3 -end. Sau3A. 3 -end TAA TAA. ~9.1kb SalI TAA. ~12.6kb. HindШ ATG 5,860bp TAA. ~12.7kb
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1 Supplemental Data. Chen et al. (2008). Badh2, encoding betaine aldehyde dehydrogenase, inhibits the biosynthesis of 2-acetyl-1-pyrroline, a major component in rice fragrance. A pcam-cah/cah Sau3A Promoter ATG 2,499b ~6.3kb TAA 3 -end Sau3A Backbone of vector Coding region from Suyunuo BAC clone Coding region from NJ11 BAC clone 1kb Promoter ATG 2,523b TAA 3 -end ApaI ~5.2kb SmaI B pcam-mccc2/mccc2 ATG Promoter 6,506bp TAA 3 -end SalI ATG Promoter ~9.1kb 6,509bp TAA SalI 3 -end SalI ~9.1kb SalI C pcam-badh2/badh2 ATG Promoter 5,846bp TAA 3 -end HindШ ATG Promoter 5,860bp ~12.6kb TAA 3 -end HindШ HindШ ~12.7kb HindШ Supplemental Figure 1. Maps of three Fgr constructs derived from the non-fragrant cv. Nanjing 11 and three fgr constructs derived from the fragrant cv. Suyuno. (A) pcam-cah/cah : The intact Cah gene (>2kb promoter, ~2.5kb coding region, and >1.5kb 3 -UTR.) was extracted from the Nanjing11 BAC clone ( N11-17-M18) by Sau3A partial digestion, and then cloned into the BamHI-digested vector pcambia1302. The Suyunuo BAC clone (Su-39-E2) was digested with ApaI and SmaI and the digested fragment containing the intact cah gene was then cloned for sequencing. (B) pcam-mccc2/mccc2 : The Nanjing11 BAC clone (N11-17-M18) and Suyunuo BAC clone (Su-39-E2) were separately digested with SalI. The digested fragments containing the intact Mccc2/mccc2 genes (~1.4kb promoter, ~6.5kb coding region, and ~1.2kb 3 -UTR) were cloned into the SalI-digested vector pcambia1302. (C) pcam-badh2/badh2 : The Nanjing11 BAC clone (N11-16-F20) and Suyunuo BAC clone (Su-39-E2) were separately digested with HindIII. The digested fragments containing the intact Badh2/badh2 genes (~1.3kb promoter, ~5.8kb coding region, and ~5.6kb 3 -UTR.) were cloned into the HindIII-digested vector pcambia1302.
2 Supplemental Figure 2. Characterization of nucleotide acid sequences and amino-acid sequences of the Badh1, Badh2, badh2-e2, and badh2-e7 alleles. (A) Nucleotide sequence variations among the Badh2, badh2-e2, and badh2-e7 alleles. The functional Badh2 allele consists of 15 exons (brown boxes) and 14 introns (black lines) as presented in three non-fragrant varieties: Nipponbare, 93-11, and Nanjing11. (B) Alignment of amino-acid sequences among the rice BADH1, BADH2, the truncated BADH2-E7, and the truncated BADH2-E2. Black blocks represent conserved residues among all the four BADHs. Pink blocks represent conserved residues among any three BADHs. Baby-blue blocks represent conserved residues among any two BADHs.
3 Supplemental Figure 3. Identification of transgenic lines using the gene-tagged markers. Because the rice transformation recipient Wuxiangjing belonged to the japonica rice subspecies and three Fgr candidates were derived from the indica cv. Nanjing11, the presence/absence of the exogenous Fgr candidates in regenerated plantlets could be investigated by using the gene-tagged markers (primers listed in Supplemental Table 4). The transgenic line gave rise to two PCR bands, one corresponded to Wuxiangjing, and the other corresponded to Nanjing 11. The non-transgenic line only showed one single PCR band corresponding to that of Wuxiangjing. P1: the japonica rice cv. Wuxiangjing ; P2: the indica rice cv. Nanjing 11. (A) The plantlets regenerated from the Cah-transformed calluses were genotyped at the Cah-tagged marker and only two of them (Lanes 1 and 2) were transgenic lines. The PCR bands in Wuxiangjing (P1) and Nanjing 11 (P2) were 187bp and 210bp, respectively. (B) The 12 plantlets regenerated from the Mccc2-transformed calluses were genotyped at the Mccc2-tagged marker. Three of them (lanes 7, 9, and 10) were proved to be transgenic lines. The PCR bands in Wuxiangjing (P1) and Nanjing 11 (P2) were 312bp and 298bp, respectively. (C) The 17 plantlets regenerated from the Badh2-transformed calluses were genotyped at the Badh2-tagged marker. All plantlets were confirmed to be transgenic lines. The PCR bands in Wuxiangjing (P1) and Nanjing 11 (P2) were 391bp and 348bp, respectively.
