Day 2 :
University of Missouri, USA
Keynote: RNA-Seq analysis reveals host plant transcriptomes in response to agrobacterium-mediated transformation
Time : 09:30-10:15
Zhanyuan J Zhang has expertise in Plant Genetic Transformation and Gene Regulation. He got his Ph.D. in the area of Plant Genetic Engineering at University of Nebraska-Lincoln, NE, USA. He has been the director of Plant Transformation Core Facility at University of Missouri, Columbia, Mo, USA since 2000. The mission of his core facility is to enhance both basic and Applied Plant Biology studies by providing Plant Transformation services and to advance transgenic technologies. He has contributed to the transformation system improvements in soybean, maize, sorghum, wheat, and switchgrass. His interest in gene regulation has led to the validation and revealing of effective RNAi in soybean and novel approach for plant gene silencing employing artificial trans-acting siRNA. His research efforts in basic study of Agrobacterium-mediated transformation has discovered the role pf the heat shock protein 90.1 in this T-DNA transfer process.
Agrobacterium-mediated plant transformation has become a predominant tool for many basic studies and biotechnological applications. Discoveries in molecular mechanisms governing this transformation process have significant implications in both basic and applied plant biotechnological applications. To date, however, knowledge about plant genes and associated pathways involved in the Agrobacterium-mediated T-DNA transfer has been very limited. Here, we employed RNA-seq to exploit Arabidopsis thaliana transcriptomes in responses to Agrobacterium transformation process. We used two contrasting Agrobacterium strains to infect Arabidopsis young seedlings using AGROBEST assay protocol. The two strains included a non-oncogenic disarmed Agrobacterium strain, A136, and At804 which is a derivative of EHA105 and contains a disarmed super virulent Ti-plasmid and a binary vector pBISN1. The strain A136 lacks Ti-plasmid and therefore is unable to deliver T-DNA and effector (Vir) proteins to plant cells. This is in contrast to At804 which is capable of transferring both T-DNA and effector proteins into plant cells. Arabidopsis tissue samples for RNA-Seq were from three different treatment conditions, i.e., mock, A136 and At804, at 6 different time points (0, 3, 6, 12, 24, and 48 hours), respectively, during the Agrobacterium infection. Total RNA samples at each time point were then subject to NGS analysis. The transcriptomic analysis results showed that many plant genes responded to Agrobacterium infection. GO (gene ontology) analysis revealed that many plant biological processes are involved during Agrobacterium-plant interactions. These processes include hormone signaling, defense response, cellular biosynthesis, and nucleic acid metabolism and so on. Key genes displaying substantial changes in their transcripts were further validated by qRT-PCR and mutant screen. More details will be presented.
Universiti Kebangsaan Malaysia, Malaysia
Time : 10:15-11:00
Roohaida Othman received her PhD in Biochemistry from University of Southampton, and joined Universiti Kebangsaan Malaysia as a lecturer immediately after in 1995. Her research interest is focused on understanding the molecular mechanisms underlying the biosynthesis of commercially important metabolites in plants and algae. Her research group has employed tools of molecular biology, biochemistry and physiology as well as genomics and transgenics technology platforms to study the enzymes involved in these pathways. They have also developed protocols for higher plant and algae RNA extraction methods and overexpression of recombinant proteins in bacterial systems. She has been Editor-in-Chief for the Journal of Tropical Plant Physiology since 2010 and has been reviewer for several journals including International Journal of Food Properties.
