CN109371155B

CN109371155B - Suitable for the fluorescent quantitation reference gene and its primer of clerodendri,folium different development stage and application

Info

Publication number: CN109371155B
Application number: CN201811441231.0A
Authority: CN
Inventors: 华雅洁; 杨秀莲; 施婷婷; 岳远征; 欧阳绮霞; 王良桂
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2018-11-28
Filing date: 2018-11-28
Publication date: 2019-09-17
Anticipated expiration: 2038-11-28
Also published as: CN109371155A

Abstract

The invention discloses the fluorescent quantitation reference gene for being suitable for clerodendri,folium different development stage and its primer and applications, the reference gene is Actin, RAN and SAND gene, wherein, the gene order of Actin gene is as shown in SEQ ID NO.1, the gene order of RAN gene is as shown in SEQ ID NO.6, and the gene order of SAND gene is as shown in SEQ ID NO.8.The application selects according to existing disclosed GeneScreen and expresses relatively stable candidate gene, its stability is assessed by software, obtain the fluorescent quantitation reference gene suitable for clerodendri,folium different development stage, and with devising real-time fluorescence quantitative PCR primer based on these genes, there is no the status of reference gene under the different leaf phases for having filled up Different Provenances clerodendron trichotomum, accurate quantification for the correlation function gene of the different leaf phases of Different Provenances clerodendron trichotomum provides strong support, and the stability, repeatability and reliability of research can be improved.

Description

Suitable for clerodendri,folium different development stage fluorescent quantitation reference gene and its draw Object and application

Technical field

The invention belongs to field of plant molecular biology, and in particular to suitable for the glimmering of clerodendri,folium different development stage Light quantifies reference gene and its primer and application.

Technical background

Clerodendron trichotomum (Clerodendrum trichotomum Thunb.) also known as clerodendron trichotomum, Verenaceae (Verbenaceae) machaka or dungarunga under Clerodendron (Clerodendmm L.) are that a kind of widely used food medicine is same Source plant has the characteristics such as drought resisting, barren-resistant, anti-pollution, saline-alkali tolerant and disease and insect resistance, can be used for afforesting discardedly, is arid lean Thin deserted mountain and Mine Abandoned Land vegetation restoration and reconstruction, be saline land greening fine tree species.Clerodendron trichotomum blade is larger, not only It is edible, it is also used as medicinal material, can be used for rheumatic arthralgia, hemiplegia, high blood pressure, migraine, malaria, dysentery, hemorrhoid Sore, ulcer, sore scabies etc..The clerodendron trichotomum of Yancheng provenance and Tai'an provenance is strong to stress resistance, and the florescence is long, and tree-like grace is value The fine provenance of afforestation must be developed in.And do not had been reported that also now concerning the research of clerodendron trichotomum molecule mechanism, according to grinding Study carefully and show the constant expression under all experiment conditions of none reference gene, common reference gene is in different plants and reality Unstable character can be showed under the conditions of testing, so that the accuracy of target gene detection is influenced, so to develop clerodendri,folium Excellent genes in piece, it must be determined that reliable and stable reference gene.

Summary of the invention

Goal of the invention: being directed to the deficiencies in the prior art, and it is an object of that present invention to provide be suitable for clerodendri,folium The fluorescent quantitation reference gene of different development stage, the gene can satisfy real time fluorescent quantitative and detect the normal transcriptional expression water in Haizhou Flat requirement improves stability, reliability and the efficiency of the research of clerodendron trichotomum gene expression analysis.Another object of the present invention It is to provide a kind of primer special of above-mentioned reference gene.Further object of the present invention is to provide a kind of above-mentioned reference gene or dedicated The application of primer.

Technical solution: in order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention are as follows:

It is Actin, RAN and SAND gene suitable for the fluorescent quantitation reference gene of clerodendri,folium different development stage, Wherein, the gene order of Actin gene is as shown in SEQ ID NO.1, the gene order of RAN gene as shown in SEQ ID NO.6, The gene order of SAND gene is as shown in SEQ ID NO.8.

The primer special of the fluorescent quantitation reference gene suitable for clerodendri,folium different development stage, SAND base The primer sequence of cause is as follows:

5 '-CCACAACTAACGCCTCCACC-3 ' of SAND forward primer

5 '-AAATTCACCCTCCGACTCCC-3 ' of SAND reverse primer.

The primer special of the fluorescent quantitation reference gene suitable for clerodendri,folium different development stage, RAN base The primer sequence of cause is as follows:

5 '-CGATTCTTGCTTCCCTCAGTA-3 ' of RAN forward primer

5 '-ACCATTTTCGCCGCCTTA-3 ' of RAN reverse primer.

The primer special of the fluorescent quantitation reference gene suitable for clerodendri,folium different development stage, Actin The primer sequence of gene is as follows:

5 '-CGATTCTTGCTTCCCTCAGTA-3 ' of Actin forward primer

5 '-ACCATTTTCGCCGCCTTA-3 ' of Actin reverse primer.

The fluorescent quantitation reference gene suitable for clerodendri,folium different development stage it is glimmering in clerodendri,folium Application during light is quantitative.

The application in clerodendri,folium fluorescent quantitation of the primer sequence of the SAND gene.

The application in clerodendri,folium fluorescent quantitation of the primer sequence of the Actin gene.

The application in clone's 18S gene of the primer sequence of the SAND gene.

The application in clone's EF-1A gene of the primer sequence of the Actin gene.

The application in clerodendri,folium fluorescent quantitation of the primer sequence of the RAN gene.

The application is steady to 17 internal references progress genes by these three softwares of GeNorm, NormFinder and BestKeeper Qualitative evaluation, integrated software analysis is as a result, the clerodendri,folium different times for filtering out Different Provenances stablize the internal reference base of expression Cause.Obtaining three altogether to stablize expression reference gene is respectively SAND, RAN and Actin gene.

The utility model has the advantages that compared with the prior art, the advantages of the present invention are as follows: the present invention is surveyed by the transcript profile of clerodendron trichotomum Sequence library, and with reference to common reference gene, the reference gene suitable for clerodendron trichotomum blade developmental stage is had devised, simultaneously These three reference gene sequences are disclosed, and devise real time fluorescent quantitative primer according to this, not only solve existing clerodendron trichotomum There is no the status of reference gene in detection, and designed real time fluorescent quantitative primer is used for clerodendron trichotomum blade growth course Primer specificity is strong when middle gene expression analysis, uses real time fluorescent quantitative to detect clerodendron trichotomum so as to greatly improve Detection efficiency when gene, and improve the confidence level of testing result.

Detailed description of the invention

Fig. 1 is the sample drawing of clerodendron trichotomum blade acquisition；

Fig. 2 is agarose gel electrophoresis detection RNA mass glue figure；Swimming lane M, 1,2,3 be respectively standard specimen, tender leaf, climax leaves, Old leaf；

Fig. 3 is the Ct value result figure of 17 candidate reference genes；

Fig. 4 is that GeNorm determines the reference gene number result figure optimal for accurate quantitative analysis；

Fig. 5 is GeNorm software to 17 candidate reference gene expression stationary value (M) sequences, and stationary value is lower, gene table Show more stabilization result figure；

Fig. 6 is that filtered out reference gene result figure is verified using ClLAC.

Specific embodiment

The present invention is described further combined with specific embodiments below.

Main material to be tested used in following embodiment are as follows: test material is using plantation in Nanjing Forestry University's Baima base The clerodendron trichotomum of Tai'an provenance on ground, Yancheng provenance.Well-grown is randomly choosed respectively in Tai'an provenance, Yancheng provenance, and And height and consistent 3 plant of growing way make sample acquisition, every plant of tree acquires sample in four direction, acquires different development stage Blade (tender leaf, climax leaves and old leaf (Fig. 1)) it is 2 each, centrifuge tube is put into after collection, rapidly use liquid nitrogen flash freezer, -80 DEG C guarantor It deposits, until using.

Embodiment 1

1, the synthesis of plant tissue Total RNAs extraction and cDNA

Total serum IgE is extracted with EASYspin Plus plant RNA extraction kit (Ai Delai Bioisystech Co., Ltd), is used Spectrophotometer (METTLER TOLEDO, Switzeriand) detects RNA concentration and quality, utilizes 1.5% Ago-Gel The total integrality of electrophoresis detection.Using Beijing Quan Shijin reverse transcription reagent box (Quan Shijin Bioisystech Co., Ltd), illustratively Book carries out purifying and synthesis first chain of cDNA of RNA.

2, the selection of reference gene and the design of primer

Go out 17 internal reference bases needed for testing according to reference gene main in literature query other plant and final design Cause, respectively Actin (SEQ ID NO.1), PP2A (SEQ ID NO.2), RPL (SEQ ID NO.3), PK (SEQ ID NO.4)、18S(SEQ ID NO.5)、RAN(SEQ ID NO.6)、APT(SEQ ID NO.7)、SAND(SEQ ID NO.8)、 PROF(SEQ ID NO.9)、MDH(SEQ ID NO.10)、EF-1A(SEQ ID NO.11)、UBC-E2(SEQ ID NO.12)、 AP-2(SEQ ID NO.13)、HSP70(SEQ ID NO.14)、TUA(SEQ ID NO.15)、UBQ(SEQ ID NO.16)、H3 (SEQ ID NO.17).Each candidate gene is found in the clerodendron trichotomum transcript profile database that the applicant has obtained first Conserved sequence is analyzed according to gene conserved sequence 5.0 design primer of Primer Premier, then with Oligo7.Primer Agarose gel and gel imaging system are utilized by regular-PCR preliminary screening primer in Nanjing Jin Sirui company synthesis (table 1) Overall view examines the band of PCR product, detects product specificities.Select stripe size correct, band specificity is good, does not have primer dimerization The primer of body selects peak figure single, without miscellaneous peak, negative control further by the further detection primer specificity of RT-qPCR Primer without peak is as final primer.

