stilbene synthase (Stemona collinsae)

1. stilbene synthase (STS) (Stemona collinasae) 2. stilbene synthase stilbenoid root tissue elicitor

Strategies

PCR based approach AAAAA Total mRNA RT-PCR

Partial stilbene synthase gene

5RACE

3RACE

AAAAA
Full length stilbene synthase cDNA

Pinus sylvestris (ACC. no. Q02323) Arachis hypogaea (ACC. no. BAA78617) Rheum tataricum (ACC. no. AAP13782), Bauhinia variegata (ACC. no. ABF59517) Vitis vinifera (ACC. no. ABV82966) Forward primer KAIQEW STS1 - 5 AA(A/G)GCIAT(N)CAIGA(A/G)TGGGG 3 Reverse primer W(I/V)(A/V)HPGG STS4 - 5 CCACCIGG(A/G)TGI(A/G)CAA(T/G)CC 3

The nucleotide & amino acid sequences of the partial of cDNA CHS/STS-like gene

Forward primer KAIQEW STS1 - 5 AA(A/G)GCIAT(N)CAIGA(A/G)TGGGG 3 Reverse primer W(I/V)(A/V)HPGG STS4 - 5 CCACCIGG(A/G)TGI(A/G)CAA(T/G)CC 3

 open reading frame

homology to the other Chalcone/Stilbene synthase superfamily

The nucleotide & amino acid sequences of 3end CHS/STS-like gene

1 2

1. 2.

1 Kb DNA marker 3 RACE product of CHS/STS like gene

~ 600 bp

The nucleotide & amino acid sequences of 5end CHS/STS-like gene

1 2 1. 2. 1 Kb DNA marker 5 RACE product of CHS/STS like gene

~ 850 bp

Full-length CHS/STS-like gene cDNA

ORF 1186 bp, 394 amino acid residues

Rheum tataricum 61% identity

Vitis vinifera 62% identity Pinus sylvestris 63% identity Arachis hypogaea 63% identity

Stemona Pinus Arachis Bauhinia Vitis Rheum Stemona Pinus Arachis Bauhinia Vitis Rheum Stemona Pinus Arachis Bauhinia Vitis Rheum Stemona Pinus Arachis Bauhinia Vitis Rheum Stemona Pinus Arachis Bauhinia Vitis Rheum Stemona Pinus Arachis Bauhinia Vitis Rheum Stemona Pinus Arachis Bauhinia Vitis Rheum