Statement of the Problem: Persicaria minor (synonym Polygonum minus). produces a broad range of secondary metabolites such as sesquiterpenes that contribute towards the unique aroma of this plant. In an effort to understand the biosynthesis of these compounds, candidate genes involved in the sesquiterpene biosynthetic pathway have been identified from an expressed sequences tags (ESTs) collection. The purpose of this study is to characterize a gene which was initially identified as an-alpha farnesene synthase gene from the EST studies. Methodology & Theoretical Orientation: The full length cDNAs from P. minus was isolated and cloned into Escherichia coli, Lactococcus lactis and Arabidopsis thaliana following standard protocols. Enzymatic assay for the recombinant sesquiterpene synthase was performed using farnesyl diphosphate as substrate and the products from the enzymatic assays were analyzed using gas chromatography-mass spectrometry (GC-MS). Findings: The full length sequence of P. minor sesquiterpene synthase cDNA clone (PmSTS) was 2035 bp in size and was expressed in E. coli as a ~65 kDa soluble protein whereas in L. lactis, the size of the recombinant protein was ~63kDa. For enzyme activity assay, the major product of the recombinant PmSTS in E. coli was α-farnesene whilst for L. lactis recombinant protein, the major product was 𝛽-sesquiphellandrene with beta-farnesene as a minor product. Subsequent expression in A. thaliana also produced transgenic lines with increased 𝛽-sesquiphellandrene production. Finally, new expression in E. coli produced a recombinant PmSTS that released 𝛽-sesquiphellandrene as a major product and 𝛽-farnesene as a minor product, similar to the L. lactis recombinant protein. Conclusion & Significance: The identity of a plant sesquiterpene synthase has been confirmed as a 𝛽-sesquiphellandrene synthase. The correct identity of the gene was finally achieved due to the updated version of the mass spectral library used in identifying the products from GC-MS.
University of Sumatera Utara, Indonesia
Time : 11:20-12:05
Yusuf Leonard Henuk is a Professor in the Faculty of Agriculture at University of Sumatera Utara (USU), Medan, North Sumatera, Indonesia. He received a Bachelor’s degree (S1: ‘Sarjana’) from the the University of Nusa Cendana in Kupang-Indonesia from 1980-1984. He obtained Master in Rural Science (M.Rur.Sc.) from the University of New England in from 1991 – 1995 and continued Doctor of Philosophy (Ph.D) from the University of Queensland both in Australia from 1998 – 2001. Prof. Henuk was a prolific writer and has published many articles in either national or international journal within the field of agriculture and mainly animal sciences. He also participated in many national in Indonesia and international seminars.
Indonesia’s biodiversity is ranked 3rd after Brazil and Zaire. It is home to 30,000 out of 40,000 medicinal herbal plants in the world. Medicinal plants or known in Indonesia as “Tanaman Biofarmaka” are defined as plant which are useful for traditional medicine. It is consumed from part of the plant, either in the form of leaf, fruit, tuber or root. The roles of medicinal plants as traditional medicine in Indonesia has always been a part of culture that has been passed down from generation to generation. By trial-and-error, the country’s early inhabitants learned how to distinguish useful plants with beneficial effects from those that were either toxic or non-active used as traditional medicine. They picked, kept and used medicinal plants to satisfy their basic needs and even experimented on combinations of plants or processing methods to gain optimal results. Throughout the centuries, Indonesia’s indigenous people developed traditional medicines from plants identified by their forefathers for curing illnesses and keeping their health. In general, there are about 30,000 species of medicinal plants owned by Indonesia, and potentially to develop herbal products which have equal quality with modern medicines. Currently, there are 15 important type of medicines plants and herbs in Indonesia. They are ginger, galangal, east Indian galangal, turmeric, zingiber aromaticum, Java turmeric, black turmeric, Chinese keys, sweet root/calamus, Java cardamom, Indian mulberry, phaleria macrocarpa, verbenanceae, king of bitter, and aloevera. These type of medicinal plants can be divided into two groups: rhizome and non-rhizome. There are three provinces known as the main producer of medicinal plants used for traditional medicine in Indonesia. In conclusion, medicinal plants and herbs in Indonesia used as traditional medicine has also contributed significantly in supporting the national economy, availability of food products, health and cosmetics, trades, construction of gross domestic product and absorption of worker.