1 reference gene of table and its corresponding primer

3, the foundation of reference gene primer standard curve

Respective standard curve is made to the primer of each pair of reference gene, calculates the amplification efficiency of corresponding primer.It will be anti- After the cDNA mixing of transcription, 5 gradients (1,1/5,1/25,1/125,1/625) is diluted to for multiple with 5, as establishing standard The template of curve.Meanwhile with dd H₂O carrys out the reagent or artificial pollution during test experience as negative control template. All samples do 3 repetitions, to ensure the confidence level of experimental data.With Applied Biosystems StepOne (Thermo Fisher Scientific, USA) carries out RT-q PCR, acquires each candidate gene using obtained data result Amplification efficiency and slope, the amplification efficiency of primer selected by fluorescent quantitative PCR efficiency requirements is between 90%-110%.

4, fluorescence real-time quantitative

RT-q is carried out using Applied Biosystems StepOne (Thermo Fisher Scientific, USA) PCR carries out quantitative analysis by template of the first chain of leaf sample, template is diluted 10 times first.It utilizesPremix Ex Taq^TM(TakaRa, Japan) carries out real time fluorescent quantitative, and reaction system is that total volume is 10uL, forward and reverse primer 0.4uL, SYBR5uL, cDNAl uL, correcting fluid 0.2uL, ultrapure water 3uL.Amplification program is 95 DEG C of initial denaturation 30s, 95 DEG C of denaturation 5s, 60 DEG C annealing 30s, 72 DEG C of extensions 45s, 40 recycle, 95 DEG C of 15s in solubility curve stage of reruning, 60 DEG C of 1min, 95 DEG C of 15s' Analysis, each sample sets 3 repetitions, and sets negative control.

5, data processing

Reference gene stability analysis, using software GeNorm, NormFinder, BestKeeper and on-line analysis software The expression of RefFinder (http: // 150.216.56.64/referencegene.php) comprehensive analysis reference gene is stablized Property.Filter out stable reference gene.

6, result

1) RNA extracts quality and primer specificity detection

The OD of each sample₂₆₀/OD₂₈₀、OD₂₆₀/OD₂₃₀Meet the requirements, agarose gel electrophoresis figure detection display 28S and 18S band is clear, and gray scale is more darker than being about the brightness of 2: 1,5S band, and no signs of degradation meets the requirements (Fig. 2).17 pairs of primers Melting curve only has apparent simple spike in fluorescence real-time quantitative, and electrophoresis detection also only has single histogram, illustrates institute With primer can each reference gene of specific amplified corresponding product, primer free dimer, and the weight of each sample to be tested amplification curve Renaturation is good, and template energy specific amplification, real time fluorescent quantitative result is accurately credible.

2) reference gene Ct value is analyzed

The expression quantity of Ct value and gene is in inverse ratio, and Ct value is bigger, and the expression quantity of gene is lower, conversely, Ct value is smaller, is represented The expression quantity of gene is higher.17 reference gene Ct average values are between 20.71-29.46, wherein Actin and HSP70 expression quantity Higher, PR0F, APT and H3 expression quantity are lower (Fig. 3).

3) software is analyzed

The analysis of GeNorm software: GeNorm software is the stability that gene is measured according to average variation degree M value, and software is silent The choice value recognized is 1.5, and the gene higher than 1.5 is not suitable as internal reference use, and M value is lower, and expression gene is more stable.Furthermore GeNorm software determines suitable reference gene number also according to the pairing difference value Vn/Vn+1 of candidate reference gene, works as Vn/ When Vn+1 < 1.5, using n reference gene number.According to software, calculate V3/V4=0.115 < 1.5, thus blade not Three reference genes (Fig. 4) should be selected with developmental stage, are SAND, RAN and RPL (Fig. 5).

The analysis of NormFinder software: NormFinder software is commented according to the stationary value for calculating candidate reference gene Valence, stationary value more high stability is poorer, and having the gene of minimum stationary value is most stable of gene.The value of SAND is 0.288, is Most stable of gene, H3 most unstable (table 2).

2 NormFinder software of table analyzes result

Ranking	1	2	3	4	5	6	7	8	9
										Gene	SAND	RAN	HSP70	RPL	Actin	PP2A	UBC-E2	TUA	MDH
Stationary value	0.288	0.322	0.324	0.43	0.444	0.534	0.575	0.598	0.619
										Ranking	10	11	12	13	14	15	16	17
Gene	AP-2	UBQ	PK	EF-1A	18S	APT	PROF	H3
										Stationary value	0.637	0.641	0.757	0.94	0.944	1.28	1.715	2.294

The analysis of BestKeeper software: BestKeeper software is standard deviation (SD) and adjusting based on reference gene Ct value Factor standard difference SD (± x-fold) judges the expression stability of gene, directly analyzes gene expression Ct value.SD value Smaller, expression is more stable, and program default threshold value is 1, when SD value is greater than 1, it is believed that the gene expression is unstable.Analyze result Show that PROF, APT and H3, SD numerical value are greater than 1, remaining gene SD numerical value is respectively less than 1, and the stability of gene is Actin > UBQ > AP-2 > 18S > PP2A > SAND > HSP70 > RPL > UBC-E2 > RAN > EF-1A > TUA > PK > MDH > PROF > APT > H3, expressing most stable of is Actin, most unstable for H3 (table 3).

3 BestKeeper software of table analyzes result

Ranking	1	2	3	4	5	6	7	8	9
										Gene	Actin	UBQ	AP-2	18S	PP2A	SAND	HSP70	RPL	UBC-E2
SD	0.27	0.34	0.39	0.4	0.51	0.52	0.57	0.61	0.64
										CV	1.24	1.27	1.58	1.53	2.21	1.91	2.74	2.5	2.83
Ranking	10	11	12	13	14	15	16	17
										Gene	RAN	EF-1A	TUA	PK	MDH	PROF	APT	H3
SD	0.65	0.81	0.84	0.87	0.99	1.19	1.33	1.9
										CV	2.37	3.43	3.36	3.61	4.03	4.04	4.62	7.46

4) reference gene stability is verified

The analysis result for comprehensively considering Ct value and Best Keeper, Ge Norm and 3 softwares of Normfinder obtains, SAND, RAN and Actin are suitable for the reference gene (table 4) being applicable in clerodendron trichotomum blade growth course.

4 overall ranking of table

ClLAC (SEQ ID NO.18) is selected to verify screened reference gene, and with the H3 gene of unstable expression It compares, discovery has a similar variation tendency with filtered out reference gene as ClLAC when internal reference, but with H3 base Because illustrating that the reference gene filtered out is accurately and reliably (Fig. 6) come when doing internal reference, ClLAC expression conditions are different.

Sequence table

<110>Nanjing Forestry University

<120>suitable for the fluorescent quantitation reference gene of clerodendri,folium different development stage and its primer and application

