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Black soybean [ Glycine max (L.) Merr.] produces isoflavones as secondary metabolites, which have many benefits for human health and plant defense system. Expression profiling can guide potential work in functional genomics of the isoflavone biosynthesis pathway. Previous studies showed the vital role of the CHS8, CHI1A, and IFS 2 genes in isoflavone biosynthesis. However, expression profiling of these genes in the local black soybean varieties is still limited. This study investigated the gene expression levels of the CHS8, CHI1A, IFS2, and CHR genes in local varieties, namely, UP106 (high isoflavone) and UP122 (low isoflavone) and its progenies, i.e., UP106xUP122 and UP122xUP106. Relative gene expression profiling was conducted on the basis of Reverse Transcriptase Polymerase Chain Reaction (RT‐PCR) with ACT2/7 as a housekeeping gene. As a result, the expression level of CHS8 in UP122 is lower than that in UP106. No significant difference in the expression level of CHI1A was observed in all samples. The expression levels of CHS8 and CHI1A in both progenies were higher than that in the parental line, whereas the expression levels of IFS2 in both progenies were lower than that in the parental line. CHS8 and IFS2 expression from UP106xUP122 was higher than that from UP122xUP106, whereas CHI1A expression from UP122xUP106 was higher than that from UP106xUP122. CHR showed a high expression in the reciprocal cross; however, this expression did not exceed from UP106. In conclusion, the crossing between parental lines did not affect the gene expression level in the isoflavone biosynthesis pathway.

Background: Human papillomavirus (HPV)-45 genotype circulates in high percentage in Bandung area - Indonesia, after HPV-16 and HPV-18. The aim of this study was to analyse variations of major capsid (L1) HPV-45 and its phylogeny. Furthermore in silico protein structure and epitope prediction was explored. Methods: L1 gene of HPV-45 was amplified, sequenced and aligned. Phylogenetic tree had been built and compared with a complete L1 HPV-45 sequence. Structure and epitope prediction of L1 protein were then developed in silico. Results: Of 5 L1 HPV-45 sequences collected, we have detected one variant of sub lineage A2 which was considered as a new variant, and two variants of B2. Superimposition of structure of these two variants with reference showed very similar structure. Furthermore, seven amino acid substitutions were found within these L1 variants of which two substitutions might change the polarity of corresponding amino acid I329T and S383G. The S383G occurred in surface loop (HI-Loop) of new L1 HPV-45 variant. Conclusion: Similar structure of Indonesian variants indicates that amino acids variations do not affect the L1 structure. However, one substitution with altered amino acid polarity found within the area of surface loop suggests a potential impact in antibody recognition and neutralization.

Pyrophosphate-dependent Phosphofructokinase or PFP is an enzyme that regulate sucrose metabolism. It consists of α- and β-subunits, which encoded by PFPα and PFPβ genes, respectively. Sugarcane PFPa has a strong function in glycolysis and has the potency to be engineered to increase sugarcane yield. Hence, the purpose of this work was to isolate, clone and characterize the sugarcane PFPα gene. Total RNA was isolated from leafrolls of TD 91 sugarcane variety. cDNA synthesis followed by DNA amplification of PFPα gene were performed using degenerate primers. cDNA and DNA fragments were ligated into pGEM-T Easy vector, which were subsequently introduced to E. coli competent cells. EcoRI were used to cut the plasmids for sequencing. Finally, homology searching was conducted using BLASTn, and then the nucleotide sequence was translated to a protein sequence using Bioedit. The results showed that PFPα cDNA fragment was 900 bp in length. The translated PFPa protein showed binding sites for fructose-6-phosphate and fructose-1,6-biphosphate, which are conserved in all family members of PFP. In silico analysis of the DNA fragment showed gene without intron. In conclusion, the PFPα gene from sugarcane has been successfully isolated, cloned and characterized.