Bauhinia variegata 65% identity

MVGVAAALRLGTQRADGPAAIMAIGTANPPNVVEQSSYPDFFFRVTNSEDKTQLKNKFKR MGGVDFEGFRKLQRADGFASILAIGTANPPNAVDQSTYPDFYFRITGNEHNTELKDKFKR --MVSVSGIRKVQRAEGPATVLAIGTANPPNCVDQSTYADYYFRVTNSEHMTDLKKKFQR --MVSVNEIRKVQRAEGPATVLAIGTANPPNCIEQSTYADYYFKITKSEHKTELKEKFKR --MAVAEELRNAPRAKGPATILAIGTATPDHCIYQSDYADYYFRVTKSEHMTDLKKKFNR --MAPEESRHAETAVNRAATVLAIGTANPPNCYYQADFPDFYFRATNSDHLTHLKQKFKR . .. *:::*****.* : *: :.*::*: * .:. *.**.**:* ICEKTMIKKRYMHLTEEILKDNPNMCPHMA-PSLDAREAILVEEVPKLGHQAAVKAIEEW ICERSAIKQRYMYLTEEILKKNPDVCAFVEVPSLDARQAMLAMEVPRLAKEAAEKAIQEW ICERTQIKNRHMYLTEEILKENPNMCAYKA-PSLDAREDMMIREVPRVGKEAATKAIKEW MCERTLIKNRHMHLTEEILKENPSICEYKA-PSLDAREDMIIREVPKVGKEAATKAIKEW ICDKSMIKKRYIHLTEEMLEEHPNIGAYMA-PSLNIRQEIITAEVPRLGRDAALKALKEW ICEKSMIEKRYLHLTEEILKENPNIASFEA-PSLDVRHNIQVKEVVLLGKEAALKAINEW :*::: *::*:::****:*:.:*.: . ***: *. : ** :.::** **::** GRDKDEITHLVFGSTMSGADMPGSDYRLIQLLGLSPSVQRVMLNHQGCFAGGTVLRVAKD GQSKSGITHLIFCSTTT-PDLPGADFEVAKLLGLHPSVKRVGVFQHGCFAGGTVLRMAKD GQPMSKITHLIFCTTSG-VALPGVDYELIVLLGLDPSVKRYMMYHQGCFAGGTVLRLAKD GQPMSKITHLIFCTTSG-VGLPGVDYQLIVLLGLDPYVKRYMMYQQGCFAGGTVLRLAKD GQPKSKITHLVFCTTSG-VGMPGADYKLANLLGLETSVRRVMLYHQGCYAGGTVLRTAKD GQPKSKITRLIVCCIAG-VDMPGADYQLTKLLGLQLSVKRFMFYHLGCYAGGTVLRLAKD *: . **:*:. :** *:.: **** *:* . : **:******* *** LAENNRDARVLVVCAEITAVFFRGPDDVHFDNLVGQTLFGDGAAALIVGADPIHGVEKPI LAENNRGARVLVICSETTAVTFRGPSETHLDSLVGQALFGDGASALIVGADPIPQVEKAC LAENNKDARVLIVCSENTSVTFRGPSETDMDSLVGQALFADGAAAIIIGSDPVPEVENPL LAENNKDARVLIVCSENTAVTFRGPSDTDLDSLVGQALFADGAAAIIIGSDPIPEVEKPL LAENNAGARVLVVCSEITVVTFRGPSEDALDSLVGQALFGDGSSAVIVGSDPDVSIERPL IAENNKEARVLIVRSEMTPICFRGPSETHIDSMVGQAIFGDGAAAVIVGANPDLSIERPI :**** ****:: :* * : ****.: :*.:***::*.**::*:*:*::* :*.. FEMASAAQCILPDSEGTLEGHLREVGLIFQLLSQVPTLIANNIEKCLVKAFKPLGISDWN FEIVWTAQTVVPNSEGAIGGKVREVGLTFQLKGAVPDLISANIENCMVEAFSQFKISDWN FEIVSTDQQLVPNSHGAIGGLLREVGLTFYLNKSVPDIISQNINDALSKAFDPLGISDYN FEMVSTDQKILPGSQGATGGLLREVGLTFYLNKSVPDMISQNIKEALVKAFNPLGISDYN FQLVSAAQTFIPNSAGAIAGNLREVGLTFHLWPNVPTLISENIEKCLTQAFDPLGISDWN FELISTSQTIIPESDGAIEGHLLEVGLSFQLYQTVPSLISNCIETCLSKAFTPLNISDWN *:: : * .:* * *: * : **** * * ** :*: *: .: :** : ***:* SIFWVAHPGGRAILDQIEAKLSLDPEKLRATRHVLSEFGNMSSASVFFILDEMRRNSAAK KLFWVVHPGGRAILDRVEAKLNLDPTKLIPTRHVMSEYGNMSSACVHFILDQTRKASLQN SIFWIAHPGGRAILDQVEEKVNLKPEKMKATRDVLSNYGNMSSACVFFIMDLMRKKSLEA SIFWIAHPGGRAILDQVEHKLGLKPEKMKATRHVLSKYGNMSSACVLFIMDEMRRRSAED SLFWIAHPGGPAILDAVEAKLNLEKKKLEATRHVLSEYGNMSSACVLFIMDEMRKKSLKG SLFWIAHPGGRAILDDIEATVGLKKEKLKATRQVLNDYGNMSSACVFFIMDEMRKKSLAN .:**:.**** **** :* .:.*. *: .**.*:..:******.* **:* *: * GQATSGEGLEWGALFGFGPGLTVETVVLRSVPLHE- 394 GCSTTGEGLEMGVLFGFGPGLTIETVVLKSVPIQ-- 393 GLKTTGEGLDWGVLFGFGPGLTIETVVLRSMAI--- 389 KLSTTGEGLDWGVLFGFGPGLTVETVVLRSVAV--- 389 EKATTGEGLDWGVLFGFGPGLTIETVVLHSIPTITN 392 GQVTTGEGLKWGVLFGFGPGVTVETVVLSSVPLIT- 391 *:****. *.*******:*:***** *:.

60 60 58 58 58 58 119 120 117 117 117 117 179 179 176 176 176 176 239 239 236 236 236 236 299 299 296 296 296 296 359 359 356 356 356 356

Functional analysis
CHS/STS-like gene (Stemona collinsae)

 stilbene synthase stilbenoid root tissue elicitor Semi-quatitative RT-PCR 1 2 3

FP- CHS/STS like gene 5 ATCACCCACCTCGTCTTC 3 RP- CHS/STS like gene 5 TTCCTCCGCATCTCATCC 3 FP- GAPDH gene 5 ATCACCCACCTCGTCTTC 3 RP- GAPDH gene 5 TTCCTCCGCATCTCATCC 3
1. 2. 3.

686 bp.

309 bp.
1 Kb DNA marker CHS/STS like gene GAPDH gene

 (Root culture) elicitor CHS/STS-like gene (Root culture) - MS NAA 1.0 /

 (Root culture) - MS NAA 1.0 /

 (output)
1. Chalcone/stilbene synthase Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Stemona collinsea) 2. 1

 ()
-

34,650 50,000
5,000 261,850

238,801
38,500

Functional analysis
Transformation
CHS/STS
CHS/STS

Yeast cell or E. Coli

Induction of gene expression Induced cells

Secondary metabolite analysis by HPLC