<130> 100

<160> 51

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1283

<212> DNA

<213>the Actin gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 1

ggaaagcaat tgcaataaat ccattccatt caaaacacaa tagcagaaca acgatgtaaa 60

aacgttcagc gcagtccaaa atactacagc gttaaggaca cgtctagaaa accataaacg 120

attttttttt ttagaaaagc aaatataaat agtacctagg cataccaacc aaaacaaaag 180

accatataaa ataacttcat gcactcaaaa ctgcaatcga actcacacgg actccaatag 240

aagcatgaat aaatgatacc attttcgccg ccttagaagc actttctgtg aacaatagca 300

ggacctgatt catcgtactc tccttttgca atccacatct gttggaaagt actgagggaa 360

gcaagaatcg accctccaat ccagacacta tacttccttt caggtggggc aaccaccttt 420

atcttcatac tactcggagc aagagccgta atttccttac tcatacgatc agcaataccc 480

gggaacatag ttgaaccacc actgagcaca atgttcccat acagatcctt cctaatatcc 540

acatcacatt tcattataga attgtaagtt gtttcatgaa ttccggcagc ttccattcca 600

atcagtgatg gctggaacaa cacttccgga caacggaacc tctcagcgcc gatggtgatg 660

acctgtccat ccggaagctc gtagctcttc tccaccgcag agctgttctt cgccgtctct 720

gtttcctgct cgaaatcaag tgccacataa gcgagcttct cctttacatc gcggacaatt 780

tcccgctcag cggttgttgt aaacatgtaa cctctttcag tcagaatctt catgaggtaa 840

tcggtgaggt cacgaccagc gagatccaat ctcagaatag cgtggggaag ggcgtaccct 900

tcataaatag ggacagtatg gctcacaccg tcaccagaat caagcactat acctgttgta 960

cgaccactag cataaagaga gaggacagcc tggatggcaa catacatggc cggcacattg 1020

aaggtctcga acatgatttg agtcattttt tctctgttcg ctttagggtt gagaggggcc 1080

tctgtgagga ggacagggtg ctcttcagga gcgacacgca gctcattgta aaatgtatgg 1140

tgccaaatct tctccatatc atcccagttg ctcactattc catgctcaat tgggtacttc 1200

aatgtgagga tacctctttt ggattgggct tcatcaccta cataagcatc tttttggccc 1260

attccaacca tgacacctgt atg 1283

<210> 2

<211> 1437

<212> DNA

<213>the PP2A gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 2

attcccagaa actgtcatgc aagagattgt aaaaatggtg tccataaatt tgtttcgtac 60

actctcgtcg cagcctcgtg aaaacaaagt cttggaagct tttgatgtgg atgaagagga 120

acccttgatg gatcctgcct ggccccattt gcaagttgtg tatgagtttc ttctaaggtt 180

tgtggcttca ccagagacag atgcaaagtt agcaaaacgt tacattgatc actcctttgt 240

tttgaggtta ttagatcttt tcgactctga agatcccaga gaaagggact acctaaagac 300

tgtccttcac cgcatatatg gaaagttcat ggttcatcgt cccttcatca ggaaatcgat 360

caataatata ttctaccact tcatatttga aacagagaag cataatggga tcgctgaact 420

gttagaaatt ctgggtagca ttataaatgg ttttgcattg cctttgaaag aagaacataa 480

acttttcctt gttcgggcac tcattccgct tcacaagcca aagtgcatcc ctatgtacca 540

tcaacaacta tcgtattgca taacacaatt tgtggagaag gactgcaaac ttgctgatac 600

tgtcataaga ggcttactca ggtattggcc gatcacaaac agttcaaagg aggtaatgtt 660

cttgggtgag ctagaggaag tgttggaagc aactcagcct ccagagtttc agcgttgtat 720

ggttcctttg ttccgccaga ttgctcgttg cctgagcagt tcgcattttc aggttgcaga 780

aagggctctc tttttatgga acaacgatca catcgagaac ctgatcaaac aaaaccgtag 840

agtgatccta ccggttatac tccctgcatt ggagaagaac tcaagaaccc attggaacca 900

ggcggtacag agtttgacca tgaatgtccg taaaatcttc tccgatgctg atcctgagct 960

ctttgaggaa tgtttgatca aattccaaga agacgaagca agggaagaag aaaccaaaaa 1020

gaaacgggaa gcaacatgga aacgtctaga agaaatagcc gcaatgaaag cttctagtaa 1080

tgaaccggtc cttgtttctc ccaggacaag caccaagacg ccctccggct aaacatggaa 1140

ggcttcgttt gagtcgcttg aattgtggaa ttatgtattt gcccctactc atgcttctgg 1200

ttttttttgt ccggttgggt cgagcagggt gaaggaaact aatatctttt tgggggttat 1260

gatgaagcgt tgtttttgat tgtaagaaat agtgggtgta catatagaat tgtgggggcc 1320

agatactgat gcggttttag aagtgctgat gttctatggg ggaatatttg ctggcaaatt 1380

tgccctactt tcatctttag ctctttagaa tgatgttaaa attttaagtt atatttt 1437

<210> 3

<211> 1554

<212> DNA

<213>the RPL gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 3

ttaaagcgaa gtctcactag atctgggtcg ttgtctaact gagtgataat cttatataat 60

taaccaaagc ctgcatatag aatagggttt tgagcctctg gaattttact ttgcggcggt 120

cagagtaatt aggcccaaga aactatgtct cacagaaagt ttgagcaccc aagacacgga 180

tctttggggt ttctaccaag aaagagggcc tctcgtcaca gaggaaaagt gaaggccttt 240

cccaaggatg atccaacaaa accttgcagg ttgactgctt ttcttggata caaagcaggg 300

atgacacata ttgttcgtga ggttgaaaaa cctgggtcca aactccacaa gaaggagaca 360

tgtgaagctg taactattgt tgaaacccca ccaatgattg ttgttggtgt tgttgggtat 420

gtcaaaacac cacgaggcct ccgttgcttg aatactgttt gggctcaaca tcttagtgaa 480

gacctcaaga ggaggttcta caaaaactgg tgcaagtcca aaaagaaggc tttctcaaag 540

tattcaaaga agttcgacag tgaagaagga aagaaggata tccaagccca gttggagaaa 600

atgaaaaagt actgcactgt tgttcgtgtt cttgctcata cccagattcg aaagatgaaa 660

gggcttaagc agaagaaagc ccacttgatg gagatccaag tcaatggtgg cgatgtagct 720

aagaaggttg actatgcata cagctttttc gaaaaacagg tccccattga tgctgttttt 780

gggaaagatg agatgatcga cattatcgga gtgaccaagg ggaagggtta cgagggcgtg 840

gtgacccgtt ggggtgtgac tcgactccca cgcaagaccc accgtgggct tcgcaaggtt 900

gcctgtattg gcgcatggca tcctgctagg gtttcattca ctgttgctag ggctggacaa 960

aatggttacc atcacagaac tgagttgaac aagaagatct acaaattggg caaggctggt 1020

caagagtctc actctgccat gacagagttt gataggaccg ataaagatat tacaccaatg 1080

ggagggttcc cacactatgg tgtggtgaag gatgactatc tgttgataaa gggctgctgt 1140

gtgggaccca agaagcgcgt cgtcacattg aggcagtctt tgttgaacca gacgtctagg 1200

gttgcgttgg aggagatcaa actcaagttc attgatacgt cttctaaatt cggccatggc 1260

cgcttccaga ctatggacga gaaagctaag ttttttggca agctcaaggc ttaaaatgac 1320

gtcgattggg tggggatata atggaatcgg tttttatcgt tcattttcct ttataacttt 1380

gttggtttgc cttttttccc ttgtaagctg caggtcttga aatcgtccaa agaactttaa 1440

atagattttg catttgtgtt aaacctgtac cctgcacaat tacaatcatc tacatgttct 1500

catttgcgtt cataagatgc atgtactggt aaatggctat acttaattaa gatg 1554

<210> 4

<211> 1758

<212> DNA

<213>the PK gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 4

agtgataaca gtaatctcct aaagtctcaa tcccatacca caagtaccca ccacttccaa 60

tctctctcct ccaattacac tctctcttcc acacatactc acgcactttt ctgagcgagg 120

cgcccacaca ttgaacacag tgagaatggc gtccacagct gcatcatcag ccttctgcgg 180

catccgcaag gcatcgaccg ccgctgccgc caaggcttct ctcaatgccc ctaccaccta 240

cttcctcccc aaaacccccc tccgccgcct cggattcgcc gccggagccg ccgaaccgat 300

cctatctcac catgtcgcca ctaagatgcg atcatttggg aattccggca aaccaattag 360

aggcgtggcc tccatggcga agaagagcat cggagacctt ggtgctgctg acctcaaggg 420

caagaaagtc tttgttaggg ctgacctcaa tgtgcctctt gatgacaacc agaatattac 480

tgatgatact aggattagag ctgctgtccc gacaattaag catctgatta acaatggtgc 540

taaagtcatt ctttctagtc atttgggacg accgaaaggc gtcacgccaa aatacagtct 600

ttcacctctt gttcccaggc tatctgaact tctgggcatt caggttgtga aggccgatga 660

ctgcattggt ccagaggttg aaaaattggt cgcttcactg cctgaaggtg gtgtactcct 720

gctcgagaat gtaaggtttt ataaagagga agagaagaac gagcctgagt ttgcaaagaa 780

acttgcctcg ttggctgatc tttacgttaa cgatgcattt ggtaccgcac acagagcaca 840

tgcctcgact gagggagtca caaagttctt gaagccctct gttgctggtt tcctattaca 900

aaaggagctt gactatcttg ttggggcagt ttcgaaccca aaaagaccat ttgctgccat 960

tgttggtggt tctaaggtct cctccaagat cggagtaatt gaatcacttc tagagaagtg 1020

tgatattttg ctattgggag gaggaatgat atttacattt tacaaagcac aaggactttc 1080

