Isolation And Characterization Of Genes Expressed In Early Flowering Tissues Of Teak (Tectona Grandis Linn. F)

Teak is a highly sought-after timber species in the world, and therefore has been selected as one of the timber species for forest plantation in Malaysia. However, in Malaysia teak has been observed to flower as early as three years after planting. The early flowering leads to the forking phenomenon, which lowers the quality of the timber produced. This study was initiated in order to understand the genetic control of flower development in teak with the ultimate aim of being able to manipulate this process for improvement of the species. In the observations of flower development in teak two different types of shoots were identified, flowering and vegetative shoots. The difference gave an opportunity to isolate the genes expressed in flowering shoots using the PCR-subtractive hybridization method. Based on 130 clones isolated, 22% were functionally unknown and 13% to 15% each were involved in cell structure, signal transduction and transcription. The other clones, 1% to 10% each, were involved in energy, protein synthesis, protein digestion and storage, disease and defense, intracellular traffic and metabolism.Out of the 130 clones analyzed, two were chosen for further analysis. The clones were TFS3-B7, which is similar to Late Elongated Hypocotyls (LHY) gene and TFS3-B17, which is similar to Arabidopsis Shaggy kinase-11 (AtSK-11) gene. The full-length cDNA of TFS3-B7 was 2948 base pair (bp) and potentially encoded for 768 amino acids. It was named Tectona grandis LHY (Tg-LHY), as the gene was similar to the LHY gene of some species. The level of gene expression was found to be high four hours after dawn in flowering shoots and flower, which might indicate involvement of the circadian clock system in teak flower development. Temperature might be a potential environmental cue detected by the teak circadian clock system, as the temperature was found higher within three months before the flowering season occurred. The cDNA of Tg-LHY translated into a protein of about 110 kD in a prokaryotic expression system. The gene construct of Tg-LHY in GATEWAY expression vector was also transformed into Arabidopsis. GUS assay analysis indicated successful integration of reporter gene into the Arabidopsis genome. Arabidopsis transformation will be further investigated in the future. The second clone, TFS3-B17, with its cDNA of 1705 bp in length, was potentially encoded for 410 amino acids. The gene was named Tectona grandis Shaggy kinase (Tg-SK), as it was similar to Arabidopsis Shaggy kinase-11 (AtSK-11). Analysis of the gene structure showed that it had 11 introns, similar to the number of introns found in AtSK-11. The high similarity between Tg-SK and AtSK-11 within their kinase region and structure might indicate their similar function. In Arabidopsis, AtSK-11 gene has been suggested to play a role in meristem identity fate. Higher transcription level of this gene was detected in early and later stage of flower development, which was similar with what has been reported in Arabidopsis. Gene expression analysis in a prokaryotic system showed that Tg-SK cDNA translated into a protein of about 40 kD

DNA Fingerprinting of Theobroma Cacao

Traditionally, the characterisation of Theobroma cacao is based on morphological characteristics and geographical distribution. Three major groups have been identified, namely, eriolla, Forestero and Trinitario (Wood, 1985). Crosses between these three groups have constitute the major breeding strategies used during the last few years. Currently, large numbers of cocoa clones derived from these crosses have been introduced in cocoa plantations. Two different techniques, Random Amplified Polymorphic DNA (RAPD) with random primers and Restriction Fragment Length Polymorphism (RFLP) using universal probes, has been investigated as a mean to differentiate 12 local cocoa clones with 1 imported clone as a comparison. 40 primers have been used to amplify genomic DNA from 13 cocoa clones. Agarose and acrylamide gel electrophoresis used to separate the RAPD products were further stained with ethidium bromide and sliver nitrate, respectively. NTSYS-PC computer programme was used to analyse the RAPD data. Meanwhile, two types of probe were used in RFLP study, viz M13 phage DNA and a fragment from a RAPD product. The RAPO technique was found to be able to differentiate between local cocoa clones. Combining the result with UPGMA analysis, 13 tested clones were determined to fall Into 3 main clusters. A band map has been formed as a future source of reference to determine the best combination of markers to differentiate two or more clones. M 13 DNA, as a probe in the RFLP study, failed to give a consistence results in differentiating cocoa clones. An RAPD product, as a probe however was able to hybridise to few digested genomic DNA fragments. This indicates that the product is truly amplified fragment and was confirmed to be mid-repetitive. However the probe did not produce any polymorphic bands between the clones

Protein expression of Late Elongated Hypocotyl (LHY) homolog genes of teak in Escherichia coli

Expression of an isolated gene in a system that directly translates it into a protein is an important step to study the protein encoded by the gene. The isolated gene can be expressed in vivo by a heterologous system. In this study, a bacteria system was used to translate the Tectona grandis Late Elongated Hypocotyl (Tg-LHY) gene, which was isolated from flowering tissues of teak (Tectona grandis). The gene was cloned into the pET 14b vector (Novagen) and transformed into BL 21(DE3)/pLysS and Rosetta 2 expression host cells (Novagen). Rosetta 2 host cell has been found to be a good candidate to express the Tg-LHY protein from plant origin, as it recognizes the codon that was found in plant but rarely used in bacteria. The expressed protein was about an expected size, which was 90 kD. Western blot analysis using antibody against His-tag, which was fused to the Tg-LHY protein, proved that the expressed protein was Tg-LHY protein

