Biology Reference
In-Depth Information
province, China, where plants of Opithandra dinghushanensis W. T. Wang are
mainly distributed.
Gene Cloning and Sequence Analyses
CYC-like genes were isolated from O.dinghushanensis using degenerate oligonu-
cleotide primers in 3' and 5'-RACE according to described methods [47] and the
manufacture's protocol (INVITROGEN), respectively. Two D3-type cyclin genes
were also isolated from O. dinghushanensis using the above methods. Total RNA
was extracted from the floral buds of O. dinghushanensis using the Plant RNA
Purification Reagent (INVITROGEN) according to the manufacture's protocol.
First-strand cDNAs were synthesized from total RNA with the Supertranscript™
III RNase H- Reverse Transcriptase (INVITROGEN). To examine the intron/
exon structures we isolated and sequenced the corresponding genomic DNA of
OpdCYC and OpdcyclinD3 genes from leaves.
According to the known sequence information, phylogenetic analy-
ses of CYC-like and D-type cyclin genes were conducted to identify the po-
sition of CYC-like and D3-type cyclin genes isolated herein in their gene
families, respectively. AmCYC and AmDICH are from A. majus [4,5], and
LjCYC1/2/3, TCP1/12/18 and TB1 are from Lotus japonicus [13], Arabi-
dopsis [12] and maize [34]. GCYC1A/1B are from Saintpaulia ionantha and
Streptocarpus primulifolius [43-45]. GCYC1C/1D (designated as GCYC1
respectively in some taxa) are from Chirita heterotricha [25], Jankaea helde-
ichii [43], Conandron ramondioides, Haberlea ferdinandi-coburgii, Primulina
tabacum, Ramonda myconi [44], Cyrtandra apiculata, Didymocarpus citri-
nus and Loxostigma sp. [46]. GCYC2 (2A/2B) are from Chirita heterotricha
[25], Conandron ramondioides, Haberlea ferdinandi-coburgii, Ramonda my-
coni [44], Cyrtandra apiculata [46] and Didymocarpus citrinus (Yin-Zheng
Wang, unpublished). Amino acid sequences of D-type cyclin genes are from
A. majus (Amcyclin D1/D3a/D3b) [6], Populus trichocarpa (Ptcyclin D1/
D2/D3) [48], Nicotiana tabacum (Ntcyclin D/D3) [49], Solanum tuberosum
(Stcyclin D) (accession nos.EU325650), Lycopersicon esculentum (Lecyclin
D3-1) [50] and Arabidopsis (Atcyclin D1/D2/D3) [32]. Phylogenetic analy-
ses with the neighbor joining method and p-distance were carried out using
PAUP*4.0b4a [51] and bootstrap was estimated with 1,000 resampling rep-
licates. The DNA sequences of genes reported in this paper, i.e. OpdCYC1C,
OpdCYC1D, OpdCYC2A, OpdCYC2B, OpdcyclinD3a and OpdcyclinD3b
have been deposited in the GenBank database (accession nos. FJ710518,
FJ710519, FJ710520, FJ710521, FJ710522 and FJ644637).