agtggggtcg tctctagtag aagaagacaa gctaaatcta gcaacatcgc tccttgagaa 1140

ggcaaaggcc aagggggtga gcctcttgct gcccactgat ttggttatag ctgacaagtt 1200

cgctcctgat gctaatagca agattgtggc agcatctgct attccagaag gatggatggg 1260

attggatatt ggcccggatt ctataaagac attcagtgat gcactgggga ctacacaaac 1320

tgttatttgg aatggaccga tgggagtgtt tgaatttgac aagtttgctg ttggcacaga 1380

ggcgattgca aacaagctag cagaacttag tgggaaggga gtgacaacaa ttattggtgg 1440

aggagattca gtagcggcag tggagaaagt tggagttgct aatgtgatga gccacatatc 1500

gactggtggt ggtgccagtt tggagctctt ggaaggcaaa gaactgccag gtgttcttgc 1560

ccttgatgaa gccacgcctg ttgccgtgta gtgtgtaaaa aacaccattc ttctcgatta 1620

ttttaacacc catctctaat aatgtctcat tactagcttg taaacgagaa atttcttggt 1680

atacgtggtc atgctataga tgttttacct tgcctccttt tcttgtcatg ttaatatcac 1740

accatggaat ggatccgt 1758

<210> 5

<211> 1308

<212> DNA

<213>the 18S gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 5

cgaatgctgt taaatttgaa tggtttaaat aaaaacatat taagagagat tacgcaagct 60

taaatctctg gcaacaatca actttcctcg aataattgta aagggtgaag aagaaaaaaa 120

aatcaaaaag caaaggccaa aatttggtat gaatagcaag tagcaacaca agcaaatttg 180

cttgtgtgga cctaacatct tctgtgccac aatttccata attggatcgc ttgcgcgact 240

atagagattc ttattagaac cgagcttttc ttttccgagc agtgtatttt gaatctggtg 300

gcactatatt cccctttctg cgcatttgtt ccttcttacg cagaacccat tctttcccct 360

tcaccttctt gtttagtttt tgccttttcc ttgctctctg ccggtctgat acaaaaactg 420

tctgttcttc atcatcactg ctatcttcat cagaactctc tccatcttca cctttcgcct 480

ttggagtcgc actactcaga gatggtgggc cacaagtgag cacgaggtat tctttcctcg 540

atttcgtact gttaccaaga agaagtatta cagttaacgt gacaacttat cagaactcca 600

gtgcagggaa gttgattcaa acctccaagg aatttacaag aataaaaccc tatgcggata 660

atcaactact acaccaccag aaaacccagc acgcatagca aatcccaaga tcagctcacg 720

ttgagccaaa ttttcaggat atacctgtag cactgcccta gctcctcgtg ctaaacatct 780

gtataatgac ccaaagaagg ccttcagtct caagcgtggc tcatgagaag atttgtcagc 840

attgcataac cactgaacag ctgagatact tattgcgcca tcaataactc cagggcgaag 900

ccctaagccc tgacccatgt caccaagtat aaggtcaccc tcagcttcac gctccaatgc 960

aatatctagc attgatcctg atatatctaa accgatccat tggtgcccat ttccagccaa 1020

tgtctcccca ctaagtccag atccacaacc gatatcgaga agtaatcttg gaacgccatc 1080

atcaggcaaa gcgagaagct ccaatgccct ctcggaaagt ttagcctgaa tttcgatgat 1140

acgagaagaa gaggtgtact tacgagcttc ctcgtcgtta taaaatatct caggcggtgc 1200

ttgtagctct ggtcgagagg ccattgtttc tttccttgtt cttcgaaaca gtactcagag 1260

ggtggctgaa ggctgctttg ttcggtgatt gtccggtttt gtctgatg 1308

<210> 6

<211> 2008

<212> DNA

<213>the RAN gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 6

ccccaacatg tgggacccat aaattagaat atgcatacga agtaacagtg aattaccaaa 60

agtaccctca gctgatacat aatgcaaaat aatcaaaagt agagagatca agggcaaaaa 120

tgtcacataa aatcaaacac agcacacttt tggcaagcag ttggcttcag gcttaaacag 180

cgccatctcc tgctcaaatc tagcagaccc accaaagaca tcaaactaaa acctaatgat 240

ctaaatgaca aaactatctt aatctttgtc ttcttcattg acatcaagat tcttgagctt 300

cttttccaat tcatcctgac taccttcgtc ttcatccccc ttgtcgtcaa aatcgtttcc 360

ttcagggtcg ttctcatcca acggcccaag tttctcagga caatctttaa agattatcct 420

caactcatcg agatcttcct cagaaatgaa gttcccgttt acatccaata gcttgaaacc 480

aggcaaacga accaaagcca gagccaactt cttaaccgag gtagttagtg agttctggct 540

catatcaact tccctcaatt gctcttgccc atcttcaaat gctttgctta tctgaatcgc 600

accttcatcc ttaaggtcat tctctgagag gttcaacttg acaatagttt tctttctaga 660

gatgcaggcc gccaagctag gagcagcttc agcagttata tcatttccag ccatctccaa 720

tactgcaagt gatggtgctg actccttgag agcgtcggct attgctattg ctccatcatc 780

ttcaagattc gagtagctta aataaatctc agttagattc ccatgtttgg agagagcgtc 840

gcttagcttc aaaccagcct ccactccaaa catattgtcc cgaagatcga ttttcttcag 900

attcttacat aattccaggg cttcagtcaa cgcaacgcct ccttcagaac caatacgggt 960

agatgagcat cggaaatcct ccaataaagg gcaacgtttc agtatctcgg aaatggcaat 1020

agcgccttca tctcctgtca tattgttatg aaaatggagg attctaagcg aatctgtaga 1080

aggaaccaac tcacaaacag cttgtgcggc ttccttcgat atcccatcat tcatcagata 1140

aagttcctct aagctagtct gagatttcaa gagcttcccg aatgctctga ctcccttctc 1200

acctaaggca ttatcggaga tattgagata tttcaaatga gaaccttcta gagcttcaga 1260

gaagatcttc atgacatcaa gagcttctgc ttctggtcgc cccgcaacaa aatcagacag 1320

atcgacttcc ttcagttggt tcttgattga cgccaagata ggtccagcaa cattggctgc 1380

cccaagacca aagcttcgat tgctaaagca aatcttggtg taagaattcc caggttcttt 1440

caaggggctt aaaagtgttt gagcctcatc tgcttcaata aatgctcgct gacccttgga 1500

aatatcgaaa aatgtctcct gcaaaaccga attgacctca gatttcagta cctgcttttc 1560

ctctgtccca gagcctccct tgagaacatc cagtattagc ttgctgcatt ccttggcata 1620

ccattgtaca gcagaactgc catcaccatc gggttcttgt tcataatgct gatttgccga 1680

agaaaatgct gattcttcaa tttgtttggc atgttttgaa gcctcagctt gactaagagt 1740

gccatacttg cgggtgaaaa ctgttggcgt cgagagattg tttgtcattc gctctacaag 1800

catcaacctc gtgttctggc tagggggcca cagtttaatg gttatgtgcc ggcgttctcc 1860

atttggtgtt ttagcatcca tcgcacccaa actgtcgtct ccttcctgac gaatttttca 1920

atttgcagct cttcggactt cgactgtgta ctgcctgtgt atgtataggc aagaggggag 1980

aagtgaagga aaactaaaat gaaatgag 2008

<210> 7

<211> 1149

<212> DNA

<213>the APT gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 7

aaaatctaga gatttaggca aatccattag aaaacaagca tcctgtcttc aattgattct 60

aaaaatccca ctaatgtacg ctgtatctgt atctaagcca tctctctccc tatcaagtga 120

tgtcattgat taaacaaatc ttgagtttta ggcaaatcca ttagaaaaca agcaccctca 180

tggacttcaa aaatcccact aatgtatgct gcatctgtat ctaagctatc cctcaaactc 240

ctcaaggcta gagttgtctc accaaagtac ttgcgcagtg ttaatttctg cttacctcac 300

accgtagcga acactccgct gacctacgtc ggatcaattg gtggcgcagc cgtaccgaaa 360

cgtcggccgg tgttcgtttc aaacatggca tcctcaaatg ctgaaagcat tgatgagcgc 420

atccggcgga ttgcctccac gattcgggtc atacccgact ttccaaaacc tgggatcatg 480

tttcaagata taacgacgtt gttgcttgat cctaaggcgt ttaaggatac gattgatttg 540

tttgttgaga gatataaatg ccaaaacatc aatgttattg ctggtgttga ggcaaggggt 600

tttatattcg gtcctcctat tgcattggct attggtgcaa aatttgttcc tatgaggaag 660

cccaaaaagt tacctgggga agtaatatca gaagagtact ctttggagta tggaactgac 720

ataatggaga tgcatgttgg agccgtacaa cctggggaac gtgcagttgt tgtagatgat 780

cttatcgcaa cagggggtac cttgaatgct gctattcgcc tacttgaacg cgttggagtc 840

catgtggttg agtgtgcttg tgttattgaa ctggagggac tgaagggtcg ggacaagtta 900

ggggagaatc cactgtttgt tcttgtgagc tcaccttgaa gaaaagagta tctacaacaa 960

caataaattg ctgttaacat ctgtatgagt tgatagcatg tgcttgttcc tttctatggg 1020

atatacacat ttcaaatgga tgatggtgtg ttgtatgttg agtgaaattg cttttctttt 1080

ttttttggat gagtcatgtt caggccaccc tctcctgctt gcggattgtt actcaacctg 1140

tcaaataag 1149

<210> 8

<211> 2473

<212> DNA

<213>the SAND gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 8

gtaatttgtt tctgtctccg ccatccgtga ctggcagatg cgagtgcgac accatcactg 60

aacttcaaat gttttccttt aaactctaat ttcttagcga ttatccaatc caattctcgt 120

tcgggattgt agattgattg acaatgtctt ccaattcaaa tatttcatct gcgagttcct 180

ctactaatgg agatgacggg aaccctaatt cgtatacaaa aaaggagccg gagaatctgc 240

