Optimization of primer and polymerase chain reaction conditions to amplify COI locus for identification of Purnajiwa (Euchresta horsfieldii (Lesch.) Benn.) collected from Bedugul, Bali

The polymerase chain reaction (PCR) has been used for molecular research to amplify DNA fragments, especially from a small amount of genetic material. PCR has been applied for numerous researches, including for plant molecular identification. A good PCR product is dependent on good amplification of the DNA segment in optimum condition. This research was conducted to optimize the primer combination, and the annealing temperature for DNA barcoding application of Balinese rare medicinal plant (Euchresta horsfieldii (Lesch.) Benn.) collected from Bedugul. COI is a suitable primer to identify unknown species to higher taxa levels and species with high phenotypic plasticity. The range of annealing temperatures was tested to amplify the combination of five COI primers: GWSF and GWSF5 for forwarding primers and GWSR, GWSR3, and GWSR5 for reverse primers. The annealing temperature was 52, 54, 56, 58, 60°C for 30 seconds. The result showed that GWSF-GWSR, GWSF-GWSR5, GWSF5-GWSR, GWSF5-GWSR5, and GWSF5-GWSR3 produced multiple bands, the double band that cannot be cut, multiple bands, faint amplification band for GWSF5-GWSR5 and GWSF5-GWSR3, respectively. Primer combination of GWSF-GWSR3 with an annealing temperature of 52°Cproduced double bands that were available to have proceeded for sequencing. In conclusion, the combination of GWSF-GWSR3 that is annealed at 52°C produced the best amplification band. The primer combinations and conditions can be used for further identification of Purnajiwa collected from Bedugul.

Expression of Nitrite Transporter Gene to Increase Nitrate Absoprtion in Rice Plant (Oryza Sativa Cv Nipponbare) as Sole Nitrogen Source

Incorporated of CsNitr1-L-transgene in rice plant was showed to active and transport NO2 - from cytosol into chloroplast. With a PCR method screening of CsNitr1-L gene were confirmed which the CsNitr1-L-transgene have amplification band, while the plant were not contained CsNitr1-L gene, there are no amplification band. Some strain were obtained and got a seeds. Almost CsNitr1-L-transgene was raising in all parameter test, such as plant height, tillers number, respectively. The accumulation of NO2 - in the leaves of CsNitr1-L-transgene was lower compared with wild type. CsNitr1-L is a cucumber NO2 - -transporter gene that located in chloroplast membrane have been transformed into japonica rice cv. Nipponbare. A series experiments was carried out to understand the expression- CsNitr1-L-transgenic rice. The seeds were cultivated to observe the activity of CsNitr1-L-transgene with medium containing NO3 - or NO2 - as inducer. Agronomical test, like tillers number and plant height were observed and compare with non CsNitr1-L- transgenic rice. While the NO2 - content in leaves were analyzed with capillary electrophoresis method

Characterization of a tandemly repeated subtelomeric sequence with inverted telomere repeats in maize

In this study, two complementary telomere primers were applied to a single-primer PCR. A clear amplification band was obtained with one primer, while a smear pattern was seen with the other primer. Sequence analysis of the isolated clones from this specific amplification band revealed that a 412 bp clone designated as MTAS1 shared high homology with a reported subtelomeric sequence (382 bp) from maize ( Zea mays L.), which indicated that this clone was possibly present at subtelomeric regions. The clone MTAS1 displayed a novel structural feature flanked by the forward and inverted telomere repeats. Southern hybridization revealed a ladder of hybridization bands, suggesting that MTAS1 was a tandemly repeated sequence. Fluorescence in situ hybridization results showed that the strong MTAS1 signals were present at the ends of short arms of several long chromosomes, confirming that MTAS1 was a subtelomeric sequence and the high brightness of signals further indicated this cloned sequence was a highly and tandemly repetitive sequence in maize. Fluorescence in situ hybridization with telomeric DNA and MTAS1 as probes on metaphase chromosomes and extended genomic DNA fibers showed that hybridization signals of this clone located adjacent to or overlapped with signals of telomere tandem repeats distributed heterogeneously in subtelomeric regions of several chromosomes and even exhibited differences in two subtelomeres of a single chromosome.