4 A B Supplemental Figure 4. Chromatogram of rice leaf tissue extracts. The GC-MS (Gas-Chromatography/Mass-Spectrometry) method was used to determine 2AP levels. Arrows indicate the 2AP peaks in Suyunuo and 3037 (A) Analysis of the fragrant rice variety 'Suyunuo' (B) Analysis of the non-fragrant rice variety '3037'
5 Supplemental Figure 5. The genotypes at the exogenous Badh2 gene in T 1 progeny. The transgenic line was self-pollinated to generate T 1 progeny. Progeny were examined by PCR using the Badh2-specific primers listed in Supplemental Table 4. M: DL2000 Marker P1: The non-fragrant rice cv. Nanjing11 from which the Badh2 gene was derived. P2: The fragrant rice cv. Wuxiangjing which was the target of transformation. Lanes 1 to 19: The randomly selected T1 progeny.
6 Supplemental Figure 6. Maps of the three Badh2 transcripts and locations of the primers and probes used for investigation of Badh2 transcription. The complete Badh2 transcript with the 1512-bp CDS was predicted from the Badh2 gene (on the top). The partial Badh2 transcript with the 1182-bp CDS accounted for the majority of the 5 -RACE products (in the middle). Another partial Badh2 transcript with the only 1095-bp CDS was found in the Gramene database (at the bottom). Three different forward primers and one common reverse primer were designed to investigate the relative abundances of the three Badh2 transcripts. The forward primer FP1 corresponds to the leader sequence of the complete Badh2 transcript and the primer pair FP1/RP could specifically amplify the complete Badh2 transcript. The forward primer FP2 is close to the start codon of the partial Badh2 transcript (middle) and the primer pair FP2/RP could amplify both the complete and partial Badh2 transcripts. The forward primer FP3 was designed using the leader sequence of the short Badh2 transcript and the primer pair FP3/RP could therefore amplify all possible Badh2 transcripts. The probe A covers the region from -74bp to 244bp and this probe could exclusively hybridize to the complete Badh2 transcript. The probe B corresponds to the segment from 367bp to 1325bp, which could hybridize to all three Badh2 transcripts.
7 Supplemental Figure 7. Sequence alignment between BADH2 and ALDH2. The template ALDH2 (the human mitochondrial aldehyde dehydrogenase) was obtained by screening the protein database. The alignment was first obtained from the Swiss-Model program and then refined using ESPript. The secondary structure elements for ALDH2 are shown on the top of the sequences. α-helices are represented by squiggles; while, β-strands are represented by arrows. Identical residues are highlighted in white letters with a red background. Residues with similar physico-chemical properties are shown in red letters. Alignment positions are framed in blue if the corresponding residues are identical or similar. The catalytic residues interacting with the substrate oxygen are labeled with red pentacles, while those residues forming the substrate binding pocket are labeled with green triangles.
8 Supplemental Figure 8. In vitro expression of the Badh2/badh2 cdnas in E. coli cells. The complete and partial Badh2/badh2 cdnas from both the non-fragrant cv. Nanjing 11 and the fragrant cv. Wuxiangjing were cloned into the vector PET43.1a. These constructs were expressed in vitro as Nus-BADH2 fusion proteins in E. coli expression strain BL21(DE3). Before and after induction with 0.5M IPTG, the E. coli cells were lysed and analyzed by electrophoresis on a 12% SDS-polyacrylamide gel. Lanes 1 and 2: The vector PET43.1a (negative control). The Nus/His/S-tag protein of ~66KDa (arrowed) was observed after induction with IPTG (lane 2), but not before IPTG induction (lane 1). Lanes 3 and 4: The expression vector PET43.1a-NFCcDNA. The Nus-BADH2 fusion protein of ~116KDa (arrowed) was present after IPTG induction (lane 4), but not before IPTG induction (lane 3). Lanes 5 and 6: The expression vector PET43.1a-FCcDNA. The Nus-BADH2 fusion protein of ~88KDa (arrowed) was present after IPTG induction (lane 6), but not before IPTG induction (lane 5). Lanes 7 and 8: The expression vector PET43.1a-NFPcDNA. The Nus-BADH2 fusion protein of ~106KDa (arrowed) was present after IPTG induction (lane 8), but not before IPTG induction (lane 7). Lanes 9 and 10: The expression vector PET43.1a-FPcDNA. The Nus-BADH2 fusion protein of ~76KDa (arrowed) was present after IPTG induction (lane 10), but not before IPTG induction (lane 9).