cggaacccgc actcaacacg agtccggact tgatcgacca gtcgcttgat gcccttgaga 300

atcaattggc ctcaatatca atgtcctccc gttccccgac ggcgactctt gacgatgaag 360

agttcgtcga agaagaagag cagtacctgg agcatccaac aatcataaat ggatcgttga 420

gtgaagaaaa tggagccgga ggatcaagtg atgagcatat tgagtgttat ttgaagggag 480

agtcttcggg agtgataaaa gagaccttgt cctctgtgtg gaggaattat ttagaggcag 540

atgaagttga gcggccgttg agtccgacca gcagcggcta cgctggtgag atggggagca 600

ccagcggcgg tgatgaaatc aatgatgatg acgaagatat taatgaaatt aaagaggtcg 660

aaaacgactt tgtttcgggg tcccatgatc agtgggtttc aggaaaacgc cacccagatg 720

aggatgatgc ttccgtttct tggaggaaaa ggaagaaaca tttctttatc ctgagtcact 780

ctgggaaacc catatattcg agatatggag atgaacacaa gctagctgga ttctctgcaa 840

ctttgcaagc cattatttcc tttgttgaaa atgggggaga ccgggtcaaa ttggttagag 900

ctggtaaaca ccaggtgatt tttcttgtga aaggacccat atacttggtt tgcataagtt 960

gcacggaaga accttatgaa tcactaaaag accagatgga gctactctac ggtcagatga 1020

ttctcatctt gacaaagtct gtaaatcgtt gttttgagaa gaatccaaag tttgatatga 1080

catcattgct ggggggaact gatgctgtct tctcttctct catccattct ttcagttgga 1140

atcctgccac ttttctacat gcctactcat gtcttcccct tgcttatcca acacgtcaag 1200

ccgcaggtgc catcttgcaa gatgtagctg gttcgggagt cctatttgcg cttttgatgt 1260

gtaaacacaa ggttatcagt cttgttggcg cccaaaaagc atctcttcat cctgatgata 1320

tattattact gtcaaatttg ataacgtcct ctgaatcttt taggacatct gaatccttct 1380

caccggtttg cctgccaaga tacaatccca tggcatttct ttatgcttat gtgcattact 1440

ttgatattga tacatacctg atcttgctca ccacaagttc cgatgccttc tatcatttaa 1500

aagattgcag gattcgaatt gaaaacgtac ttttgcagtc aaatgtactg agtgaagttc 1560

aaagatcctt ggtggatggc ggtatgcata tagaggattt gcttgctgac cccacatctc 1620

gtcctggatc cgtttcttct catttaggtc aaccagggcc tggtacaggt tccccggggc 1680

gacttacaga tggatttgtt ggaattggtg gtccagctgg gctatggcat ttcatataca 1740

gaagtattta tcttgaccag tatgtgtcat ctgaattttt atcaccaatc agcagcttga 1800

gacagcaaaa aagattgtat agagcgtacc aaggacttta tgcatccatg catgacaaag 1860

gaattggacc tcacaaaact cagtttagaa gagatgaaaa ttatggtgac atttactaaa 1920

gcaaccaaat tcttcaatta tatgcaagac tggaactctg gaagatagtt tacatcgaga 1980

ttctatgggg tagagccata aattgaagcc atgctactct gctgggtcac acaagatttt 2040

gaactatatg cagcatttga tccactagca gacaaggcac ttgctataaa ggtgtgcaac 2100

cgggtttgtc aatgggtgag agacgttgaa aatgaaatat ttttattggg agtaagccct 2160

ttttcgtggt gattatctca agtattctgt acactggcat gtattctagt ctttccgttc 2220

gactattact taactaccgc ttcctatatg aaattttgcc ttatttttgt tgtacggcct 2280

ctatgttggt ttgcagttcg gatctctatc ctattttttc ttcaccactt gtatctaact 2340

ataaaaacga ataaattgaa gttctatagt gagcttaatt gtagttgttg taattggatg 2400

tttataaaaa ggtaacgttt taacagtgag acataagtgg tatatgggga agtatgattt 2460

tagaggatac cga 2473

<210> 9

<211> 1456

<212> DNA

<213>the PROF gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 9

actaacttga taccaatacg cttgcgatta attgttatct ccgccgagag agaagctctc 60

gcatctttct aagaaagaaa ttttcacttt agatctattt gttttcactc tccatcctca 120

cctccctatc tttccgacgt tgaagtctct tcctcacttc ttgatcctat actaactcca 180

gtttctacat ttgaaatgaa gatggaagat ctttttaggt ctagacgcaa ccagagttgt 240

gtgacagcag cagcggcaag attagggcga ctgcagaagg tggatgtagt tccaccatgg 300

cagtgaccct tgatgtgtgt atttccaaat tttctttagt gtagttttac gttgttttca 360

tgttcatcct cgatctggac tggttctcaa ggtgctagtt ttagggtgct ttatactagc 420

tagtcaagtt gggacagtga cctgaatctc tctctaccaa tgtccttgaa tagggttttg 480

gtccggtcaa gctttcttta ttttgtctcc tttagtacga gtggtttttc ttccatcttt 540

gggattgacc gttcattttg tacatttagt cagttggcta tgtattccac atgtatgtga 600

agttttgggg gaagaagata actaagggga aaaaatttca aagccaattc tgtatttgta 660

aagatgtcca taaaatttga acatctatca gagaaccaac caaaaataca taatttactc 720

acatggaatc ataaacccaa gttttcacaa cgagaaaatc aaaagacaag ccctgtttca 780

attcagatag ggacataatc taacacctct cactcaaaac accacattct ttttgccata 840

accaacaacc cacgctgcaa atcattgaca aaaaaagcag taaaggatgc cgaatttaat 900

aatattaccc aaaaaattat taatttataa ttatataatg aatattgatt attaaatgaa 960

atgatttgga agacttacca atcaatttca gacaccctgt tcaataagat agtcaccaat 1020

cttctcaaca accatgttgc actggcctgc agtcattggt tcatcataga taccgatgat 1080

caaggccaaa ttggtctttt tgacagtggc accaccagat cccttcttcc ctcggatgac 1140

agcatttggt tcaccttgaa tcaccatgta tttagtaccc ccaatgtaca atccagtagg 1200

agctaatgat cccggattat caaaatcatt cataatagct gttacttcct caggcttgaa 1260

ctgagggaag ttggaactct gtgcccaaac gctgccgtca tgaccgacaa ttgcggcgga 1320

ggtgagatgg caaccgtcga cgtcggccat caagtggtca tctatgtagg attgccacga 1380

catcgtttcg ataggcggaa ggaatcaata ccaggaaggt ttggagaatg ggtgaggact 1440

gatgatagcg tgaaaa 1456

<210> 10

<211> 1516

<212> DNA

<213>the MDH gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 10

tttataagaa ttggtaacat ccattggtat atgatattta tctgtcagca aacgggttca 60

ttacatatct agaattgcaa taaccagaac aactcacgta gcataaggta caacatacac 120

taccgcttat tccaagatgt gtgatcttga atctccaaga ttatttcctt acgaaggaga 180

cccccttatc aatgcttcca tccaactctt tctttgcctt ctccagccca accctttcgt 240

attcatttag tggaccaaga gggaagatct cctcgacccc gttacgtcca agccgtaccc 300

ttgaagcaaa gaatggaagt tccgttacct gggaagaaac aaatgcacat tcaacaacac 360

cggaatctcc cctcaagccc ttcaggcaag catcggcaaa tttcactgct gcatatgcca 420

tggaaagagt tgcagagcct gtacccgctt tggcctcaac aacttcagtc ccaccattct 480

ggatgcgaga tgtcagatac tcagtttctt ctttggtgaa agagcatgca ggtgtaatct 540

gcgagaggag aggtaaaatt gtcacaccag catggcctcc tacaactgga acattaactt 600

cccgaggatc tagccccaaa acttcagcca caaacgtatt agctctgact acatcaagca 660

tcgtgactcc aaggagtcgc cttggatcgt aggtcccagc cttcttgaaa acctctgcag 720

caattggaac tgtggagttc actggattac ttattaagtt aacaatggcc ttagggcagc 780

acttagcaac tccttcacaa agtgtcttta ctattcctgc attaatgttg aaaagatcat 840

ccctcgtcat tccaggtttc ctgggaacac cagcaggaat gattacgagg tccatgccag 900

ttagtgcatc cttcaactgg ttttgaccca gaaaaccacg taccacagca ccagtgttca 960

tatggctgat atctgctgta accccaggag tattaactac atcataaaga tgaagcaccg 1020

aaaccagtgg gttcatcttc atcagcattg ctaaaggctg gcctatgccg ccagctgccc 1080

ccaataccgc caccttgaaa cctggtgacc ctccttttgc tcggcaatcg gaaagccata 1140

aactcccctc cacctgcagc tgattagtag gaggatcgag gtgagctgaa attctggaaa 1200

ttcttcgatg aatatcccca ccctgctgca tactataact cgaacgaatt cttctccaat 1260

atcagaaagc agcaaattcc ttttggaata atgagaaatg aaatgatgga agataacttg 1320

aacggcagaa tagtggagat acccgtattt atacggtaga aaattggaat taattgtatt 1380

ttatggcaca gcacataata aaataaataa atacgtcaga ggctcataga aatgcgttaa 1440

tgtatttttg ggtgcatagc cttatcttat acgtacacgc ttcggcaaga aaattacaaa 1500

ttatcaccat aattca 1516

<210> 11

<211> 1351

<212> DNA

<213>the EF-1A gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 11

gaaattcgag accaccaaat attactgcac agtcattgat gcacctgggc atcgtgactt 60

catcaagaac atgattactg gaacctcaca ggctgactgt gccgtcctca tcattgactc 120

cacaacaggt ggttttgaag ctggtatttc caaggatgga cagacccgtg agcatgctct 180

gctggctttc actcttggtg tcaagcaaat gatctgttgt tgtaacaaga tggatgccac 240

tacaccaaaa tactccaaag caaggtatga tgaaattgtg aaggaagtgt cctcttacct 300

