Cloning of full-length cDNA
One μg was taken from each of all the total RNA samples, mixed, and used as template for the
synthesis of the 1st strand of cDNA by using PrimeScriptTMⅡ1st Strand cDNA Synthesis kit (TaKaRa,
Japan). A pair of degenerate primers (5'-GTCGGAGAGRCKTTTCCRTGG-3'/
5'-TGCACAWGCDCCWGGATTR-3'; where D=A, G or T; K=G or T; R=A or G; W=A or T) was
designed according to the alignment between the putative amino acid sequences of the LOS5/ABA3
gene and the predicated MCSU genes from the genomic sequences of other leguminous plants, and
used to amplify the core sequence of the AnMCSU gene from the prepared 1st strand cDNA sample.
The product was cloned into pMD19-T vector (TaKaRa, Japan), and sequenced at Invitrogen (USA).
The first strand of the cDNA for 3'-RACE was reverse transcribed from the mixed total RNA
sample by using the 3'-RACE adaptor with 3'-Full RACE Core Set Ver.2.0 (TaKaRa, Kapan), and used
for the first amplification of the nested PCR with the specific outer primers
(5'-GCATGGCGATGGATCTAGCATGTGCAT-3') designed according to the core sequence, and the
3'-RACE outer primer (5'-TACCGTCGTTCCACTAGTGATTT-3') provided in the kit. The product was
used as template for the second amplification of the nested PCR with the specific inner primer
(5'-GGCTCTTGGTATGGATACCGTGAAGTGG-3') designed according to the core sequence, and the
3'-RACE inner primer (5'-CGCGGATCCTCCACTAGTGATTTCACTATAGG-3') provided in the kit.
The product was cloned into pMD19-T vector (TaKaRa, Japan), sequenced at Invitrogen (USA).
The 5'-RACE was conducted using First Choice® RLM-RACE Kit (Ambion, USA). The mixed
total RNA sample was dephosphorylated with calf intestinal alkaline phosphatase, decapped with
tobacco acid pyrophosphatease, ligated with the 5'-RACE adaptor provided in the kit, and used for the
reverse transcription of the first cDNA strand with the random decamer primer provided in the kit. The
product was used for the first amplification of the nested PCR with the specific outer primer
(5´-TTCACCAAGTCAAGGCCAAATCTC-3´) designed according to the core sequence, and the
5'-RACE outer primer (5'-GCTGATGGCGATGAATGAACACTG-3') provided in the kit. The product
was used as template for the second amplification of the nested PCR with the specific inner primer
(5'-GCTGATGGCGATGAATGAACACTG-3') designed according to the core sequence, and the
5'-RACE inner primer (5'-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3') provided in
the kit. The product was cloned into pMD19-T vector (TaKaRa, Japan), sequenced at Invitrogen
(USA).
The full-length cDNA sequence of the AnMCSU gene was assembled with the sequenced results
of the core sequence, the 3'-RACE, and the 5'-RACE amplifications.
Fig. S.1 Expression vectors of the AnMCSU gene. a Expression vector pC2300-35S-AnMCSU-eGFP
for subcellular localization; b Expression vector pC2300-35S-AnMCSU for Arabidopsis transformation.
The AnMCSU-eGFP or AnMCSU gene is under the control of CaMV 35S promoter and octopine
synthetase terminator OCS. And the selection marker gene npt II was under the control of CaMV 35S
promoter and poly A terminater.
Fig. S.2 Amplified fragments of AnMCSU gene by RACE. a The core sequence of 960 bp amplified by
the degenerate primers. b. The 1170 bp fragment at the 3'-end amplified by the inner primers c The 753
bp fragment at the 5'-end amplified by the inner primers.
Fig. S.3 Full-length cDNA sequence and amino acid sequence of AnMCSU gene. The frames indicate
the start codon and the stop codon, respectively.
Fig. S.4 Phylogenetic tree among the putative proteins in A. nanus and other plants.
Fig. S.5 Specific PCR amplification of T3 plant lines. a AnMCSU. b 35S promoter. M: DNA maker;
CK+: positive control (pC2300-35S-AnMCSU); CK-: blank control; WT: untransformed wild-type
control; L4-25: T3 plant lines.