Protein degradation can occur through Ubiquitin 26S-Proteosome System (UPS). The degradation can be mediated by
the SCF E3 ubiquitin ligase complex consisting of Skp1, Cullin, and F-box protein as the main components. The F-box
protein at the C-terminal domain functions to recognize the targeted protein to be ubiquitinated and degraded via UPS.
A stress-responsive F-box gene, PmF-box1 from Persicaria minor was categorized in the F-box containing kelch repeat
(FBK) family; a family that specific to plant kingdom. To identify the targeted protein of PmF-box1, yeast-two hybrid system
(Y2H) was used. In the Y2H screening process, mating efficiency is very important to fish out the interacting proteins.
Therefore, one modification was conducted to increase the mating efficiency. In this screening, PmF-box1 was used as a
bait to screen for the Y2H library which was constructed using RNA from plant samples treated with abscisic acid (ABA)
and polyethylene glycol (PEG)-8000 and control sample. Autoactivation and toxicity tests of bait were performed before
the Y2H screening. Tests on PmF-box1 showed that it is not toxic to the yeast and cannot autoactivate the yeast reporter
genes. Mating efficiency was improved from 2.07% to 9.15% after addition of PEG-4000 in the mating culture compared
to the original protocol, which it also increased the colony number in the screening step afterward. Additionally, bands
of gene with different sizes were observed on electrophoresis gel after colony PCR analysis from the improved technique.
Those genes may code for potential interacting proteins that needs further identification and confirmation.
Kapsaisinoid merupakan alkaloid yang memberikan ciri kepedasan pada cili serta khusus pada genus Capsicum. Sebatian kapsaisinoid terdiri daripada dua komponen utama iaitu kapsaisin dan dihidrokapsaisin. Dalam kajian ini, pengklonan cDNA Kapsaisin sintase (Cs) telah berjaya dilakukan menerusi kaedah transkripsi berbalik PCR (RT-PCR) dan klon cDNA tersebut dinamakan CUKMCS yang bersaiz 981 pb. Pencarian homologi menggunakan program blastx dan blastp yang terdapat pada pangkalan data NCBI mendapati CUKMCS mempunyai persamaan yang sangat tinggi terhadap Cs pada Capsicum frutescens, Capsicum annuum dan Capsicum chacoense. Saiz ramalan protein CUKMCS diangggarkan sekitar 36 kDa. Penentuan pengekspresan transkrip Cs pada 5 tisu yang berbeza mendapati transkrip dikesan pada tisu plasenta, mesokarp dan biji manakala tiada transkrip Cs dikesan pada daun dan akar
Kajian ke atas kaedah inokulasi eksplan menggunakan vektor penandaan tanpa promoter, pCAMDIN ke atas tomato varieti tempatan MT1, bagi tujuan mutagenesis penyelitan T-DNA menggunakan kaedah infiltrasi agro telah dijalankan. Bahagian daun muda disuntik dengan larutan yang mengandungi Agrobacterium tumefaciens, 100 mg/l Str, 50 mg/l Kan, 10mM MES dan 100 μM Acetosyringone. Infiltrasi dilakukan ke atas sampel daun dengan menggunakan medium LB, bahagian permukaan daun dilukakan dan infiltrasi agro dilakukan ke atas sampel daun pokok tomato yang berada di dalam rumah kaca. Tisu yang diinfiltrasi tidak mengalami kecederaan dan kekal hijau selepas 5 hari. Infiltrasi yang
mudah dilakukan dan pengekspresan transien GUS yang dapat dicerap berlaku pada sampel yang dianalisa adalah antara kebaikan kaedah ini. Seterusnya, fragmen gen gus telah berjaya diamplifikasi. Kejayaan integrasi gen gus ke dalam tomato juga dapat dilihat melalui penghibridan Southern. Berdasarkan keputusan ini, dapat dicadangkan bahawa penyelitan transgen daripada vektor rekombinan pCAMDIN telah berlaku dalam genom tomato.
Agrobacterium-mediated transformation of indica rice is undoubtedly a challenging task due to the rice recalcitrant nature to transformation process. Therefore, optimization of the transformation protocol is important for specific indica rice cultivar to ensure effectiveness of the transformation. In this study, crucial parameters affecting Agrobacteriummediated transformation were optimized to obtain transgenic rice of local rice cultivar (indica MR219). Embryogenic calli were chosen for inoculation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pH2GW7ABP57 containing gene of interest, Auxin binding protein 57 (Abp57). The parameters that have been optimized were the immersion time, co-cultivation period, acetosyringone concentration and co-cultivation temperature. A total of four days co-cultivation period and 30 min immersion of embryogenic callus are optimum for the transformation of MR219 with transformation efficiency of 26.4% and 16.0%, respectively. Acetosyringone at 200 μM and co-cultivation at 28°C also gave the highest transformation efficiency (14.4 and 18.4%, respectively). Meanwhile, inclusion of 20 g/L maltose+20 g/L sorbitol into the regeneration media has significantly improve the transformed somatic embryos growth and increase the regeneration efficiency up to 40.0%. The results of polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) indicated that the transgene was successfully integrated and overexpressed in transgenic rice of MR219. In conclusion, significant improvement in transformation efficiency for rice cv. MR219 has been obtained by using the optimised protocol for transformation and regeneration developed in this study.
