cDNA Actin Isolated From Pandanus Sp.

Actin is one of the reference genes that is often used as an internal control in gene expression analysis. This study aimed to isolate actin cDNA from Pandanus sp originated from Riau. Fresh leaves Pandanus sp. Lake Kajuik, Langgam District, Pelalawan Regency, Riau Province. Isolation of RNA, synthesis of total cDNA, amplification of actin genes used McDowell's designed degenerate primer (PlAc46S-20/PlAc245N-20), electrophoresis, sequencing, and data analysis. Actin cDNA fragments obtained were 353 pb in size, registered at GenBank and encoded 117 amino acids. Actin cDNA fragment consists of two exons and one introne. Specific actin primers from Riau Pandanus sp. can be designed based on sequences obtained for the purpose of analyzing certain gene expressions.

Aktivitas ACCase mesokarp kelapa sawit dan kloning fragmen gen penyandi ACCase subunit biotin karboksilase ACCase activity of oil palm mesocarp and cloning of gene fragment encoding biotin carboxylase subunit of ACCase

Summary Genetic engineering to produce high yielding oil palm might be done by over expressing gene encoding key enzyme for oil biosynthesis in the oil palm mesocarp, one of which is ACCase. The objective of this research was to analyze ACCase activity of mesocarp from several developmental stages of fruit and to clone conserved region cDNA of gene encoding biotin carboxylase subunit of ACCase (BC-htACCase) from oil palm mesocarp. Activity of ACCase was analyzed by HPLC. Amplification of cDNA was done by means of reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate heterologous primer on several annealing temperature and MgCl2 concentration. The cDNA fragment of RT-PCR product was cloned, sequenced and analyzed to confirm that the cloned cDNA was conserved region of BC-htACCase. The result showed that ACCase activity increased from the 14 week to the 20 week-old fruit, and then decreased. Using heterologous degenerate primers, cDNA fragments of BC-htACCase conserved region (469 bp) can be specifically amplified at 60 oC annealing temperature with 2 mM MgCl2 concentration.The result of BlastX analysis showed that the sequence of cloned cDNA fragment was highly homologous with the conserved region of BC-htACCase from Glycine max, Arabidopsis thaliana, Nicotiana tabacum, and Brassica napus with 243, 237, 236, 231 bit score, and E. value 2e-63, 1e-61, 2e-61 and 5e-60, respectively. Ringkasan Rekayasa genetika untuk menghasilkan bibit kelapa sawit berdaya hasil tinggi dapat ditempuh dengan meningkatkan ekspresi gen penyandi enzim kunci biosintesis minyak pada kelapa sawit, salah satunya adalah ACCase. Tujuan penelitian ini adalah menguji aktivitas ACCase mesokarp beberapa tahap perkem-bangan buah sawit dan mengklon fragmen cDNA daerah konservatif gen penyandi ACCase heteromerik subunit biotin karbok-silase (BC-htACCase) dari mesokarp buah sawit. Aktivitas ACCase dianalisis dengan HPLC. Amplifikasi cDNA dilakukan dengan teknik RT-PCR menggunakan primer degene-rate heterologus pada berbagai suhu penempelan dan konsentrasi MgCl2. Fragmen cDNA hasil RT-PCR diklon, disekuen dan dianalisis untuk mengkonfirmasi bahwa cDNA terklon adalah daerah konservatif BC-htACCase. Hasil penelitian menunjukkan bahwa aktivitas ACCase meningkat dari buah berumur 14 minggu hingga buah berumur 20 minggu, kemudian menurun kembali Dengan primer degenerate heterologus, fragmen cDNA daerah konservatif BC-htACCase (469 pb) dapat diamplifikasi secara spesifik pada suhu penempelan 60 oC dan konsentrasi MgCl2 2 mM. Hasil analisis BlastX dari sekuen DNA fragmen terklon menunjuk-kan bahwa sekuen tersebut mempunyai homologi tinggi antara lain dengan gen penyandi BC-htACCase dari Glycine max, Arabidopsis thaliana, Nicotiana tabacum dan Brassica napus, masing-masing dengan skor 243, 237, 236, 231 bit, dan E. value 2e-63, 1e-61, 2e-61 dan 5e-60.

Aktivitas ACCase mesokarp kelapa sawit dan kloning fragmen gen penyandi ACCase subunit biotin karboksilase ACCase activity of oil palm mesocarp and cloning of gene fragment encoding biotin carboxylase subunit of ACCase

