）Set up the checking of enzyme gene in verbascoside biosynthesis pathway：According to the enzyme disclosed out in above-mentioned approach and During which is numbered, find（5）Described in 19 enzymes in have 9 enzymes to have 13 Congtigs or Unigenes（Gene）Coding （Table 1）.According to the corresponding relation between enzyme, ORFs and Congtigs or Unigenes, the ORFs of the gene of codase is searched （Table 1）；According to the base sequence of ORFs, bioinformatics software synthetic primer is used（Table 2）, candidate is verified for RT-PCR method Gene（Fig. 9）.
Table 1 is excavated and 8 verbascoside biosynthetic enzymes of checking and its corresponding gene
2 RT-PCR of table verifies the size of 8 gene the primers and amplified production
The detailed process of RT-PCR method checking candidate gene experiment is as follows：
（1）The extraction and detection of total serum IgE
Appropriate Radix Rehmanniae temperature 85-5 plant tuber is taken, and total serum IgE is extracted with plant total RNA extraction reagent box.Take 1 μ of RNA solution of dilution L, using the DDH2O without RNase as control, the ultraviolet absorption value of RNA of the measure at A260 and A280, obtains corresponding OD values, When the numerical value of A260/A280 is in 1.8-2.1, purity is higher.The carried RNA concentration of record and OD values.Take 5 μ L of RNA solution 6 × Loading Buffer point samples of 1 μ L are added, electricity under 1 × TAE buffer, the agarose concentration of 1wt%, 120V voltages Swimming, after electrophoresis is placed on gel under the multi-functional ultraviolet transmission reflectometer of Bole and observes and take a picture, and detects the integrity of RNA.
（2）The preparation of the first chains of cDNA（Press kit specification）
1. following reactant mixture is added in the centrifuge tube of the nuclease free of 1.5mL ice baths：Total serum IgE（5μg）5 μ L, Oligo （dT）18 2 μ L, dNTP Mix（2.5mM each）2 μ L, mend the μ L of RNase-free ddH2O to 13.5；
2., in water-bath after 70 DEG C of heating 5min, 2min is cooled down rapidly on ice.Brief centrifugation collecting pipe reactant liquor；
3. following ingredients are added in each reaction：5 × First-Strand Buffer, 4 μ L, 0.1M DTT 1 μ L, Rnasin （40U/μL）0.5 μ L, M-MLV（200U/μL）1μL；
4. in 42 DEG C of temperature bath 50min in water-bath；
5. 5min terminating reactions are heated in 95 DEG C in water-bath；
6. reaction system is diluted to 50 μ L with RNase-free ddH2O, puts；
7. pcr amplification reaction, the 14.1 μ L containing distilled water, 10 × buffer in 25 μ L reaction systems（Mg is not contained2+）2.5 μ L, dNTP Mixture 2.5μL（Each 2.5mmol/L）, Mg2+2.5μL（25mmol/L）, 0.4 μ L of Taq enzyme（5U/μL）, primer each 1 μL（10μmol/L）, template cDNA of 1 μ L（Table 3）.Above-mentioned reactant liquor is placed in PCR test tubes, 94 DEG C of denaturations 5min, 94 DEG C Degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, circulate 30 times, last 72 DEG C of extensions 10min, 4 DEG C of preservations.
3 pcr amplification reaction system of table
8. 5 μ L PCR primers are taken to mix with appropriate sample-loading buffer, 10 μ L mixed liquors are taken, point sample carries out 1.5wt% agaroses Detected through gel electrophoresis.
Ultimate principle, principal character and the advantage of the present invention is embodiment above describes, the technical staff of the industry should Understand, the present invention is not restricted to the described embodiments, the original that the present invention is simply described described in above-described embodiment and description Reason, under the scope without departing from the principle of the invention, the present invention also has various changes and modifications, and these changes and improvements each fall within In the scope of protection of the invention.