N owadays , t he development of molecular biology techniques has enabled to engineer drought tolerant sugarcane to accelerate thebreeding program. Dehydrin (DHN) that belong to the g roup II l ate e mbryogenesis a bundant (LEA) family is known to have an important role in plant response and adaptation to abiotic stresses (drought, high salinity, cold, heat, etc.). Literature study and bioinformatics analysis reported that DHN 1 gene on sugarcane showed high homology sequences with sorghum DHN. The e xpression of DHN 1 gene on sugarcane var. PSJT 941 treated with various period of drought stress had been conducted using semi-quantitative reverse transcriptase (RT)-PCR method . The results showed that the expression level of DHN 1 gene increased along with the increased period of the treatment. The highest expression level of DHN1 gene was resulted from plants that had been subjected to drought for 25 days. Amplification of DHN 1 gene from plants with the highest gene expression, resulted an amplicon with a size of 465 bp which represent s a full length coding sequence (CDS) of DHN1. Identification using Blast analysis showed that DHN 1 sequences from sugarcane var. PSJT 941 shared high homology with DHN gene on sugarcane and sorghum. The alignment results also revealed a conserved motif that characterized DHN genes. [Key words: drought stress, dehydrin, DHN 1 gene, sugarcane ] Abstrak Dengan berkembangnya teknik biologi molekuler saat ini, maka perakitan tanaman tebu yang toleran kekeringan lebih diarahkan melalui teknik rekayasa genetika untuk mempercepat program pemuliaan tanaman. Protein dehydrin (DHN) yang termasuk ke dalam kelompok II famili LEA ( L ate E mbryogenesis A bundant )diketahui berperan penting dalam respon dan adaptasi tanaman terhadap cekaman abiotik (kekeringan, salinitas tinggi, suhu dingin, panas, dll). Studi literatur dan analisis bioinformatika menunjukkan bahwa gen DHN1 pada tanaman tebu memiliki homologi yang tinggi dengan gen DHN pada sorghum. Analisis ekspresi gen DHN 1 pada tanaman tebu varietas PSJT 941 yang diberi cekaman kekeringan telah dilakukan menggunakan semi-kuantitatifreverse transcriptase (RT)-PCR dan terlihat bahwa ekspresi gen DHN 1 meningkat secara nyata sejalan dengan semakin lamanya waktu pemberian cekaman. Tingkat ekspresi gen DHN1 paling tinggi diperoleh dari tanaman yang mengalami cekaman kekeringan selama 25 hari. Amplifikasi gen DHN 1 pada tanaman dengan tingkat ekspresi yang paling tinggi menunjukkan pita dengan ukuran 465 bp yang merepresentasikan full coding sequence (CDS) gen DHN1 . Identifikasi menggunakan analisis Blast menunjukkan bahwa sekuen gen DHN1 dari tanaman tebu varietas PSJT 941 yang diperoleh memiliki homologi yang tinggi dengan gen DHN pada tanaman tebu dan sorghum. Hasil penjajaran sekuen protein juga menunjukkan adanya motif lestari yang mencirikan gen DHN . [Kata kunci: cekaman kekeringan, dehydrin , gen DHN1 , tebu]

Increasing world sugar demand might be fulfilled with land extensification which include the use of dry area. Development of drought tolerance and high productivity sugarcane variety could be achieved by plant genetic engineering. Under drought condition, proline will be accumulated and functioned as an osmoregulator in plant cells. ∆ 1 - pyrroline -5- carboxylate synthase (P5CS) is one of the important enzymes in proline biosynthesis. This enzyme is encoded by P5CS gene family. We cloned two homologous P5CS genes from sugarcane, SoP5CS1 (Accession Number : KF178299) and SoP5CS2 (Accession Number : KF178300), which encode 729 and 716 amino acid polypeptides. The identity between these two genes was 74% based on nucleotide sequences. The SoP5CS1 gene had 98% identity with SbP5CS1 (Accession Number : GQ377719.1) and SoP5CS2 had 99% identity with SaP5CS (Accession Number : EF113257.1). In this experiment, sugarcane plantlets were exposed to medium containing PEG 6000 (40%) for 12, 24, 48, and 72 hours. Proline concentration was measured after treatment and genes expression were analyzed by real time-qPCR. The results showed that the proline concentration was increased 12 folds (9.8 umol.g -1 ) after 48-hours stress treatment. The highest expression of SoP5CS1 occured at 24-hours treatment with approximately 16 times from plant without PEG (control plant) and decreased gradually at 48 and 72 hours treatment. The highest expression of SoP5CS2 occured at 24-hours drought stress with approximately 3.6 folds compared to control. In drought treatment, the expression level SoP5CS1 was higher than SoP5CS2 and has increased significantly at 12-hours treatment. It is suggested that the SoP5CS1 gene contributes more significantly to the production of proline during drought stress than SoP5CS2 . Hence, SoP5CS1 could potentialy be used as a marker to screen sugarcane variety for drought tolerance and for the development of transgenic plant tolerant to drought. Keywords: cloning, drought, expression, P5CS, sugarcane