Summary Genetic engineering to produce high yielding oil palm might be done by over expressing gene encoding key enzyme for oil biosynthesis in the oil palm mesocarp, one of which is ACCase. The objective of this research was to analyze ACCase activity of mesocarp from several developmental stages of fruit and to clone conserved region cDNA of gene encoding biotin carboxylase subunit of ACCase (BC-htACCase) from oil palm mesocarp. Activity of ACCase was analyzed by HPLC. Amplification of cDNA was done by means of reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate heterologous primer on several annealing temperature and MgCl2 concentration. The cDNA fragment of RT-PCR product was cloned, sequenced and analyzed to confirm that the cloned cDNA was conserved region of BC-htACCase. The result showed that ACCase activity increased from the 14 week to the 20 week-old fruit, and then decreased. Using heterologous degenerate primers, cDNA fragments of BC-htACCase conserved region (469 bp) can be specifically amplified at 60 oC annealing temperature with 2 mM MgCl2 concentration.The result of BlastX analysis showed that the sequence of cloned cDNA fragment was highly homologous with the conserved region of BC-htACCase from Glycine max, Arabidopsis thaliana, Nicotiana tabacum, and Brassica napus with 243, 237, 236, 231 bit score, and E. value 2e-63, 1e-61, 2e-61 and 5e-60, respectively. Ringkasan Rekayasa genetika untuk menghasilkan bibit kelapa sawit berdaya hasil tinggi dapat ditempuh dengan meningkatkan ekspresi gen penyandi enzim kunci biosintesis minyak pada kelapa sawit, salah satunya adalah ACCase. Tujuan penelitian ini adalah menguji aktivitas ACCase mesokarp beberapa tahap perkem-bangan buah sawit dan mengklon fragmen cDNA daerah konservatif gen penyandi ACCase heteromerik subunit biotin karbok-silase (BC-htACCase) dari mesokarp buah sawit. Aktivitas ACCase dianalisis dengan HPLC. Amplifikasi cDNA dilakukan dengan teknik RT-PCR menggunakan primer degene-rate heterologus pada berbagai suhu penempelan dan konsentrasi MgCl2. Fragmen cDNA hasil RT-PCR diklon, disekuen dan dianalisis untuk mengkonfirmasi bahwa cDNA terklon adalah daerah konservatif BC-htACCase. Hasil penelitian menunjukkan bahwa aktivitas ACCase meningkat dari buah berumur 14 minggu hingga buah berumur 20 minggu, kemudian menurun kembali Dengan primer degenerate heterologus, fragmen cDNA daerah konservatif BC-htACCase (469 pb) dapat diamplifikasi secara spesifik pada suhu penempelan 60 oC dan konsentrasi MgCl2 2 mM. Hasil analisis BlastX dari sekuen DNA fragmen terklon menunjuk-kan bahwa sekuen tersebut mempunyai homologi tinggi antara lain dengan gen penyandi BC-htACCase dari Glycine max, Arabidopsis thaliana, Nicotiana tabacum dan Brassica napus, masing-masing dengan skor 243, 237, 236, 231 bit, dan E. value 2e-63, 1e-61, 2e-61 dan 5e-60.

IDENTIFICATION OF CDNA FRAGMENT TIGHTLY LINKED TO NV AND TM-2 LOCI IN TOMATO

Tm-2 is a resistance gene in tomato to Tomato Mosaic Virus (ToMV), located in heterochromatic region of chromosome nine. Since map based cloning difficult to perform for identify the gene on that region, we apply differential display approach by using two near-isogenic tomato lines (NILs), one without Tm-2 and the other with Tm-2 to identify cDNAs of the transcripts from the region surrounding the Tm-2 locus. Among the 150 combinations of three anchor primers and fifty arbitrary primers, 10 combinations generated cDNA polymorphic bands. Out of them, only one combination of CA6, exhibited polymorphic band under southern blot analysis, subsequently a genetic experiment showed that the CA6 locus tightly linked to the Tm-2 locus. The CA6 fragment also hybridized to genomic DNA fragments from a tomato line carrying Tm-2a, a line of L. peruvianum from which Tm-2a originated, and a tomato line carrying another Tm-2-like gene. A northern hybridization blotting result suggested that the gene corresponding to CA6 fragment was constitutively transcribed.

Molecular Cloning and Expression of β-1,4-endoxylanase Gene from Chaetomium cupreum in Yeast Saccharomyces cerevisiae

The gene encoding an endo-β-1,4-xylanase (XynCC) fromchaetomium cupreumwas amplified using PCR. The nucleotide sequence of a 690 bp cDNA fragment was determined. Based on the nucleotide sequence, calculated molecular mass of the enzyme was 24.7 kDa. The XynCC gene was inserted into the pYES2 vector and transferred into the cells ofS. cerevisiaeH158 for heterologous expression.

Study on Characteristics of Chemokine CXCL10 Gene Cloned from cDNA Expression Library of Ujumqin Sheep

Chemokines were a major regulator of body’s inflammatory and immune responses. In this study, the cDNA fragment of chemokine CXC ligand 10 (CXCL10) was cloned from the Ujumqin sheep ear marginal tissue cDNA expression library; the CXCL10 gene had 103 amino acids and a molecular weight of 11.47 kDa, and it shared a high homology among cattle, sheep, and goat, while a low homology compared with mouse. The CXCL10 protein had 4 conservative cysteine residues, located in 28, 30, 55, and 72 sites. The expression pattern and intracellular distribution of recombinant CXCL10 proteins in Ujumqin sheep fibroblast cells showed that there were green fluorescence signals both in cytoplasm and nucleolus after 24 h of transfection, the number of positive cells was increased with time, the peak level of fluorescence signal was reached after 48 h of transfection and the transfection efficiency was 33.3%; there was a significant decrease in fluorescence intensity after 72 h of transfection. Expression of recombinant CXCL10 gene inEscherichia colihad a time- and temperature-dependency on the amount of protein expression, and a small quantity of inducer was needed.