SEQUENCE LISTING
<110>He'nan Normal University
<120>Verbascoside biosynthesis pathway and its synzyme related gene
<160> 13
<170> PatentIn version 3.3
<210> SEQ ID NO:1
<211> 432
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:1
atgcacggct cgaaactccg cgtcgcctac caaggcgtcc ccggcgcgta cagcgaagcc 60
gccgccggaa aggcttaccc gaattgcgag gccatcccct gcgaccaatt cgaagttgca 120
ttccaagccg tggagctctg gatagctgat agagccgttt tgccggtgga gaattcctta 180
ggcggctcaa tccaccggaa ctacgacctc ctcctccgcc accgcctcca catagtcgga 240
gaagtccaac tccccgtcca ccactgcctc ctagctctcc caggcgtcag aaaagagtac 300
ctcacgcgcg tcatcagcca cccacaagcc ctctcccaat gcgagcacac actcaccaaa 360
atggggctaa acgttgcccg cgaagcggtc gacgacaccg ccggcgcggc ggagtacatc 420
gcgacgaaca at 432
<210> SEQ ID NO:2
<211> 498
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:2
atggagcttc tgatcgctca gtcttacagt aaaaatttgg ggctctatgc agaaaggatt 60
ggagcaatta atgttgtttg ttcgacatct gaagttgcaa aaagggtaaa aagccaattg 120
aaaaggattg cacggccaat gtactcaaat cctcccatac atggagctag gattgtcgca 180
aatgttgtgg ggaattcaga tctcttcaat gaatggaaag aggagatgga attgatggct 240
ggaaggataa tgagtgtcag aaagaaacta tatgatagtc tgagtgccaa ggacaacagt 300
gggaaggact ggtcctttat tctcaaacag atcggcatgt tctcatttac aggcttgaac 360
aaagctcaga ctgagaacat gaccagcaag tggcacgtat acatgaccaa ggatggaaga 420
atatccttag ccggattatc ttcagccaag tgtgaatacc ttgctgatgc tattattgat 480
tcattccata acgttagc 498
<210> SEQ ID NO:3
<211> 618
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:3
atgatagctg acataaaggc agctccagaa ggaagttttg tgttacttca tggttgtgcg 60
cacaacccaa ctggcattga tcccactcca gcacaatggg aaagaatcgc tgatgtcatt 120
cagaaaaaga gtcatattcc gttctttgat tttgcctatc agggttttgc atgtgggtgc 180
cttgacaaag atgcattatc gatgagattg tttgctgcac gtggcatgga gcttctgatc 240
gctcagtctt acggtaaaaa tatggggctc tacgcagaaa ggattggagc acttaatgtt 300
gtttgttcgt catctgaaga tgcaaaaagg gtaaaaagcc aattgaaaag gattgcacgg 360
gcaacgtact caagtcctcc agtacatgga gctaggattg tcgcgaatgt tgtggggaat 420
ccagatctct tcaatgaatg gaaagaggag atggaattga tggctggaag gataatgagt 480
gtcagaaaga aactatatga tagtctgatt gccaaggaca acagtgggaa ggactggtcc 540
tttattctca aacacatcgg catgttctca tttacaggct tgacagaagc acagattgag 600
aacatgacca gcaagtgg 618
<210> SEQ ID NO:4
<211> 345
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:4
atgccttatt tttctcaaat ggttttgact aatataagat cctcaagttt caaatgctgg 60
actgcatatg gtccattaat tgtatgtaag gagcatatgc taattatatt actccattct 120
ggttcatatg atctttttgt ccctggcctt tacatctttc aatggctatt tatcctagtt 180
cccagaggca aatttccaca tgttgctttc tggcatgcga cattgttgtc cagcctcaca 240
aaccttgtcc aatcatccga tgcgattagc tattatgcta cattatatct gcagaaagag 300
gttcaatcct atttttttat ttttttttgt aattacggct ttgat 345
<210> SEQ ID NO:5
<211> 636
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:5
atgtacctaa gccaccctgc acaaccttcc atgcntaaaa tgacatgcct gttctccgtc 60
aggctcgccn ccgaccgcga atcgcatcac gtagacgccc accaaccacc gcgtgggtca 120
tgtaaatctt ancccgattc gtttatcgtc tccaacagnc ttcgcgttga gattgttcgc 180
cgcgtcctcc gacngagacg atctggtggt ttccggcgaa ctccgccggn nnnnnnnnnn 240
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 300