Ten Elaeis oleifera microsatellite markers were developed and characterised from 1500 sequences of the E. oleifera genomic library. The markers were utilised to assess the genetic diversity of E. oleifera germplasm collections from four South American countries (Colombia, Costa Rica, Panama and Honduras). The number of alleles per-locus varied from 2 to 11 and the observed and expected heterozygosity ranged from 0.0685 to 0.9853 and 0.1393 to 0.8216 respectively. Majority of the markers showed transferability to Elaeis guineensis while two markers showed transferability across Arecaceae taxa. These E. oleifera microsatellite markers are expected to become useful tools to determine the population structure and conservation of E. oleifera populations.
Metalotionin (MT ) merupakan protein pengikat logam berberat molekul rendah dan kaya dengan sistein yang hadir dalam pelbagai jenis organisma termasuklah bakteria, kulat, tumbuhan dan haiwan. MT tumbuhan dipercayai mengambil bahagian dalam metabolisme dan penyahtoksikan logam dengan cara pengkelatan ion-ion logam berat. Fungsinya yang unik ini telah mendorong minat untuk memencilkan gen MT daripada rumput sambau, Eleusine indica. DNA pelengkap (cDNA) eiMT 1 telah diklonkan ke dalam vektor binari pBI121 untuk ditransformasikan ke dalam pokok tembakau melalui perantaraan Agrobacterium tumefaciens. Penyaringan pokok tembakau transgenik dengan PCR dilakukan menggunakan 3 pasang pencetus yang direka khas iaitu pasangan CMT F dan CMT R, 35SF dan PMT R, dan pasangan pencetus khusus-gen MT FS2 dan MT RS2. Ketiga-tiga pasangan pencetus ini berjaya menghasilkan saiz serpihan DNA jangkaan iaitu masing-masing 270 pb, 1.1 kb dan 170 pb. Penjujukan terhadap serpihan bersaiz 170 pb dan analisis jujukan menunjukkan persamaan 100 % dengan eiMT 1. Kajian pengekspresan gen melalui pendekatan transkripsi berbalik-PCR membuktikan bahawa transgen eiMT 1 telah berjaya diekspreskan dalam 11 daripada 19 pokok transgenik yang dikaji.
F-box proteins containing variable C-terminal domains make an essential part of SKP1-Cullin-Ring box-F box (SCF)
complex. SCF complex catalyzes the final step to link the ubiquitin tag with the target protein, destined for degradation,
through F-box protein that confer overall substrate specificity to the complex. In this study, we analyzed the role of
At2g02870, a Kelch containing F-box protein from Arabidopsis thaliana, by using reverse genetics strategy. At2g02870
loss of function mutant lines (at2g02870) were analyzed and compared with wild type plants for the expression of genes
and products of hydroperoxide lyase (HPL) branch of oxylipin pathway. We found that the at2g02870 plants have enhanced
expression of HPL pathway genes and produce more green leaf volatiles (GLV) than the wild type plants. Our results
suggested that the gene is involved in the regulation of HPL pathway, possibly through the degradation of enzymes or/
and the regulatory factors of the pathway.
DOG(R)1, which encodes 2-deoxyglucose-6-phosphate phosphatase, has been used as a selectable marker gene to produce transgenic plants. In this study, a transformation vector, pBIDOG, which contains the DOG(R)1 gene, was transformed into oil palm embryogenic calli (EC) mediated by Agrobacterium tumefaciens strain LBA4404. Transformed EC were exposed to 400 mg l(-1) 2-deoxyglucose (2-DOG) as the selection agent. 2-DOG resistant tissues were regenerated into whole plantlets on various regeneration media containing the same concentration of 2-DOG. The plantlets were later transferred into soil and grown in a biosafety screenhouse. PCR and subsequently Southern blot analyses were carried out to confirm the integration of the transgene in the plantlets. A transformation efficiency of about 1.0% was obtained using DOG(R)1 gene into the genome of oil palm. This result demonstrates the potential of using combination of DOG(R)1 gene and 2-DOG for regenerating transgenic oil palm.