Status of root knot nematode disease on kenaf cultivated on BRIS soil in Kuala Terengganu, Malaysia

A field survey was carried out to investigate kenaf plants cultivated in Telaga Papan, Terengganu, Malaysia with respecting to their performance, root galling and growth characteristics. In this investigation, the aims were to assess the status of nematode infection on kenaf planted on this sandy BRIS area and estimate the incidence and severity of infection. Inspection of kenaf plants showed decolorating, drying and wilting of their leaves along with developing of galls on their root. Leaf colour and plant performance were found to be significantly different between infected and control plant. Infection with nematode was detected to reduce height, stalk diameter and number of node in kenaf plants. Root galling severity and percentage of active galls in kenaf roots were calculated as well. They showed to be closely correlated to each other. However, the relation between root galling severity and height was higher than with diameter and number of node

Effect of root knot nematode on growth and agronomic traits of Hibiscus cannabinus L. varieties

The purpose of the current study was to determine the effect of root-knot nematode infection on the growth of kenaf. Seeds of sixteen (16) varieties of kenaf, comprising of eight from Australia, three local and five accessions from Bangladesh were planted under controlled condition and inoculated with four level of 0, 1000, 5000 and 10000 RKN eggs per plant. The growth parameters of height, stalk diameter and number of node of plants were measured at interval of 30, 60, 90 and 120 days after planting. The results of this investigation showed that all kenaf plant cultivars studied here, differed in growth parameters (P= 0.05) both in the absence and presence of nematode infection. M. incognita race 1 was found that can infect all of these cultivars. The damage due to nematode; however; differed based on cultivar, level of inoculation and month of growth. Reduction of plant height and stalk diameter was observed in all inoculated varieties. Number of internode, however; was increased instead. In different growing time, the values for these parameters were shown to be different as a function of growing age and nematode infection. When infected with RKN, a superiority of varieties KK60 (M), G4 (AUST) and entry 3740 concerning to others were observed for height, stalk diameter and internode respectively. The growth parameters of resistant varieties treated with RKN was significantly better over the time as compared to the susceptible one

Function and expression pattern of Terminal Flower 1 (TFL1) homolog genes in dicot plants.

Terminal floweringl (TPLI) is a key gene in charge of flowering time inArabz'dopsz's thaliana. During the past decade, genetic studies have found out several TFL] like genes in dicot plants. The main issues addressed in this paper are current advances in TFL] homologs isolated from different dicot species and their function in flowering. Taking advantage of previous studies of cloning, in this paper we have evaluated function of these genes in regulating flowering time. It will then go on to place or parts of plants in where these genes express. Moreover, similarity and differences between them and other known genes, have been compared

Isolation and characterization of LHY homolog gene expressed in flowering tissues of Tectona grandis (teak)

Floral initiation of teak through molecular biology approach is being studied for better understanding of teak flower development. Through PCR subtractive hybridization method, LHY homolog gene has been isolated from teak flowering tissues. The full-length cDNA of the gene was 2948 base pair (bp) and potentially encoded for 768 amino acids. It was named Tectona grandis LHY (Tg-LHY), as the gene was similar to the LHY gene of some species. Amino acid sequence alignment revealed that Tg-LHY was similar to LHY of Castanea sativa, LHY ofPhaseolus vulgaris and LHY of Arabidopsis thaliana. The highly conserved region found in Tg-LHY was the MYB protein, which is the DNA-binding protein responsible in negative feedback loop reaction of central oscillator in plant circadian clock system. The level of gene expression was found to be high four hours after dawn in flowering shoots and flower. This paper reported the isolation and characterization of the gene

Analysis of expressed sequence tags derived from inflorescence shoot of Tectona grandis (teak)

Teak Inflorescence Shoot Stage 4 (TIS4) shoots bearing the floral meristems were used to construct a cDNA librariy with insert size range of 1500 - 5000 bp. The titer of the library was 7.5 x 105 pfu/ml (primary) and 4.5 x 109 pfu/ml (amplified). EST generation and analysis were performed using the cDNA library where a total of 1384 plaques were randomly picked and their inserts PCR-amplified using T3 and T7 universal primers. Only 1125 plaques generated single amplified fragments, each which were purified and sequenced using the SK universal primer. The generated raw 5’ ESTs were filtered and clustered. A total of 674 nonredundants (69 consensus sequences and 605 singletons) were generated and their identities searched through BLASTX. Of the 674 nonredundants, 107 of them (15.9%) showed no hits or no identity. All the 567 nonredundants identified through BLASTX were categorized into their functional categories and were further analysed using InterProScan to detect their protein signatures and to assign their GO numbers. From all the sequences analysed, only 186 (32.8%) sequences were given the GO numbers and grouped into the three GO main categories namely biological process, cellular component and molecular function. Several important ESTs were highlighted based on their functional categories. There were five sequences found to be related to flowering and light induction