nnnnnnnnnn nnnnnnttgt aatcgacgac ctgtttcgtt tccgaggctt tgtttctcaa 360
atactctgga tatgtcngat agcgctttta ccagagctcc agggtccttc acccaaaggc 420
aacagcaatc taaagtcngt caaaaaccac ttatgcgcgt taaagctgaa cgagtgcgct 480
ttctcgnact ccgtcgagan aaatggcggt actcggggca gatacacgcg cttccggcgt 540
acgccgcgnt cgacgtgcac cncagatccc gtactcctcc gccacgtcac acagcgggcc 600
cagcggatcc naccgcggtg gacgacgtcg ttccaa 636
<210> SEQ ID NO:6
<211> 306
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:6
atgcatatat ccacggccct gttcttcgtg ttnatacacg agttttctac cacgccggct 60
gcatattctc aaattcaatg gacattcctt caanttggtg tagcgtctcg acccagtact 120
tttcgaccga tgagaacccg attttggagg tatgnaatca tctttgttct cttcggacat 180
ctgaatttaa tcgctacaaa ccatctcgat gccggntcca atcgtaccta ctcctaccgc 240
tatagcctga gtcaccaata tcaccgtagt cgaccanctt gtaatccgac tcccctgtgc 300
aaaacg 306
<210> SEQ ID NO:7
<211> 2335
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:7
atggccacaa ctgcgaaaaa ggcgacgctt cctgctccga agacggctgc ttgttgcgct 60
cccgcggccg ccggcgattc agccgccgta gtccgccgtg agtctgcatc tgccaccgct 120
gctgcagact ggaaggtttc acccgtcgcc gcggaagatc agcagagcaa gaaagctgct 180
gccgttgcgt ccttaattag accggagcct tcttccaatg ccaccactaa agggatccag 240
atcatgacaa gggcgcaaac gaaacatcct ttggatcctt tatctgctac tgaaatctct 300
gtggctgtgg gaacagttag agcagctgga gccacccctg aggtcagaga tagcatgaga 360
tttattgaag ttgttctgtt ggaaccagag aagaatgtgg tggcactggc agatgcttat 420
ttctttcccc cttttcaacc atcattgttg cttagaacga aaggaggacc ttcaattcct 480
agcaagctcc caccgaggag agccagacta gttgtctaca ataagaagtc caatgagact 540
agtttgtgga ttgttgagtt gacggaagta catgcaacaa cacgaagtgg acatcatcga 600
ggaaaagtca tttcctctac aattgtccct gatgttcagc caccaatgga tgctgcagaa 660
tatgccgaat gtgaagctgt tgtcaaagat taccctccat ttattgaggc aatgaagaag 720
aggggtattg atgatatgga cttagtcatg gttgatcctt ggtgtgttgg ttaccacagt 780
gaggctgatg ctcctagtcg cagacttgca aaaccactta tattttgccg gacggagagt 840
gactgcccac tggaaaatgg ttatgcgcga cccgttgaag gaatttatgt gcttgttgat 900
atgcagaata tggtggtaat tgagtttgaa gatcgtaagc ttgttccttt gccgccagct 960
gatccactga gaaattatac tcctggtgaa acaaggggag gggtagatag aagtgatgtg 1020
aaaccccttc aaattattca gcctgaaggt ccaagctttc gaatcaacgg acattatgtt 1080
gagtggcaga agtggaactt tcgcattggc ttcactccga gggagggttt ggtcatccat 1140
tctgttgcct atgttgatgg cagtcggggt aggagaccta tagcccatag gttgagtttc 1200
gtggagatgg ttgtgcccta tggggatcct aatgaacctc attacaggaa aaatgcattt 1260
gatgctgggg aagatggatt ggggaaaaat gctcattctc ttaagaaggg atgtgattgt 1320
ttgggttata taaaatactt cgatgctcat tttacaaact tcactggagg agttgaaact 1380
attgaaaact gtgtatgctt gcatgaagaa gattatggaa ttctatggaa gcatcaagac 1440
tggagaactg gcctcgctga agttcgaaga tcaaggcgtc tcactgtttc tttcatttgc 1500
actgtggcta attatgaata tggattttac tggcactttt atcaggatgg gaaaattgaa 1560
gcggaagtta aacttactgg aattcttagt ttaggggctc tgcaacctgg agaatataga 1620
aaatatggta caacaattgc accaggacta tatgccccag ttcatcaaca cttttttgtt 1680
gctcgcatgg atatgtcagt cgactgtaaa cctggagaaa tgcacaatca ggttgttgaa 1740
gtgaatgtta gaatcgaaga acctggaaag gacaatgttc acaataatgc attctatgct 1800
gaggaaactt tacttagatc tgaattagaa gccatgcgtg attgtgatcc gttatcagcc 1860
