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Title:	Pemencilan dan analysis fungsi cDNA a-Farnesen Sintase daripada Polygonum minus
Authors:	Tan Ee Fun (P48559)
Supervisor:	Roohaida Othman, Prof. Madya Dr.
Keywords:	Antisense DNA Laboratory manuals
Issue Date:	29-Jun-2012
Description:	Polygonum minus (kesum) menghasilkan pelbagai jenis metabolit sekunder seperti sesquiterpen termasuk a-famesen yang menyumbang kepada aroma unik tumbuhan ini. a-famesen sintase (EC 4.2.3.16) daripada tumbuhan dipercayai memainkan peranan penting dalam tapak jalan metaboiisme sekunder, iaitu mengkatalisiskan perubahan 2- trans,6-trans-famesil difosfat (FPP) kepada a-famesen dan difosfat dengan kehadiran ion divalen sebagai kofaktor. Jujukan lengkap cDNA AFS daripada P. minus bersaiz 2035 pb dengan rangka bacaan terbuka bagi 562 asid amino telah beijaya diperoleh dan berat molekul protein dijangka adalah 65 kDa. Analisis BLASTx menunjukkan jujukan lengkap a-famesen sintase P. minus mempunyai kepadanan jujukan nukleotida dan asid amino yang tinggi dengan jujukan sesquiterpen sintase Santalum album (44%) dan Santalum spicatum (44%), E-beta-famesen sintase Artemisia annua (40%), delta-kadinen sintase Gossypium arboreum (40%) dan Gossypium hirsutum (39%) serta (E,E)-alfa-famesen sintase Vitis vinifera (41%). Selain daripada itu, terdapat juga jujukan terpulihara DDxxD dan motif RxR pada jujukan asid amino AFS yang merupakan motif bagi kebanyakan jujukan terpen sintase yang dipercayai bertindak sebagai tapak pengencaman bagi pengikatan logam difosfat Analisis Southem menunjukkan bahawa gen ini adalah ahli kepada famili multigen. Hasil tindak balas berantai polimerase kuantitatif masa sebenar (RT-qPCR) menunjukkan pengekspresan transkrip AFS adalah lebih tinggi di bahagian daun berbanding dengan batang dan akar.Seterusnya, jujukan gen AFS telah diperoleh dan menunjukkan ketiadaan intron manakala kotak TATA gen AFS dengan jujukan TATAAAT didapati berada pada jujukan ke-30. Untuk mengesahkan bahawa klon AFS yang diperoleh mengekod enzim a-famesen sintase, pengekspresan protein rekombinan pAFS telah dilakukan dengan mengunakan vektor pQe-2 dalam sistem pengekspresan Escherichia coli. Hasil analisis SDS-PAGE dan pemblotan westem menunjukkan protein dalam fraksi terlarut yang bersaiz 65 kDa telah beijaya diekspres. Pengasaian aktiviti enzim bagi protein recombinan pAFS menggunakan kaedah GC-MS menunjukkan AFS P. minus dapat mengkatalisis FPP kepada a-famesen. Pencirian gen dan cDNA bagi afamesen sintase memberikan maklumat baru mengenai enzim yang terlibat dalam tapak jalan biosintesis a-famesen yang merupakan salah satu sebatian yang dihasilkan oleh P. minus.,Polygonum minus (kesum) produces a broad range of secondary metabolites such as sesquiterpenes including a-famesene which contributes to the unique aroma of this plant. a-Famesene synthase (EC 4.2.3.46) from plant is believed to play an important roie in secondary metabolism pathway, in which it catalyzes the conversion of 2- trans,6-trans-famesyl diphosphate (FPP) to a-famesene and diphosphate in the presence of divalent ion as cofactor. The full length sequence of a-famesene synthase (AFS) cDNA clone from P. minus was obtained with the size of 2035 bp encoding predicted protein containing 562 amino acid residues with the expected molecular mass of 65 kDa. BLASTx analysis showed that the deduced amino acid sequence of the cDNA was highly similar to sisquiterpene synthase from Santalum album (44%) and Santalum spicatum (44%), E-beta-famesene synthase from Artemisia annua (40%), delta-cadinene sintase from Gossypium arboreum (40%) and Gossypium hirsutum (39%) and (E,E)-alpha-famesene sintase from Vitis vinifera (41%). In addition, the AFS amino acid sequence was found to contain the conserved DDxxD and RxR motifs which are motifs for most terpene synthases believed to act as a recognition site for binding of metal diphosphate. Southem analysis showed that the gene AFS is a member of a multigene family. Real time quantitative polymerase chain reaction (RT-qPCR) results showed that the AFS transcripts were expressed highest in leaves compared with roots and stems. Subsequently, AFS gene sequence was obtained and showed the absence of any intron in the sequence and TATA box was located on position -30 with the nucleotides sequence of TATAAAT. To conftrm that the AFS clone encodes a-famesene synthase enzyme, recombinant expression of AFS was performed in Escherichia coli using pQe2 vector. SDS-PAGE and westem blotting analyses showed that a ~65 kDa solublc protein was successfully expressed. For enzyme activity assay, the result from GC-MS headspace proved that the AFS P. minus was capable of converting FPP to a-famesene. The characterization of the gene and cDNA for a-famesene synthase provides new infonnation regarding the enzyme involved in the biosynthetic pathway of a-famesene which is one of the compounds produced by P. minus.,Master/Sarjana
Pages:	148
Call Number:	QP624.5.A57T343 2012 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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