caagaaggtt ggatacaacc cggacaagat accatttgtc cccatttctg gttttgaggg 360

agataacatg attgagaggt ctaccaacct agattggtac aagggcccca cgctactcga 420

tgcacttgac cagattaacg agcccaagag gccaacagac aagccactcc gtctcccact 480

tcaggacgtt tacaagattg gtggcattgg tactgttcca gtaggccgtg tggagactgg 540

tgttctaaag cctggtatgg tggtcacctt tggcccgtct ggtctcacca ctgaagttaa 600

atcagttgag atgcaccacg aagccttgca ggaggctctt ccgggtgaca atgtcgggtt 660

caacgtcaag aatgttgctg tcaaggatct aaagcgtggc tttgtggcct ccaactcaaa 720

ggacgaccct gctaaggaag ctgcgaactt cacctctcag gtcatcatca tgaaccaccc 780

tggccagatc ggaagtggct atgcacctgt tctcgactgc cacacctccc acatcgctgt 840

taagtttgct gagttagtca ccaagattga taggcgttcg ggtaaggaga ttgagaagga 900

gccaaaattc ttgaaaaatg gtgatgctgg attcgtgaag atggttccga ctaagcctat 960

ggttgtcgag actttctctg agtacccacc tctgggtcgt tttgctgtac gagacatgcg 1020

acagactgtt gctgttggcg ttattaagag cgtggagaag aaggacccga gtggtgctaa 1080

ggtgaccaaa gctgctgcta agaagggtgc taagtgaacg ctggtgactc cgttttatca 1140

ataaattttg tttttataat gcagtttatg attaatgatt attgctttta ctgctgtatc 1200

tctagcctct tgtaagagga tattggagag tggagcaact tggttttgat tcttgttgct 1260

tcatgtgcag tgatgaaagc agctctgttt ggatattgtc aaatttttat tttccttgtg 1320

gattttgttt tcttgaaatt gttacttagt g 1351

<210> 12

<211> 1214

<212> DNA

<213>the UBC-E2 gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 12

aaattgtttt gcaaaaacct cgattttcat agtactcgtt cgtcggtgtg cgatctgagg 60

agatggcgga agataagtat aacaggaaga accctgctgt gaagaggatt ttgcaggagg 120

tgaaagagat gcaatccaat ccttccgatg atttcatgag ccttcctctc gaggagaaca 180

tatttgaatg gcagttcgca ataagagggc caagagactc ggagttcgag ggaggaatat 240

atcatggaag aattcagttg cctgctgaat acccctttaa gccaccttca tttatgttat 300

tgacgccaaa tggtagattt gaaacccaaa ccaagatatg cttgagcatt tcaaatcatc 360

accctgaaca ctggcaacca tcctggagtg ttcgaactgc gctggttgca ttgattgcat 420

ttatgcctac tagcccaaat ggtgcgctag gatctttgga ttacacaaaa gaagagaggc 480

gagctcttgc agtaaaatct cgcgaagcag ctccaaaatt tggatcacct gaaaggcaaa 540

ggctgattga tgagattcat gagtatatgc tcagcaaagc gctgcctgtg cctcaaggca 600

acctcacctc tgaagcacac actgggagtg aaaaaatcca gcagaccacc caagacaatg 660

ttgaggaagt tacagaagca cctccccaca cagctgaaga cgacgggatc atacaagaac 720

caagtgaagc agtagcacct tctactaata caaatgttgc tcagacatcg cagcctgttc 780

ctcccgggcc gacgacgagc cagcagcagc tgttgcataa gcccgaacca agggttccta 840

aaccagcaga tgatcgtctc ttcacatggg ctgctttcgg gctcaccata gctatagtag 900

ttcttctgtt gaagaagttc ttgaaggcta atgggcacgg tgctgtcttt atgaatgagt 960

cgtaagtggg acctggtatg ttgtttatgc tgatgcagaa ggtgaatgag gggaaaacct 1020

gctttctgta atggagtgcc ttaaatttta aggggaacat atctatatat atattatacg 1080

tatacttatc tttctactta tggagtatat tgtatagttt aaataattgt taggggtaat 1140

gttctgaagc tttttaagaa gctgaactat tttaatagaa gcagttctgt aattattgct 1200

tgcatttgtg agag 1214

<210> 13

<211> 2673

<212> DNA

<213>the AP-2 gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 13

ttaatcaccg atgtcttcag aaccaatacg caactaattg acaaacagat gctgtaattg 60

ttacaaaggg tgcagttttc attatgtcac ttgcattaca gaaaccaatg aagaagggtg 120

tatttggcaa gcgctaaata aagaggaaaa aaaatctcac caaggcacaa tctgattgtg 180

cgaaagaatt atcaaaactt caaatccaaa ttctaacact atgccgatta catacaaaat 240

tacaaattca ccctccgact cccccagctc ttgcaccata ttagcgggga tccctccagt 300

acatacgagc aggtgctata tgaacattgg ttaaacaatt ttgaacagcc ttgggtttgt 360

tttatcactc aaagcaaacc agcaagaata gctccaggat ctgatgtaga tgtaggtgga 420

ggcgttagtt gtggttgtgg ttgaggttga ggttgaggtt gaggttgagg tggcgtaggt 480

gcaggtgctc gagaagggat gggaatggtg attaattgtt ctttaatgaa ttctttcaat 540

tcgtatgtta gttttggatc tccagatgcg atggtcatcc gtaattgcgt tctatctgct 600

gggtctgtct cgattcttat caaacataac atggctcgtg tactttcaga gtagaaagtt 660

gtgctcgcaa ccaaattatt agcatttgga tcaagcccag ggcaaaccat caggtgcaaa 720

ctgctaaata agtttgccat ctctgctagc agcattggtc ttacacctct aaccacttct 780

tgcagtttca atggaggtcc tgatagtgat ctccattgtg ggaaaaattc ttcagcagaa 840

actgtaattg actgaagaaa tttgttcaaa acagcaggaa ggcgaagttt tacattgacc 900

aaatgggttc caaacttgta cgagaagtcc agtactgcta aatctctgct cggttggagg 960

atgattactt caagtgggca ttgaacctgt gcccttggag gaatagtctc aggtactaaa 1020

gatagttcca tctttagatg tgatggaggc aatattaaag cttgaactga aacgagtgga 1080

gcagtatttt tatttcccaa gaagagaaca actcgcccat gatgggttcg ccattcagct 1140

tttatgccaa tctggatata ggcatcttca tatagcacac cactatcctt caggcacaaa 1200

acacgaaatc tttccactat atcaccaatc ggctggaccg tattaacttg ctcttcaaca 1260

ggtgctatgg ctaatgcatc tgtttccaaa ccggaagcaa cccttggatc agtttgggga 1320

gcaatgcctg gtgggccctc aatagccagt ggccctaaaa gatctccaag gagatcagga 1380

gaaggtgcac tgtgtggttg aggatccact acactcaggg caccattagc acgagtcaac 1440

ccttgctcag cagtgctctg atccgcatta ctcatactag gcaccttgac aagaccaagc 1500

tgggtcccag atggtgttcc atttgcaggg cgttgatgat cggtaagtac caaagcatta 1560

gaattctgct gctgtgcccg taattttatt gcactttgct cagcagtatc agcttcagca 1620

tcttccgctt tttttatcaa tgatgactgt ctctcaggga atttcggcat ttcagcaagt 1680

atatccacta gagcagcacc tttcatgctc agagcaagat actcaacagc tcgttgctgt 1740

atttcagcat caatgcaact ttcatacttt ttgaatatca cccatatttg cttctgtagc 1800

tcctggctgg gtggttgagt gtgcatcaaa atttttgcat atgtggaaag aaggatagga 1860

attgttgatg tcgagacagt gggaagcttc tcatgtatca agttgaaaat ttcctttgga 1920

ctgcacccag gtcgcctagc tagaagatga ctgtattctc caaggatata tgcactcacc 1980

ttcaccatgg tctcgtgaat cgcaggctta tcaagatact ctttggcttt cagagctgca 2040

taaggctgta aatcctcgtt gtttgtaacg aactgcacga ctcgaaacca aatgtcatca 2100

ccgacaaaat cccctgcctt gtcaattagc tgaagaatca catccacata ccatgacagg 2160

tcgggggcaa acttctctgc gagaatagca gctttaagtg acaattcttc ccgcatagca 2220

aagtcagctg agctcagata ctgtaataat tcttcaacta tgtcctttgc atttgaaaca 2280

tcacacattc catatagcaa atcaagggca cgccttctga tactgatatc aggatccttc 2340

agggaagtaa tgatctgagc ttgatgtctt ttgataattt catgcacatc ggcgaccatc 2400

aacattcgag tcatgttctc caagccaagg tatcgtatat taggttcacg aacagcaata 2460

aattttccca gcaaagcaac acactgagac atcatttctt tctctgcatc aagatgcatg 2520

acaagagcaa gagcttcaaa aagaacagca tgtgatgcat tattcttgtt cacatttttc 2580

accacatcag ttcccatgag tatccgttgc agaacctcaa acaatgatct tcttgtattt 2640

ggatcctcaa ctgttggaaa atactggaga gcc 2673

<210> 14

<211> 2071

<212> DNA

<213>the HSP70 gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 14

tgttagtttt atttcattat tgttctttga cttgagcaaa attctctggt cgatttagat 60

ctgcgattag ttttgcgagc aatggccgga aaaggagaag gtccagcgat cgggattgat 120

ctcggaacga cgtactcttg tgttggcgtt tggcaacacg accgtgttga aatcatcgcg 180

aatgaccagg gtaacagaac gacgccgtct tatgttggct tcaccgattc agagaggctt 240

attggtgatg ccgctaaaaa tcaggtcgcc atgaacccga cgaacactgt ttttgatgcc 300

aagaggttga ttggaaggcg atttagcgat gcttctgttc aaagcgacat caaactatgg 360

cccttcaagg tcattgccgg acctggtgag aaacccatga ttgttgtcaa ctataagggt 420

gaagacaagc agtttgcagc tgaggagatt tcatctatgg tcctgatgaa aatgaaggaa 480

atagcagagg cctaccttgg ttccacaata aagaatgctg tcgtcaccgt tcctgcatac 540