cgtcattgga ttataaggaa cactagaacc gtcaatcgga gtggacaact gacaggctac 1920
aaattggtac ctggttcgaa ttgtttgccg ttggctggtc ccgaggctaa atttctgaga 1980
agagctgcct ttttgaagca taatctatgg gttacacaat atgcacgcgg ggaggatttt 2040
cctggaggag agtttcccaa tcagaatcca cgtgctgggg aagggttggt ttcatgggtg 2100
aaacagaatc gccctctcga agaaaatgac atagttctct ggtatgtttt tggcattaca 2160
catgttcctc gactggaaga ctggcctgtt atgcccgttg aacacatagg gtttgtgctt 2200
cagccgcatg gattcttcaa ttgttctcct gctgttgatg ttccgccgag tacttgcgac 2260
atggatgcaa aagagaatga tgtgaaagag aatggggtcg ccaagccgag ttcgtctggt 2320
ctaatagcaa agctc 2335
<210> SEQ ID NO:8
<211> 381
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:8
atgatgtaca ataatatgta ccggattatg ttcgacagaa ggtttgagag tgaagatgat 60
cctttgtttt tgaagttaaa ggcgttgaat ggagagagga gtcgattggc tcagagcttt 120
gagtataatt atggtgattt tattccaatt ttgaggccct ttttgagagg gtatctcaag 180
atctgcaagg aggtgaagga gaagaggtta cagctgttca aggactattt tgttgatgag 240
agaaagaagc ttgcaagcac aaaggcaacc gacaacgata gcctaaaatg tgctattgat 300
cacattcttg aagcccaaca gaagggagag atcaatgagg acaatgttct gtacattgtt 360
gagaacatca atgttgctgc a 381
<210> SEQ ID NO:9
<211> 723
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:9
atggtcactg ctgatcaaga ttttgctggg gaaatgcaca attcatttct gtgtgttttg 60
cctatgttcc atgtgtttgg tctggcggtg atcatgtatg cgcagctgca gcgtggcaat 120
tccgttgttt cgatgtcgaa attcgatttg gaattgattt tgaagacggt ggagaagtac 180
ggtgtctcac atatgtgggt tgtgccgcca attatactgg gcttggctaa aagtcctgtg 240
gtcaagaagt ataatttatc ctctttgagg cagattggat caggggcggc ccctcttgga 300
agggaattga tgcaggannn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360
nctcgacatt ctggttcgac tggaatgctt gttccaggag tggagagtca gattgttagt 420
gttgagaaac tgaagcctct tcctcctggc cagttggggg aaatatgggt acgagggccc 480
aatatgatgc aaggttattt caacaannnn nnnnnnnnnn nnnnnnnnnt gagggtcagc 540
tttttgttgt tgaccgtatt aaagagctca tcaagtacaa aggttttcag gttgctccag 600
cggagcttga agggctgctt gtgtctcacc ctgagatttc agatgctgtt gttatcccgt 660
ttcctgatgc tgaagctggt gaagtcccgg ctgcttatgt tgtccgctcc cctaatagct 720
cac 723
<210> SEQ ID NO:10
<211> 528
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:10
atgaaaatta ttgatatcga aactggtgcg tctttagggc gtaaccaacc tggagaaatt 60
tgtattagag gtgaccaaat tatgaaaggt tatttgaacg atccagaatc gacagagagc 120
acaatagaca aaaatggatg gttacacacg ggtgatatag gtttcattga tggtgatgat 180
gagctattca tcgttgatcg gttgaaggaa ataataaagt acaaaggctt ccaagttgca 240
cctgctgagc tcgaggccct cctcctcaac cacccttacg tctctgatgc tgcagttgtc 300
tccatgaaag atgagcaagc aggagaagta cccgttgctt ttgttgtgag atcaaataat 360
ggttccacca tcactgagga tgaaatcaag caatttatct ccaagcaggt ggttttctac 420
aagagaataa accgtgtgtt tttcattgat gccattccca agtctccatc gggcaaaata 480
ttgagaaaag atttgagagc aagattagca tctggtgatg ttacaaat 528
<210> SEQ ID NO:11
<211> 300
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:11
atggcgctcc tccagaactg cgctgaattt gttttcattt tcatgggagc ttcgatgatt 60
ggggcgatta ccaccaccgc gaatcctttc tgcactgcca aagaaatttt caagcaattc 120
aatgcctcca aatcaaaatt aatcgtcacg caatcgcagt acgtggataa gctccgcgat 180
acaggggata attccgtggt gtttggcgag gatttctccg tcgtcacgat cgacaatccg 240
