No abstract

Capsicum frutescens L. cv. Cakra Hijau is a local cultivar that has been widely cultivated in Indonesia due to the pungency. Pungent on Capsicum is generated by capsaicin compound encoded by Pun1 gene. The sequences of Pun1 gene containing with two exons that located on the upstream and downstream, which are separated by introns in the middle. This study aimed to synthesis of cDNA of Pun1 gene from isolated total mRNA using two primers: F1/R1 (F1 5'-ATG GCT TTT GCA TTA CCA TCA-3'; and R1 5'-CTT AGC TCG AAG TGC ATC TA-3') to synthesis the exon-1 sequences and F2/R2 (F2 5'-GAA GGT GGC AGA AGA ATC AG-3'; and R2 5'-TTA GGC AAT GAA CTC AAG GA-3') to synthesis the exon-2 sequences. The cDNAs resulted from RT-PCR were visualized on 1.5% agarose gel electrophoresis. From this study we obtained a 1323 bp fragment cDNA.

Capsicum frutescens cv. CakraHijau is a local cultivar that has been widely cultivated in Indonesia due to its several advantages, including its pungency. Pungent taste of Capsicum is generated by capsaicin compound encoded by AT3 gene. Recently, 404 bp fragment of AT3 gene had been isolated. This research aimed to isolate upstream and downstream fragments of AT3 gene. PCR method used two pairs of primers: F2/R2 (F2 5'-TCT CCA TGC TGA CAA CAA CA-3', and R2 5'-CGA TGA AAG ATA GCT TGT G-3') and F3/R3 (F3 5'-GCA TCT CTT GCA GAG AGC ATA G-3', and R3 5'-TGT ACG CAC TCG TTG AGA CT-3'). F2/R2 primers amplified 326 bp upstream fragments, while F3/R3 primer amplified 261 bp downstream fragments. The alignment of those two fragments with one previously obtained produces a 675 bp partial sequence with 230 bp located upstream of presumed start codon. ClustalX analysis reveals that this fragment is located upper half compare to C. frutescens cv. Shuanla AT3 gene. Further primer design is necessary to obtain downstream of AT3 gene.

Capsicum frutescens L. is one of chili peppers with high pungency. Capsicum frutescens has several cultivars, one of those is C. frutescens cv. Cakra Hijau. This cultivars is known resistance to pests and diseases as well. Pungency is due to the accumulation of capsaicinoids. Pun1 is an important gene responsible for pungency. The full-leght genomic sequence of Pun1 is 1897 bp, containing two exons of 738 bp and 590 bpand one intron of 348 bp in between. This study was aimed to isolate Pun1 gene that free from intron. mRNA was isolated with TriReagent furthermore RT-PCR method used Qiagent One-Step RT-PCR and two pairs of primer : F1/R1 (F15'-ATG-

Pun1 gene is the one of candidate gene that responsible to determine pungency in Capsicum. In previous researches, 1 310 bp fragment of 1 671 bp Pun1 gene from Capsicum frutescens L. cv. Cakra Hijau had been isolated. The purpose of this research was to isolate of 3'-end fragment and get full length of Pun1 gene from C. frutescens L. cv. Cakra Hijau. DNA isolation was done using modified procedure. The method used to isolate the gene was PCR with a pair of primer, forward primer 5'-GAA-GGT-GGC-AGA-AGA-ATC-AG-3' and reverse primer 5'-TTG-TTG ACC-GTA-AAC-TTC-CG-3'. The result successfully to get 715 bp length DNA fragment. The assembly of this fragment into previous research produced a full length of 1 671 bp Pun1 gene from C. frutescens L. cv. Cakra Hijau consist of 738 bp first exon fragment, 348 bp intron fragment, and 585 bp second exon fragment.