Amplification of putative potBCD genes from Serratia rubidae was conducted by PCR using a pair of primers FERC and RERC. A fragment with ~1800 bp size was ligated to pGEM-T Easy vector then cloned to competent Escherichia coli DH5α. The recombinant plasmid was sequenced using SP6, T7 and two internal primers, FPC and RPC. A sequence similarity search and analysis was performed with the BLASTN program. The sequence was found to have 83% similarity to potABCD genes from E. coli. Those genes encoded a spermidine-preferential uptake system that consists of four kinds of protein: PotA is a membrane-associated ATPase, PotB and PotC are transmembrane proteins that form channels, and PotD is a periplasmic substrate-binding protein. Alignment analysis showed that the isolated clone consisted of potB (partial), potC (full length) and potD (partial). The sequences for potBCD genes from S. rubidae are not available in the NCBI database. Furthermore, we have submitted this sequence, potBCD from S. rubidae , on GeneBank with Acc. number FJ447342.

Human papillomavirus (HPV) type 52 is the most prevalent type for causing cervical cancer in Indonesian population. Cervical cancer becomes the most common cancer suffered by Indonesian women. Prevention of HPV infection can be achieved using HPV virus-like particle (VLP) vaccine derived from L1 major capsid protein. This study aimed to clone and analyze HPV-52 L1 gene. DNA obtained from biopsy of a cervical cancer patient was amplified using specific primers designed from Asian originated HPV-52 L1 gene available in the GenBank. The isolated HPV-52 L1 gene sequence was submitted to GenBank with accession number [KF225497]. Expression of HPV-52 L1 gene was performed using pRSET/EmGFP Escherichia coli expression vector. We analyzed and compared the HPV-52 L1 gene expressions from recombinant E.coli BL21 (DE3) that had been induced for 3 hours with 1 mM IPTG and without induction. The protein was expressed in insoluble form. We performed the following bioinformatic analyses: construction of phlyogenetic tree, T-cell epitopes prediction and 3D proteins structure modelling. We utilized the following softwares: MEGA5 for phylogenetic tree, IEDBann for MHC prediction, CLC DNA Workbench 6.5 for hydrophobicity analysis and PDB-Viewer Deep for 3D protein structure analysis. The phylogenetic tree which was developed based on [KF225497] sequence showed that it shared a branch with Asian countries (Philippines and Thailand). The deduced amino acid sequences of the predicted epitopes that were consistent in all of the programs were 259GTLGDPVPGDLYIQGS274 and 345KKESTYKNE353. This information may be useful to design diagnostic strategies and vaccine suitable for Indonesian population.

Transgenic plant technology is an efficient tool to study the function of gene(s) in plant. The most popular and widely used technique is Agrobacterium-mediated transformation in which cocultivation was done by immersing the plant tissues/organ in overnight bacterial cultured for about 30 minutes to one hour under in vitro condition. In this experiment, we developed more easier technique that omitted the in vitro step during cocultivation with Agrobacterium, namely in planta transformation method. Pollinaria (compact pollen mass of orchid) of Phalaenopsis amabilis and Spathoglottis plicata orchids were used as target explants that were immersed into bacterial culture for 30 minutes, then dried up the pollinaria, the transformed pollinaria was used to pollinate orchid flowers. The T-DNA used for this experiments were Ubipro∷PaFT/A. tumefaciens GV3101 for P. amabilis and MeEF1α2 pro∷GUS/ A. tumefaciens LBA 4404 for S.plicata. Seeds that were produced from pollinated flowers were grown onto 10 mg/l hygromicin containing NP (New Phalaenopsis) medium. The existance of transgene in putative transformant protocorm (developing orchid embryo) genome was confirmed using PCR with specific primers of either PaFT or GUS genes. Histochemical GUS assay was also performed to the putative transformants. The result showed that transformation frequencies were 2.1 % in P. amabilis, and 0,53% in S. plicata. These results indicates that in planta transformation method could be used for Agrobacterium-mediated genetic transformation, with advantage easier and more secure work from contaminants than that of the in vitro method.

Klebsiella oxytoca produces sucrose isomerase which catalyses the conversion of sucrose to isomaltulose, a new generation of sugar. From the previous study, palI gene from Klebsiella oxytoca was succesfully isolated from sapodilla fruit (Manilkara zapota). The full-length palI gene sequence of Klebsiella oxytoca was cloned in E. coli DH5α. The deduced amino acid sequence shows 498 residues which includes conserved motif for sucrose isomerisation 325RLDRD329 and 97% identical to palI gene from Klebsiella sp. LX3 (GenBank:AAK82938.1). This fragment was succesfullly ligated into the expression vector pET-32b using overlap-extension PCR and cloned in Escherichia coli BL21 (DE3) pLysS. DNA sequencing result shows that palI gene of Klebsiella oxytoca was inserted in-frame in pET-32b. This is the first report on cloning of palI gene from Klebsiella oxytoca.

