Isolation of Flower Development Regulator Gene SEPALLATA 1 in Phalaenopsis amabilis ( L . ) Blume

Phalaenopsis amabilis (L.) Blume is an indigenous orchid species in Indonesia. This orchid has a white large flower. The large flower is caused by the existence of gene that has an important role in flower development. One of the genes is SEPALLATA 1. This gene is a member of superfamily MADS-Box gene. SEPALLATA 1 gene is a marker of primordial flower organ. This study aimed to isolate SEPALLATA1 gene from Phalaenopsis amabilis (L.) Blume by PCR using forward primer 5’-GCT-GGA-GCG-GATCGA-GAA-CA-3’and reverse primer 5’-TCA-TGC-AAG-CCA-ACC-AGG-TG-3’. This study successfully amplified 691 bp lengths of SEPPALATA1 fragment, lacking 20 bp upstream which consist its start codon.


Introduction
Phalaenopsis amabilis (L.) Blume is a member of the family Orchidaceae.This orchid has unique flower form [1,2].The sepal is white-colored like the petal, but it has a different form.The sepals are ellipse to acute shaped while the petals are widened circular with a small base and dull top, one of petal modified to be labellum form.The labellum has a pale yellow color to dark yellow with red stripes on the inside [3,4].
There are stamen and pistil in the gynostemium [5].
The flowers begin to develop from primordial organ on apical and axillary shoots [6].In the initial primordial stage, the sepals are smaller than the leaves and petals and stamen is even smaller.Size reduction of the primordial form goes along with its formation change.Primordial on the top will conduct the phyllotaxis.There are three flower circles with 120 ∘ difference between flower sections in monocotyledons [7].
Flower development encoded by morphogenesis genes.One of the morphogenesis encoding genes is SEPALLATA (SEP).SEP is a member of superfamily MADS-box [8,9].

DNA extraction
Total DNA was extracted from young leaves using Geneaid Genomic DNA Mini Kit (Plant) protocol with some modification in incubation duration (about 4 h) and Proteinase-K addition.

SEP1 gene amplification
The primer was designed based on the conserve region in P. equestris.The pair of oligonucleotides was Forward F1: (5'ATG GGA AGA GGG AGA GTG GA-3'), Forward F2: (5'GCT GAA GCG GAT CGA GAA CA-3') and Reverse R1: (5'TCA TGC AAG CCA ACC AGG TG-3').PCR reactions were carried out in a total volume of 50 L.SEP1 gene was amplified using Qiagen Rotor-Gene Q. DFR gene amplification was done with 40 cycles of PCR which was initiated by template DNA initial denaturation at 94 ∘ C for 5 min, then followed by denaturation 94 ∘ C for 20 s, annealing at 56 ∘ C for 20 s, extension at 72 ∘ C for 50 s, and final extension at 72 ∘ C for 5 min.PCR product was examined using 1% agarose gel electrophoresis then checked using UV Transilluminator.DOI 10.18502/kls.v3i4.687

DNA sequence analysis
The sequencing of SEP1 gene was carried out in First BASE Laboratories, Malaysia.
DNA sequence were analyzed with FinchTV to read the chromatogram of sequencing product, DNA Baser to make a consensus sequence, Basic Local Alignment Search Tool (BLAST) to check the compatibility between target gene and query from Gene Bank, ClustalX to make multiple alignment between SEP1 gene in P. amabilis, P. equestris and other species.

Result and Discussion
The targeted SEP1 sequence from P. amabilis could not be amplified using F1/R1 oligonucleotides pair.Nevertheless, this study acquired SEP1 sequence of 691 bp length using F2/R1 pair (Figure 1).
The acquired DNA sequence then compared to SEP1 gene from P. equestris to determine their similarity index.BLAST analysis showed no similarity between P. amabilis and P. equestris.Mega 6 analysis showed 22.45% similarity between P. amabilis and P. equestris.SEP1 encodes of the sepals, petals and labellum.The low similarity index caused the different shape of sepals, petals and labellum between P. amabilis and P.  equestris (Figure 2).The similarity index of SEP1 sequences between P. amabilis and P. equestris is shown in Figure 3.
Alignment results showed that there are many differences between SEP1 bases from P. amabilis and SEP1 bases from P. equestris, including some discovered gaps.There are some gaps in 19th to 21st, 65th and 99th to 104th base of SEP1 sequences between P. amabilis and P. equestris, and another gaps in 45th, 85th to 88th and 130th base.This study could not amplify the start codon of P. amabilis due to failed F2/ R1 amplification of which the start codon be.SEP1 in P. amabilis was also compared to various species taken from Gene Bank databases, namely Arabidopsis thaliana, Camelina sativa (L.) Heynh., Eucalyptus grandis W. Hill ex Maiden, Malus domestica Mill, Nelumbo nucifera Gaertn.and Tarenaya hassleriana (Chodat) Iltis to analyze their similarity (Tabel 1).

Conclusion
The 691 bp length of SEPALLATA 1 gene was isolated from Phalaenopsis amabilis (L.) Blume F2/R1 oligonucleotide pair.The start codon of the sequence could not be obtained.The similarity index between SEPALLATA 1 gene in Phalaenopsis amabilis (L.) Blume and Phalaenopsis equestris (Schauer) Rchb.f is 22.45%.