9 Supplemental Figure 9. Purification of the fusion proteins containing the intact or partial BADH2. The Nus-BADH2 fusion protein expressed following induction by 0.5mM IPTG was purified by affinity chromatography using Ni-NTA Agarose. The purified fusion protein as well as the lytic E. coli cells before and after IPTG inductions was analyzed by electrophoresis on 12% SDS-polyacrylamide gel (SDS-PAGE). (A) The expression of the intact BADH2 protein in E. coli cells transformed by the expression vector PET43.1a-NFCcDNA. (B) The expression of the partial BADH2 protein in E. coli cells transformed by the expression vector PET43.1a-NFPcDNA. Lane 1: E. coli cell lysate before IPTG induction Lane 2: E. coli cell lysate after IPTG induction Lane 3: The purified Nus-BADH2 fusion protein
10 Supplemental Table 1. 2AP levels in each non-transgenic line and transgenic line with different exogenous Fgr candidates. Non-transgenic and transgenic lines Non-transgenic lines Cah-transgenic lines Mccc2-transgenic lines Badh2-transgenic lines Codes of plantlets regenerated from Genotypes at the exogenous Fgr 2AP levels (ng/g) transformed calluses candidates 021 /* / / / / / / / / / / Cah Cah Mccc Mccc Mccc Mccc Badh Badh Badh Badh Badh Badh Badh Badh Badh Badh Badh /*: The absence of the exogenous Fgr candidate
11 Supplemental Table 2. 2AP levels in transgenic and non-transgenic lines using the four fragrant rice recipients containing the badh2-e2 alleles. Fragrant rice recipients (badh2-e2) Xiangjing111 Non-transgenic lines Guanglingxiangjing Xiangjing111 Guanglingxiangjing Transgenic lines Wuxiangjing9 Zhenxiangjing5 Codes of Genotypes at the 2AP levels regenerated exogenous Badh2 (ng/g) plantlets gene /* / G-1 / G-2 / Badh Badh Badh G-4 Badh G-5 Badh W-2 Badh W-3 Badh W-10 Badh Z-1 Badh Z-2 Badh Z-10 Badh /*: The absence of the exogenous Badh2 gene
12 Supplemental Table 3. 2AP levels in each non-transgenic line and transgenic line with the different Badh2 coding sequences (CDS) driven by the CaMV35S promoter. The exogenous Badh2 CDS Partial Badh2 CDS (1095-bp) Partial Badh2 CDS (1182-bp) Complete Badh2 CDS (1512-bp) Non-transgenic lines Codes of plantlets Genotypes at the Badh2 CDS 2AP levels (ng/g) bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS bp Badh2 CDS K-1 /* K-2 / K-3 / K-4 / K-5 / K-6 / K-7 / K-8 / K-9 / /*: The absence of the exogenous Badh2 CDS
13 Supplemental Table 4. List of all primers used in the present research. Types Names Sequences (5-3 ) Sizes (bp) Indica/Japonica L02 Forward: CATCGGATAGTTCTCGGCAA 497/ Reverse: GATACGTCGGTGTCGGTCAA (HaeⅢ) Forward: GATGAACGCAGAAGCAGTAG L03 488/484 Reverse: CGAGTTGTCCTATAACCATG Markers developed Forward: CCTACAAGCCTAAGCTGCCA for fine-mapping of L04 210/187 Reverse: GCCGAACTGATCGTCTTCCT the fgr gene Forward: CTTGCAGCCATGAATGTTCC L05 369/322 Reverse: GTCCAATCGTCCATGATTCG Forward: GCAAGTGACGGAGTACGCCT L06 Reverse: GCTAACTTCCGCTCACGCAA 348/391 Cah-TM Forward: CCTACAAGCCTAAGCTGCCA Reverse: GCCGAACTGATCGTCTTCCT 210/187 Gene-tagged Forward: CAGCGTTGCTATCCATCTTG Mccc2-TM markers Reverse: TCTTCCTGACCGTGCCAGT 298/312 Forward: GCAAGTGACGGAGTACGCCT Badh2-TM Reverse: GCTAACTTCCGCTCACGCAA 348/391 Forward: TTATGGTCTGGCTGGTGCTGT Badh2-RP Real-time RT-PCR Reverse: TGCTTGACGCTTAGGTAGTTGT 133bp primers Forward: GTACAGTGTCTGGATTGGAGGAT Actin-RP Reverse: GGGTCCGAAGAATTAGAAGCA 198bp Badh2-specific primer Badh2-SP5 Reverse: GAGCACCGGCTTCAGAACCTAATCCAGT for RACE Reaction Badh2-SP3 Forward: GGATTAGGTTCTGAAGCCGGTGCTCCTT Primers used for FP1 (-74) evaluation of FP2 (+283) various Badh2 FP3 (+367) transcripts RP (+579) Probe A Northern blot analysis Probe B Forward: CCATCTCCGTATCTCTCACC Forward: CTAGAGACGCTTGATTGTGG Forward: GATCTTGCAGAATCCTTGGA Reverse: AGTCACGGAAGCCAATTCAG Forward: CCATCTCCGTATCTCTCACC Reverse: TCACTTGCTTCCGTTGAGGCGATTGCGCGGAGGTACTTG Forward: GATCTTGCAGAATCCTTGGA Reverse: GCATCGATCTCCTCAGTTAATCTCTGGCATCGCTC
Badh2, Encoding Betaine Aldehyde Dehydrogenase, Inhibits the Biosynthesis of 2-Acetyl-1-Pyrroline, a Major Component in Rice Fragrance W
The Plant Cell, Vol. 20: 1850 1861, July 2008, www.plantcell.org ª 2008 American Society of Plant Biologists Badh2, Encoding Betaine Aldehyde Dehydrogenase, Inhibits the Biosynthesis of 2-Acetyl-1-Pyrroline,
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