ccggatggat gcttgcattt ctcggtgctt tcggaagcaa acgagaagga tgcgccggcg 300
<210> SEQ ID NO:12
<211> 843
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:12
atgcaacacc atgtggccga cggcttctcc ggcctccatt tcatcaacac atggtccgat 60
atggcccgcg ggctcgacat caccgtcccg ccattcatcg accgtaccct cctccgagcg 120
cgcgacccgc ccctgccgca attcaaacac atcgaatacc agcccgcgcc cgccatgaaa 180
accaacacca acgccaacaa aatccccgaa acagccgtct cgatattcaa gctaacgcgc 240
gatcagctca acgccctcaa ggccaagtcg aaggagaacg gcaacacggt cacgtacagc 300
tcgtacgaga tgctagccgg gcacgtctgg cgctcggcct gcatggctcg cgccctgccc 360
gaggaccagg acacgaagct ctacatcgcg actgatggcc ggtccaggct taggccgtcg 420
ctccctcagg ggtactttgg gaacgtgatc tttacggcca cgcctatcgc cgtgtcgggc 480
gatctcgagt cgaagcccgt ttggtacgcg gcgagtaaga tccacgacgc gttggcgcga 540
atggacaacg agtacttgag atcggcgctg gattatttgg agctccagcc cgatctcaag 600
gcgctcgtcc gcggggccca cacgtttcgg tgcccgaacc tcgggatcac gagttgggtc 660
aggcttccga tccatgatgc tgatttcgga tggggccggc ccatttttat ggggccgggt 720
gggattgctt atgaagggtt gagctttgtg ttgccaagcc caacaaatga tgggagctta 780
tcggttgcta tttcattgca aagtgagcat atgaaggttt tcgagaagtt gctttatgac 840
att 843
<210> SEQ ID NO:13
<211> 849
<212> CDNA
<213>Artificial sequence
<400> SEQ ID NO:13
atggccgagc atatcccatg gctgcgttgg atgttcccgc tggacgaaga ggcatttgcc 60
aaacatggag cacgtagaga tcgtcttaca cgggatataa tggaagagca caccattgct 120
cgccaaaaaa gtggaggagc caaacaacac ttttttgatg ctttgctgac actgcaagat 180
aaatatgatc taagtgagga caccataatc ggccttcttt gggacatgat cactgcagga 240
atggacacaa ctgcaatatc cgttgaatgg gccatggcag agttgatcaa gaatccaagg 300
gtccaaaaaa aggcacagga cgagctagac cgtgtaatcg gacacgaacg tgtattgacc 360
gaactcgact tctcaagcct tccctatctg caatgtgtag ccaaagagtc tttgagattg 420
catcctccga cccctctaat gctccctcat cgtgccaacg ccaacgttaa gattggtggg 480
tacgacgtcc ccaagggttc aaacgtgcac gtcaacgtgt gggccatagc acgtgaccct 540
gcggtatgga agaatccctc agaatttagg ccagaaaggt tcctcgagga ggatgtggat 600
atgaagggac atgattatcg acttcttccg tttggtgccg ggagaagagt gtgtccaggt 660
gcacaactag gcatcaattt ggccacatct atgattggcc accttttgca ccacttcgat 720
tgggtgagta cacaagagat tgacatggga gagaatcctg gtttagttac ttacatgagg 780
actccattgg aggcggtacc tactcctaga ttacccgcga atctgtatag gcgtatggcc 840
gtcgacatg 849

Claims (3)

1. verbascoside biosynthesis pathway, it is characterised in that detailed process is：Tuber with 6 different developmental phases of Radix Rehmanniae For material, its total serum IgE is extracted and detected, transcript profile sequencing is combined to which with Illumina HiSeq2500 platforms, obtains 149.8 Million reads, from the beginning assembling obtain 96961 Unigenes sequences；Above-mentioned Unigenes sequences are compared with KEGG data bases Carry out metabolic pathway analysis, obtain 280 KEGG approach, so by metabolic pathway with based on cold labeling precursor hello The verbascoside route of synthesis comparison that test speculates is supported, 5 candidate's KEGG approach are filtered out；The verbascoside for relatively speculating Route of synthesis has the verbascoside biosynthesiss of 19 gene code enzyme catalysiss with 5 candidate's KEGG approach, foundation is filtered out Approach.