First Report of Passiflora virus Y Infecting Macroptilium atropurpureum in Taiwan

Macroptilium atropurpureum (siratro plants) is a perennial wild legume plant introduced to Taiwan as a forage crop in 1961 (3) and has become a naturalized weed found all over the island. In 2010, siratro plants with virus-like symptoms of mosaic and leaf deformation were observed on the campus of Da-Yeh University in central Taiwan. Flexuous virus-like particles about 750 × 12 nm were observed in the crude sap extracted from symptomatic leaves with a transmission electron microscope. Crude sap was mechanically inoculated to Chenopodium quinoa and local lesions can be observed on inoculated leaves 4 to 5 days after inoculation. Virus was purified from the leaves of inoculated C. quinoa with modified protocols of Gonsalves and Ishii (2). The virus coat protein (CP) consisted of a single major peptide with relative molecular weight of approximately 33 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral RNA extracted from the purified virus was used as a template and was primed with several primer sets corresponding to potyviruses and carlaviruses in reverse transcription-PCR to amplify possible corresponding cDNA fragments. After several attempts, a cDNA fragment of about 1,300 bp could be amplified with the degenerated primer set of BCMNV-F (5′CCDTGGACDGTWGGVATGAC3′) and BCMNV-R (5′CACCAHACCATRAARCCATTCAT3′), which were designed on the basis of the conserved region of the nuclear inclusion b (NIb) and CP genes of some potyviruses including Bean common mosaic necrosis virus, Soybean mosaic virus, Blackeye cowpea mosaic virus, East Asian passiflora virus, and Passion fruit woodiness virus. BLAST analyses showed the amplicon was highly homologous to that of Passiflora virus Y (PaVY). Together with oligo dT, a specific primer (5′GATGACACTCAAATGGCTG3′) corresponding to PaVY CP was used to amplify the cDNA fragment of the most 3′ region of the viral RNA (about 800 bp). The assembled cDNA fragment of 1,958 bp (Accession No. AB679294) contains a partial NIb gene (877 nt), a complete CP gene (819 nt), and the 3′ noncoding region (262 nt). The CP gene shared sequence identities of 89.4 to 98.9% and 92.7 to 98.9% in nucleotide and amino acid, respectively, to that of documented PaVY isolates. PaVY has also been found to be infecting Vigna trilobata, Rhynchosia minima, Clitoria ternatea, and Passiflora foetida in Australia (1). Here we present the first report to our knowledge of PaVY and its infection of siratro (M. atropurpureum) in Taiwan. Additional work is needed to investigate the spread of PaVY and its interaction with other legume plants in Taiwan. References: (1) B. A. Coutts et al. Arch. Virol. 156:1757, 2011. (2) D. Gonsalves and M. Ishii. Phytopathology 70:1028, 1980. (3) Y. Y. Lai et al. J. Taiwan Livestock Res. 42:19, 2009.

Lessons Learned from the Transgenic Huntington's Disease Rats

Huntington's disease (HD) is a fatal inherited disorder leading to selective neurodegeneration and neuropsychiatric symptoms. Currently, there is no treatment to slow down or to stop the disease. There is also no therapy to effectively reduce the symptoms. In the investigation of novel therapies, different animal models of Huntington's disease, varying from insects to nonhuman primates, have been created and used. Few years ago, the first transgenic rat model of HD, carrying a truncated huntingtin cDNA fragment with 51 CAG repeats under control of the native rathuntingtinpromoter, was introduced. We have been using this animal model in our research and review here our experience with the behavioural, neurophysiological, and histopathological phenotype of the transgenic Huntington's disease rats with relevant literature.

In silico analysis of a putative SPIRAL gene related to strawberry ripening

The aim of this study was to characterize a gene associated with ripening in strawberry, a non-climacteric fruit. Differently expressed transcripts of candidate genes functioning in fruit development and ripening were identified from strawberry ( Fragaria × ananassa Duch.) in four ripening stages using the cDNA-AFLP method. The cDNA fragment designated C11M32M003 was selected from the putative ripening-related genes for further analysis. This transcript accumulated in the green receptacle, and the achene, but gene expression decreased in both tissues in parallel with the progress of ripening (Balogh, 2006). In silico analysis revealed that both the cDNA-AFLP fragment (C11M32M003) and the full-length cDNA AY695666 showed over 60% homology at the nucleotide level with two gene groups found in various plant species, including Arabidopsis thaliana . One of the candidate groups consisted of NITRILASE sequences thought to be related to auxin biosynthesis. As an alternative, a lesser known gene group named SPIRAL was suggested. The results of the detailed bioinformatic comparisons presented in this paper prove that the strawberry sequence analysed belongs to the SPIRAL gene family.