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Title:	Functional study on sesquiterpene synthase gene identified from transcriptome profiling of salicylic acid and methyl jasmonate treated Polygonum minus
Authors:	Ee Su Fang (P53391)
Supervisor:	Zamri Zainal, Prof. Dr.
Keywords:	Sesquiterpene Polygonum minus Polygonum
Issue Date:	Sep-2014
Description:	Tumbuh-tumbuhan menghasilkan metabolit sekunder untuk membolehkannya beradaptasi terhadap nic ekologi tertentu serta bertindak balas terhadap pelbagai jenis kepayahan persekitaran. Metabolit-metabolit sekunder ini adalah berbeza dari sifat kimianya. Oleh itu, pengetahuan tentang tapak jalan metabolisme sekunder dan pegawalaturannya adalah amat penting. Kajian ini bertujuan untuk mengumpul data transkriptom Polygonum minus dengan menggunakan strategi pemprofilan transkriptom cDNA-AFLP serta menjalankan kajian kefungsian pada gen yang dikenalpasti terlibat dalam proses metabolit sekunder. P. minus merupakan tumbuhan aromatik tempatan yang mengandungi banyak metabolit sekunder, terutama terpenoid. Melalui pemprofilan transkriptom cDNA-AFLP, sebanyak 98 serpihan transkrip (TDFs) telah menunjukkan tahap pengekspresan lebih tinggi apabila P. minus dirawat dengan asid salisilik (SA) dan metil jasmonat (MeJA). Ini termasuk 46 TDFs dan 52 TDFs daripada sampel terawat SA dan MeJA. Dua set gen berbeza yang dikenalpasti telah mengesahkan bahawa laluan isyarat yang dirangsang oleh SA dan Meja adalah berbeza. Sebanyak 16% daripada TDFs tergolong dalam proses metabolik sekunder, di mana gen yang mengekodkan sesquiterpena sintase daripada P. minus (GenBank: JX025008) telah dipilih untuk kajian kefungsian gen. Sesquiterpena sintase merujuk kepada semua enzim yang dapat menukarkan farnesil difosfat (FPP) kepada sesquiterpena yang berbeza sebagai produk. P. minus sesquiterpena sintase mengandungi rangka bacaan terbuka 1689 pb yang mengekod 562 residu amino asid. Seperti yang terdapat dalam terpena sintase lain, P. minus sesquiterpena sintase mempunyai dua domain yang besar iaitu domain N-terminal dan domain C-terminal ikatan logam dengan tiga motif terpelihara iaitu DDXXD, NSE/DTE dan RXR. Pemodelan 3D protein untuk P. minus sesquiterpena sintase dibina dengan menggunakan 5-epi-aristolochene sintase (TEAS) sebagai templat. Analisis filogenetik berjaya menunjukkan bahawa P. minus sesquiterpena sintase adalah tergolong dalam subfamili sesquiterpena sintase angiosperma (Tps-a). Untuk menentukan fungsi P. minus sesquiterpena sintase dalam tumbuhan, gen ini telah diekspresskan dalam Arabidopsis thaliana. Konstruk T-DNA yang membawa gen P. minus sesquiterpena sintase telah berjaya diintegrasikan ke dalam genom tumbuhan dengan melalui kaedah transformasi pencelupan bunga. Sepuluh titisan transgenik berjaya dijanakan. Dua tumbuhan transgenik yang dinamakan OE3 dan OE7, telah digunakan untuk kajian selanjutnya. Analisis separa kuantitatif RT-PCR telah menunjukkan bahawa P. minus sesquiterpena sintase diekspreskan di bawah kawalan promoter CaMV35S dalam tumbuhan transgenik. Analisis GC-MS menunjukkan P. minus sesquiterpena mempunyai padanan pada 90.1% dengan β-sesquiterpena dalam perpustakaan NIST/EPA/NIH. Hasil menunjukkan bahawa A. thaliana berpotensi sebagai platform alternatif dalam kajian kefungsian gen-gen P. minus, khususnya gen sesquiterpena sintase.,Plants produce a wide range of secondary metabolites in order to adapt to a specific ecological niche and to respond to various environmental stresses. Majority of these metabolites are very different in their chemical nature. Therefore, the knowledge on the plant secondary metabolite biosynthetic pathway and its regulation is very important. In this present study, we aimed to gather the transcriptomic data of Polygonum minus by employing the cDNA-AFLP transcriptome profiling. We also aimed to functionally characterize a gene that was found to be involved in the secondary metabolite biosynthesis process. P. minus is a local aromatic plant that contains highly abundant secondary metabolites, especially terpenoids. Through cDNA-AFLP transcriptome profiling, a total of 98 transcript-derived fragments (TDFs) were up-regulated upon elicitation with salicylic acid (SA) and methyl jasmonate (MeJA). These included 46 TDFs from SA-treated and 52 TDFs from MeJA-treated P. minus leaf samples. The two different sets of genes confirmed that SA and MeJA mediate different signalling pathways. Sixteen percent of the annotated TDFs were found to be mainly involved in the secondary metabolic processes. Among these, a sesquiterpene synthase cDNA from P. minus (GenBank: JX025008) was found. Sesquiterpene synthases refer to enzymes that convert farnesyl diphosphate (FPP) to different sesquiterpenes The P. minus sesquiterpene synthase cDNA consists of a full open reading frame (ORF) of 1689 bp encoding 562 amino acid residues. Similar to other terpene synthases, P. minus sesquiterpene synthase predicted polypeptide sequence has two large domains i.e. the N-terminal domain and the C-terminal metal binding domain with three conserved motifs namely the DDXXD, NSE/DTE and RXR regions. A three dimensional protein model for P. minus sesquiterpene synthase was built using 5-epi-aristolochene synthase (TEAS) as a template. Phylogenetic analysis showed that P. minus sesquiterpene synthase was classified into the angiosperm sesquiterpene synthases subfamily (Tps-a). To determine the function of P. minus sesquiterpene synthase in plant, the gene was heterologously expressed in Arabidopsis thaliana. The T-DNA constructs containing P. minus sesquiterpene synthase gene was successfully integrated into the plant genome using the well established floral-dip transformation method. Ten transgenic lines were produced. Two transgenic lines, designated as OE3 and OE7, were further characterized. Growth retardation was observed in these two transgenic plants. Semi-quantitative RT-PCR analysis showed that P. minus sesquiterpene synthase gene was constitutively expressed under the control of CaMV35S promoter in the transgenic plants. Gas chromatography-mass spectrometry (GC-MS) analysis of the transgenic plants showed that P. minus sesquiterpenes exhibited a good match of 90.1% to β- sesquiphellandrene in NIST/EPA/NIH library. This result demonstrated the potential of A. thaliana as an alternative platform in functional characterization of P. minus genes, specifically the sesquiterpene synthase genes.,PhD
Pages:	170
Call Number:	QK495.P78E347 2014 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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