ttcaacgact cccagcgtca agccaccaaa gacgctggcg taatttcagg tctcaatgtt 600

atgcgtatca tcaacgaacc tacagctgcc gccattgctt atggtcttga caagaaggcc 660

tcgagtgtag gtgagaagaa tgtcttgatt tttgatcttg gtgggggcac ttttgacgtc 720

tctctgttga cgatcgagga gggtattttc gaagtgaagg ccactgctgg cgacacccat 780

cttggaggtg aggactttga caacagaatg gtgaaccact ttgttcagga attcaagagg 840

aagagcaaga aggatatcac cggtaacgct agagctctac gaaggctgag aactgcttgt 900

gagagagcaa agagaaccct ttcatccaca gcccaaacaa caatcgagat agactctcta 960

tacgaaggaa tcgatttcta ctccaccatc acccgtgcaa ggttcgagga gcttaacatg 1020

gatctgttcc gcaagtgtat ggaaccagtt gagaagtgcc tgcgcgatgc caagatggac 1080

aagagcacgg tgcatgacgt tgtgctcgtc ggtgggtcca cccgtatccc caaggtgcaa 1140

cagctgcttc aggacttctt caacgggaag gagctttgca agagcatcaa cccggatgaa 1200

gccgtggcct acggcgccgc cgtccaagcc gccattttga gtggtgaggg taacgagaag 1260

gttcaagacc tattgcttct cgatgtcact cccctctccc ttggtttgga aactgccgga 1320

ggtgtaatga ctgttttgat tcccaggaac accaccattc ctactaagaa agagcaggtg 1380

ttctctacct actcggacaa ccaacccggc gtcttgatcc aggtctacga gggtgagcgt 1440

acgaggacga gggacaacaa cctgctcggg aagtttgagc tctccggcat tccccccgcc 1500

cctcgtgggg tcccgcagat cactgtgtgc ttcgacatcg atgctaacgg cattctaaac 1560

gtgtcggccg aggacaagac gacgggccaa aagagcaaga tcacaattac aaacgacaag 1620

gggaggctgt caaaagacga gatagagaag atggtacagg aggcggagaa gtacaagtcg 1680

gaggacgagg agcataagaa gaaggtggaa gcgaagaatg cgctggagaa ctatgcgtac 1740

aacatgagga acacggttaa ggatgaaaag atcgggtcga agctgcccgg tgctgataag 1800

aagaagattg aggatgcggt tgagtcgacg attcaatggc tcgatgggaa ccagctcgcg 1860

gagtcggatg agtttgagga taagatgaag gagttggagg gcatttgtaa cccgattatt 1920

gccaagatgt atcagggtgc ggggggtgcg gcggctatgg atgacgatga cggtctggcg 1980

gcggcgcctg gtggtgctgg tcctaagatt gaagaggttg actaagttgt ggcccctttt 2040

ttggtggttt tttttttttt tttttttttt g 2071

<210> 15

<211> 1802

<212> DNA

<213>the TUA gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 15

atctgagccc tagagagaga aagtgaaatt acaccttcat tttctaccaa ctgctcttat 60

aaaatcccct ttcaccacga attaagaact acctattttc atccagagac gtcttcgtat 120

acgccctggt atttctctcc tttgatctcc gatcgaaatc catttcagat tttaatcttt 180

caaacaaaaa gcgaaaatga gagagtgcat ttcgatccac attggacaag ccggaatcca 240

ggtcggcaat gcctgttggg agctgtactg ccttgaacat ggcatccagc ctgatggaca 300

aatgcctggt gatcacactg tcggaggagg tgatgatgcc ttcaacacat tcttcagcga 360

aactggcgcc gggaagcatg ttccccgtgc agtgttcgtc gatctggagc ctactgtgat 420

tgatgaggtg cgcactggta cataccgtca gctattccac cctgaacaac taatcagcgg 480

caaagaagac gctgccaaca actttgccag aggtcattac actattggca aagaaatcgt 540

ggatctgtgc cttgatagga tccgcaaact cgctgacaac tgtactgggt tgcaaggctt 600

cctcgttttc catgctgtgg gtggtggtac cggatctggc ctcggatctc tgttgctcga 660

gaggctctcc gtcgattatg gtaaaaagtc gaagctgggt ttcactatct acccttcccc 720

tcaagtctca actgccgttg ttgagcctta taactctgtg ctttcaactc attcccttct 780

tgaacacact gatgttgctg ttcttcttga taatgaggcc atctatgata tttgccgcaa 840

atctctggac atcgagaggc ctacatacac caatttgaac aggctcattt ctcaggtgat 900

ttcctcctta actgcatccc tgagatttga cggagcattg aacgtcgatg taaacgaatt 960

ccaaaccaac ctcgtcccat acccaagaat ccacttcatg ctttcatcat acgcccccgt 1020

aatctcagct gagaaagcct accacgagca gctctccgtc gctgagatca ccaacaccgc 1080

ctttgagcca tcttccatga tggtgaagtg cgatccgcgc cacggcaagt acatggcgtg 1140

ctgcctcatg tacagaggcg atgttgtccc caaagatgtg aacgcggccg ttgccacaat 1200

caagacaaag aggacgatcc agtttgtcga ctggtgccca actggcttca agtgtggcat 1260

caactaccag ccgccgactg tggtccctgg tggtgatctt gctaaggtgc agagggcagt 1320

ttgcatgatt tcaaactcga ctagtgttgc tgaggtgttc tcgaggatcg atcataagtt 1380

tgatttgatg tatgctaaga gggctttcgt gcattggtat gtgggtgagg gtatggagga 1440

aggggagttc tcggaggcga gggaggattt ggctgctttg gagaaggatt atgaggaagt 1500

tggggctgaa tctgctgaag gtgaagatga tgagaatgaa gagtactaga gtagggacca 1560

attggctcgt tggtttagtg ctgaactttt ttccttatgg ttgaacttct tgtctggtgt 1620

tgaactgctt atggattctc tgtgtgtagt tcgtattgat tatggtatgt gttgaaaagg 1680

ggctttatgt tcatttcgta ttgattatgc ttgtgtaatt cgtatgtgtt gattattagt 1740

tattccctag gtcatactcg tattttatat gtttaaaatg ccaagataat tttttttttt 1800

tt 1802

<210> 16

<211> 1670

<212> DNA

<213>the UBQ gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 16

gaattgttct cataaatttt cagttttttc gtctctcaat cgtcaagatg caaatcttcg 60

tgaaaactct caccggcaag accatcaccc ttgaggttga aagctccgat accattgaca 120

acgtcaaggc taagatccag gacaaggaag gtatcccccc agaccagcaa aggctgatct 180

tcgcaggaaa acagcttgag gatggccgta ccctcgccga ctacaatatt cagaaggaat 240

caactctcca cctggtcctc cgtctccgtg gaggtatgca gatattcgtg aagaccctaa 300

cgggcaagac catcaccctc gaggttgaaa gctccgacac gatcgataac gtgaaggcca 360

agatccagga caaggagggt attcccccag accagcagag gctgatcttc gccggcaagc 420

agctcgagga cggacgtacc ctcgccgact acaatatcca gaaggagtcc accctccatt 480

tggtcctccg tctccgtggt ggtatgcaga tctttgtcaa gaccctcacc ggcaagacca 540

tcaccctcga ggttgaaagc tccgacacga tcgataacgt gaaggccaag atccaggaca 600

aggagggtat tcccccagac cagcagaggc tgatcttcgc tgggaagcaa ctcgaggatg 660

gacgcacgct tgctgattac aacatacaaa aggaaagcac acttcatttg gttctgaggt 720

tgagaggtgg aatgcaaatc tttgtcaaga ctctcacggg gaagaccatc accttggagg 780

ttgagagttc cgacaccatt gataatgtga aagccaagat acaagacaag gaaggtatcc 840

caccagacca gcagaggctc atatttgcag ggaagcagct ggaggatgga cgcaccttgg 900

ctgattacaa cattcagaag gagtctactc ttcatttggt gttgaggttg aggggaggga 960

tgcagatatt tgtcaagaca ttgactggga agaccatcac tttggaggtg gagagttcag 1020

acactattga taatgtgaag gccaagatcc aggataagga gggcatccca cccgaccagc 1080

agaggttgat cttcgccggg aaacagctgg aggatgggag gactctcgct gattacaata 1140

tccagaagga atccaccctt caccttgtcc tcaggctccg tggtggtttc tagagcttct 1200

ggttttcttg ttgtttgaat gttctggttt tatgttctgt ttgttgattt tacatgattc 1260

ctgttttccc gtgtgtgtgt gcgtgtgttg aaacttaagt atggtactgt tatgttgtcg 1320

tcttgctgac attctcagtt gtcattggaa taaaattggg attttatccc ccctataata 1380

aatttccggt gagtgcataa ttggctgtaa tagttttttc aatattgttg ctgttgagaa 1440

ggtgctacgt aatgttctac cttcttcatc ggttgctttt ctggagattc ttgtatgcct 1500

cggtaatgtt tggtgattat atttatcttc tttaattact gaaagaagtt ggatccgatg 1560

cactatccca ggagaggtcg aagcgatgta ttcctaggtg tttttctgtt agtggagttt 1620

acttcgttgc atgttgtccc gtcaaccatt ttcgtaaggg attaatgttt 1670

<210> 17

<211> 588

<212> DNA

<213>the H3 gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 17

actgatcagt tttactacaa aacagacaaa ggttaaacat tcaaagccac aagtgacacc 60

tcgaaacaac cacatattta ccatcggaaa aaactatccc ttagacccaa atcatccaga 120

ttacaagtta agccctttca cccctaattc tcctcgcaag ctggatatct ttgggcataa 180

ttgtaaccct cttagcatga atggcacaga ggttagtatc ctcaaacaat ccaacaagat 240

aggcctccgc tgcctcttga agagccgcca cagcagagct ctggaacctc agatccgtct 300

tgaaatcctg agcaatctca cggacaagcc tctggaaagg aagttttcgg atcaaaagct 360

cagtggattt ctggtacttg cgaatttcac ggagagcgac ggttcccggg cgaaaacggt 420

gaggcttctt cactccgccg gtggcggggg ctgatttcct agcggctttg gtggccaact 480