In order to isolate an elongation factor-1α (EF-1α)gene from cassava, a lambda EMBL3 genomic library, made from a single cassava genotype (MBRA 534, from the CIAT cassava germplasm collection), was screened with a full length EF-1α cDNA clone(blt63) from barley. Six positive clones were isolated from an amplified library and 4866 bp from one clone (MeEF1) were subcloned into pGEM3zf(-) and pGEM5zf(-). The sequence has 2709 bp5' of the translation start site, 1347 bp of coding sequence split into two exons by an intron (374 bp) and 435 bp 3' of the stop codon. A 657 bpintron was also found 5' of the translation start site. The coding sequence is very similar, with 86% DNA sequence identity and 95% deduced amino acid sequence identity, to an EF-1α gene from Arabidopsis thaliana. The promoter region of MeEF1 contains 3putative control elements that are located upstream of the transcription start site. These control elements include a TEF1 box, a TELO box and two TATA boxes. The gene is expressed in early stages of development and in young tissue. Transient expression using particle bombardment shows that the promoter drives uidA gene expression in leaves of cassava and Arabidopsis. An interesting feature of the MeEF1 gene is that the presence of the 5'UTR intron affects the level of expression.

In plant genetic engineering, the identification of gene promoters leading to particular expression patterns is crucial for the development of new genetically modified plant generations. This research was conducted in order to isolate and characterize several new promoters from cassava (Manihot esculenta Crantz) elongation factor 1 alpha (EF1A) gene family.Three promoters MeEF1A3, MeEF1A5 and MeEF1A6 were successfully isolated [corrected]. Sequence analyses showed that all of the promoters contain three conserved putative cis-acting elements which are located upstream of the transcription start site. These elements are included a TEF1, a TELO and TATA boxes. In addition, all of the promoters also have the 5'UTR intron but with a different lengths. These promoters were constructed translationally with gusA reporter gene (promoter::gusA fusion) in pBI-121 binary vector to build a new binary vector using Overlap Extension PCR Cloning (OEPC) technique. Transient expression assay that was done by using agroinfiltration method was used to show functionality of these promoters. Qualitative and quantitative analysis from GUS assay showed that these promoters were functional and conferred a specific activity in tobacco seedlings (Nicotiana tabacum), tomato fruits (Solanum lycopersicum) and banana fruits (Musa acuminata). We hypothesized that MeEF1A6 could be categorized as a constitutive promoter because it was able to drive the gene expression in all transformed tissue described in here and also comparable to CaMV35S. On the other hand, MeEF1A3 drove specific expression in the aerial parts of seedlings such as hypocotyl and cotyledon thus MeEF1A5 drove specific expression in fruit tissue. The results obtained from transient analysis showed that these promoters had a distinct activity although they came from same gene family. The DNA sequences identified here are new promoters potentially use for genetic engineering in cassava or other plants.

Musa acuminata cultivar pisang ambon lumut is a native climacteric fruit from Indonesia. Climacteric fruit ripening process is triggered by the gaseous plant hormone ethylene. The rate limiting enzyme involved in ethylene biosynthesis is ACC synthase (ACS) which is encoded by ACS gene family. The objective of this study is to identify MA-ACS gene family in M. acuminata cultivar pisang ambon lumut and to study the MA-ACS1 gene expression. The result showed that there were nine M. acuminata ACS gene family members called MA-ACS1–9. Two of them (MA-ACS1 and MA-ACS2) were assessed using reverse transcriptase PCR (RT-PCR) for gene expression study and it was only MA-ACS1 correlated with fruit ripening. The MA-ACS1 gene fragment has been successfully isolated and characterized and it has three introns, four exons, and one stop codon. It also shows highest homology with MACS1 gene from M. acuminata cultivar Hsian Jien Chiao (GenBank accession number AF056164). Expression analysis of MA-ACS1 using quantitative PCR (qPCR) showed that MA-ACS1 gene expression increased significantly in the third day, reached maximum at the fifth day, and then decreased in the seventh day after harvesting. The qPCR expression analysis result correlated with the result of physical analysis during fruit ripening. Key words: pisang ambon lumut, MA-ACS gene family, gene characterization, gene expression, quantitative PCR (qPCR)