2. verbascoside biosynthesis pathway according to claim 1, it is characterised in that comprise the following steps：
Step（1）, with the generation in the verbascoside route of synthesis that speculated based on cold labeling precursor feeding trial Step and product are thanked as reference；
Step（2）, transcript profile sequencing, take 6 different developmental phases mixing roots of a Radix Rehmanniae kind, extract total serum IgE, determining After its quality and purity, reverse transcription builds sequencing library, carries out high-flux sequence with Illumina HiSeq2500 into cDNAs；
Step（3）, sequence analysis and transcript profile assembling, carry software using Illumina and above-mentioned sequencing gained view data passed through Cross Basecalling and be converted into corresponding nucleotide sequence data, remove low quality sequence and uncertain sequence in sequencing procedure, Obtain clear reads sequences, the from the beginning assembling for carrying out transcript profile using composite software, obtain contigs sequences or Unigenes sequences；
Step（4）, transcript functional annotation and classification, by above-mentioned Unigenes sequences and Nr, Swiss-Prott, KEGG and COG Protein Data Bank is compared, and obtains GO, COG functional annotation and classified statistic and EGG metabolic pathways；
Step（5）, verbascoside biosynthesis pathway foundation, by the above-mentioned KEGG approach of comparison and be based on stable isotope The verbascoside biosynthesis pathway that labelled precursor thing feeding trial speculates, finds and includes latter reaction's step and reaction product Synzyme needed for the candidate KEGG approach and its corresponding approach of thing, sets up verbascoside biosynthesis pathway.
3. verbascoside biosynthetic enzyme related gene, it is characterised in that：By the Unigenes sequences described in claim 1 or 2 Row carry out BlastX with data base and compare, then additional ESTscan software functions, and the verbascoside biology for obtaining 13 candidates is closed Into the base sequence of enzyme gene ORFs, the base sequence such as SEQ ID NO.1 of synthase gene ORFs, SEQ ID NO.2, SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO:Shown in 13.
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CN109679991B (en) * 2019-01-18 2020-11-03 马鞍山师范高等专科学校 Transgenic plant with increased phenylethanoid glycoside content and production method thereof
CN111172255A (en) * 2019-12-24 2020-05-19 中国烟草总公司郑州烟草研究院 Screening and identifying method of CRISPR/Cas9 gene editing mutant
CN113337589A (en) * 2021-05-24 2021-09-03 华南理工大学 Method for screening genes related to synthesis of target compound and application
CN113755464A (en) * 2021-08-26 2021-12-07 中国科学院天津工业生物技术研究所 LrUGT2 protein participating in biosynthesis of cinnamic glycoside B and verbascoside as well as encoding gene and application thereof
CN113755464B (en) * 2021-08-26 2023-05-19 中国科学院天津工业生物技术研究所 LrUGT2 protein involved in biosynthesis of cinnamyl leaf glycoside B and acteoside, and encoding gene and application thereof

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