gcttccgcgg tgccttgcct ccggtggatt tgcgggcggt ttgcttagtg cgagccattt 540

ctagggtatc gatagaaagt atggaaaaaa agtgctaatt tggaattg 588

<210> 18

<211> 514

<212> DNA

<213>the ClLAC gene (Clerodendrum trichotomum Thunb) of clerodendron trichotomum

<400> 18

gccaataatt tctcagcaat ggagggctta ttttaccgtc ttctccttct tacagttcca 60

ttaatggcca tgcaagtgct tccttcttct tcatcatcca atacaaagat gtttcagttt 120

aatgttgaat ggaagagtat aacaagactt tgcaatagaa agacagtatt gacagtaaat 180

gggaagtacc cagggccaac cattgctgtg aatgaaggtg acagtgttca agtaacggtc 240

actaattggg ttgcaagaaa caccaccatc cattggcatg gagtaaggca gatgagaagg 300

ggttgggcag atggggcagc atacataaca caatgcccga ttggaggagg caaaagctat 360

agatacaact ttagtgtaga ggatcagagg ggtacactgt ggtggcatgc tcatatcggc 420

tggcaacgtg cttcggttta tggcgctttt atcatttacc ctcgcacgcc ttttccattc 480

tctctccaac tccacccact tcagattcct ctca 514

<210> 19

<211> 21

<212> DNA

<213>Actin primer sequence (Artificial)

<400> 19

cgattcttgc ttccctcagt a 21

<210> 20

<211> 18

<212> DNA

<213>Actin downstream primer sequence (Artificial)

<400> 20

accattttcg ccgcctta 18

<210> 21

<211> 19

<212> DNA

<213>PP2A upstream primer sequence (Artificial)

<400> 21

ggacaagcac caagacgcc 19

<210> 22

<211> 22

<212> DNA

<213>PP2A downstream primer sequence (Artificial)

<400> 22

tcagcacttc taaaaccgca tc 22

<210> 23

<211> 21

<212> DNA

<213>RPL upstream primer sequence (Artificial)

<400> 23

agtcaatggt ggcgatgtag c 21

<210> 24

<211> 22

<212> DNA

<213>RPL downstream primer sequence (Artificial)

<400> 24

cccttggtca ctccgataat gt 22

<210> 25

<211> 23

<212> DNA

<213>PK upstream primer sequence (Artificial)

<400> 25

ctgaaggtgg tgtactcctg ctc 23

<210> 26

<211> 21

<212> DNA

<213>PK downstream primer sequence (Artificial)

<400> 26

cgaaactgcc ccaacaagat a 21

<210> 27

<211> 21

<212> DNA

<213>18S upstream primer sequence (Artificial)

<400> 27

acaatcaccg aacaaagcag c 21

<210> 28

<211> 22

<212> DNA

<213>18S downstream primer sequence (Artificial)

<400> 28

cgagaagtaa tcttggaacg cc 22

<210> 29

<211> 20

<212> DNA

<213>RAN upstream primer sequence (Artificial)

<400> 29

ggagacgaca gtttgggtgc 20

<210> 30

<211> 19

<212> DNA

<213>RAN downstream primer sequence (Artificial)

<400> 30

tgccatactt gcgggtgaa 19

<210> 31

<211> 19

<212> DNA

<213>APT upstream primer sequence (Artificial)

<400> 31

ccacgcattc aagccattc 19

<210> 32

<211> 20

<212> DNA

<213>APT downstream primer sequence (Artificial)

<400> 32

gagcctctat cccagcaacg 20

<210> 33

<211> 19

<212> DNA

<213>SAND upstream primer sequence (Artificial)

<400> 33

gaccagtcgc ttgatgccc 19

<210> 34

<211> 22

<212> DNA

<213>SAND downstream primer sequence (Artificial)

<400> 34

ggatgctcca ggtactgctc tt 22

<210> 35

<211> 24

<212> DNA

<213>PROF upstream primer sequence (Artificial)

<400> 35

gaatgtggtg ttttgagtga gagg 24

<210> 36

<211> 18

<212> DNA

<213>PROF downstream primer sequence (Artificial)

<400> 36

atgtgaagtt ttggggga 18

<210> 37

<211> 20

<212> DNA

<213>MDH upstream primer sequence (Artificial)

<400> 37

acctggaatg acgagggatg 20

<210> 38

<211> 24

<212> DNA

<213>MDH downstream primer sequence (Artificial)

<400> 38

gccacaaacg tattagctct gact 24

<210> 39

<211> 20

<212> DNA

<213>EF-1A upstream primer sequence (Artificial)

<400> 39

aggatggaca gacccgtgag 20

<210> 40

<211> 22

<212> DNA

<213>EF-1A downstream primer sequence (Artificial)

<400> 40

aaaaccagaa atggggacaa at 22

<210> 41

<211> 23

<212> DNA

<213>UBC-E2 upstream primer sequence (Artificial)

<400> 41

gcaaaggctg attgatgaga ttc 23

<210> 42

<211> 21

<212> DNA

<213>UBC-E2 downstream primer sequence (Artificial)

<400> 42

cctcaacatt gtcttgggtg g 21

<210> 43

<211> 20

<212> DNA

<213>AP-2 upstream primer sequence (Artificial)

<400> 43

ccacaactaa cgcctccacc 20

<210> 45

<211> 20

<212> DNA

<213>HSP70 upstream primer sequence (Artificial)

<400> 45

gccattttga gtggtgaggg 20

<210> 46

<211> 23

<212> DNA

<213>HSP70 downstream primer sequence (Artificial)

<400> 46

ggttggttgt ccgagtaggt aga 23

<210> 47

<211> 20

<212> DNA

<213>TUA upstream primer sequence (Artificial)

<400> 47

gtccccaaag atgtgaacgc 20

<210> 48

<211> 20

<212> DNA

<213>TUA downstream primer sequence (Artificial)

<400> 48

aagcagccaa atcctccctc 20

<210> 49

<211> 21

<212> DNA

<213>UBQ upstream primer sequence (Artificial)

<400> 49

tgctgttgag aaggtgctac g 21

<210> 50

<211> 19

<212> DNA

<213>UBQ downstream primer sequence (Artificial)

<400> 50

cgaaaatggt tgacgggac 19

<210> 51

<211> 21

<212> DNA

<213>H3 upstream primer sequence (Artificial)

<400> 51

caagttaagc cctttcaccc c 21

<210> 52

<211> 21

<212> DNA

<213>H3 downstream primer sequence (Artificial)

<400> 52

tgctcaggat ttcaagacgg a 21

Claims

1. being suitable for the fluorescent quantitation reference gene of clerodendri,folium different development stage, which is characterized in that beActin、RANWithSANDGene, whereinActinThe gene order of gene as shown in SEQ ID NO.1,RANThe gene order of gene such as SEQ ID Shown in NO.6,SANDThe gene order of gene is as shown in SEQ ID NO.8.

2. fluorescent quantitation reference gene dedicated described in claim 1 suitable for clerodendri,folium different development stage is drawn Object, which is characterized in thatSANDThe primer sequence of gene is as follows:

SANDForward primer 5 '-CCACAACTAACGCCTCCACC-3 '

SANDReverse primer 5 '-AAATTCACCCTCCGACTCCC-3 '.

3. fluorescent quantitation reference gene dedicated described in claim 1 suitable for clerodendri,folium different development stage is drawn Object, which is characterized in thatRANThe primer sequence of gene is as follows:

RANForward primer 5 '-CGATTCTTGCTTCCCTCAGTA-3 '

RANReverse primer 5 '-ACCATTTTCGCCGCCTTA-3 '.

4. fluorescent quantitation reference gene dedicated described in claim 1 suitable for clerodendri,folium different development stage is drawn Object, which is characterized in thatActinThe primer sequence of gene is as follows:

ActinForward primer 5 '-CGATTCTTGCTTCCCTCAGTA-3 '

ActinReverse primer 5 '-ACCATTTTCGCCGCCTTA-3 '.

5. the fluorescent quantitation reference gene described in claim 1 suitable for clerodendri,folium different development stage is in clerodendron trichotomum Application in leaf fluorescent quantitation.

6. as claimed in claim 2SANDApplication of the primer sequence of gene in clerodendri,folium fluorescent quantitation.

7. as claimed in claim 4ActinApplication of the primer sequence of gene in clerodendri,folium fluorescent quantitation.

8. as claimed in claim 2SANDThe primer sequence of gene is being clonedSANDApplication in gene.

9. as claimed in claim 4ActinThe primer sequence of gene is being clonedActinApplication in gene.

10. as claimed in claim 3RANApplication of the primer sequence of gene in clerodendri,folium fluorescent quantitation.