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Title:	Pengklonan dan pencirian kefungsian gen seskuiterpena sintase daripada polygonum minus
Authors:	Chew Jin Kiat (P53444)
Supervisor:	Ismanizan Ismail, Prof. Dr.
Keywords:	Seskuiterpena Polygonum minus Pengklonan Sesquiterpenes
Issue Date:	9-Feb-2015
Description:	Seskuiterpena merupakan kumpulan sebatian metabolit sekunder yang mempunyai pelbagai kepentingan kepada tumbuh-tumbuhan dan juga manusia. Kromatografi gasspektrometri jisim (GC-MS) yang dilakukan terhadap Polygonum minus menunjukkan bahawa kebanyakan sebatian terpena yang dijumpai dalam minyak pati Polygonum minus adalah terdiri daripada sebatian seskuiterpena. Kajian ini bertujuan untuk mengklonkan gen seskuiterpena sintase daripada Polygonum minus dan mencirikan protein yang dikodkannya. Jujukan penuh bagi satu gen seskuiterpena sintase (PmSS) telah berjaya diperoleh daripada daun Polygonum minus menerusi kaedah RACE. Rangka bacaan terbuka bagi gen PmSS adalah terdiri daripada 1680 pasangan bes dan mengekodkan protein yang bersaiz 64.6 kDa. Analisis BLASTX menunjukkan bahawa germakrena D sintase daripada Vitis vinifera mempunyai tahap kehomologan yang paling tinggi (51% kesamaan, Nilai E=0) dengan jujukan rangka bacaan terbuka gen PmSS. Penjajaran di antara jujukan genomik dengan jujukan cDNA PmSS menunjukkan bahawa jujukan genomik PmSS (bersaiz 2883 pb) adalah terdiri daripada 6 intron dan 7 ekson. Corak pengekspresan gen PmSS pada tiga organ Polygonum minus yang berlainan telah dikaji dengan menggunakan qRT-PCR. Antara organ yang dikaji, tahap pengekspresan gen PmSS adalah lebih tinggi pada daun (iaitu 98 kali ganda lebih tinggi daripada akar) dan batang (iaitu 35 kali ganda lebih tinggi daripada akar). Selain itu, kajian ini juga menunjukkan bahawa pengekpresan gen PmSS adalah beresponsif terhadap rawatan asid jasmonik. Berbanding dengan sampel yang tidak dirawat, tahap pengekspresan gen PmSS dalam daun didapati meningkat kira-kira 2 kali ganda selepas dirawat dengan larutan asid jasmonik selama 12 jam. Pengekspresan protein rekombinan PmSS dilakukan dalam sistem pengekspresan Escherichia coli dengan menggunakan dua jenis vektor pengekspresan (pET28b dan pET32b) dengan ciri-ciri yang tersendiri. Selain itu, kepekatan IPTG (0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM dan 1.0 mM) dan suhu aruhan (15°C, 20°C, 25°C, 30°C dan 37°C) yang berlainan juga diuji untuk meningkatkan keterlarutan protein rekombinan bagi PmSS. Analisis SDS-PAGE dan pemblotan western menunjukkan bahawa protein rekombinan PmSS telah berjaya diekspreskan tetapi amat sedikit dalam fraksi yang terlarut. Jujukan cDNA bagi gen PmSS juga ditransformasikan ke dalam Arabidopsis thaliana menerusi kaedah floral dip. Kehadiran gen PmSS dalam Arabidopsis thaliana transgenik telah disahkan melalui analisis tindak balas rantai polimerase (PCR). Tiga titisan Arabidopsis thaliana transgenik telah dipilih untuk menganalisis kandungan sebatian seskuiterpenanya melalui GC-MS. Namun, hasil analisis GC-MS menunjukkan bahawa tiada perubahan yang ketara dari segi kandungan sebatian seskuiterpena yang dihasilkan oleh pokok transgenik dan pokok kawalan,Sesquiterpene is a group of secondary metabolites with multiple beneficial properties that are important to the plants as well as human. Gas chromatography-Mass spectrometry (GC-MS) analysis towards Polygonum minus revealed that sesquiterpene was one of the most abundant terpene compounds found in the essential oil. This study aimed to isolate sesquiterpene synthase gene from Polygonum minus and to characterize its encoded protein. Full length sequence of a sesquiterpene synthase gene (PmSS) was successfully isolated from the leaves of Polygonum minus through rapid amplification of cDNA ends (RACE) technique. The open reading frame of PmSS gene was 1680 base pairs in length which corresponds to a 559-amino acid protein with a deduced mass of 64.6 kDa. BLASTX result showed that the sequence of PmSS shared the highest identity with germacrene D synthase from Vitis vinifera (51% identity, E-value=0). Pairwise alignment between genomic sequence and cDNA sequence of PmSS gene showed that the genomic sequence of PmSS (2883 bp in size) comprised of 6 introns and 7 exons. The expression level of PmSS gene among three different organs was analyzed through qRT-PCR. Among the organs, the transcript level of PmSS gene was higher in the leave (98-fold higher than in the root) and the stem (35-fold higher than in the root). Besides that, this study have also shown that the expression of PmSS gene was responsive towards jasmonic acid treatment. As compared to the non-treated sample, the expression level of PmSS gene in the leave was shown to be increased for about 2 folds after 12 hours of jasmonic acid treatment. Recombinant expression of PmSS was done in Escherichia coli expression system using two protein expression vectors (pET28b and pET32b) with different properties. Furthermore, different IPTG concentrations (0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1.0 mM) and induction temperature (15°C, 20°C, 25°C, 30°C and 37°C) were tested to increase the solubility of recombinant protein PmSS. SDS-PAGE and Western blotting analysis showed that the recombinant protein PmSS was successfully expressed but very little in the soluble form. cDNA sequence of PmSS was also transformed into Arabidopsis thaliana through Agrobacterium tumefaciens through floral dip method. The presence of PmSS gene in the transgenic Arabidopsis thaliana was successfully confirmed by polymerase chain reaction amplification (PCR). Three different lines of transgenic plants were chosen to be analyzed for the content of terpene compound through GC-MS analysis. However, GC-MS analysis showed no significant changes in content of sesquiterpene compounds produced by the transgenic plants and control plants,Ph.D
Pages:	163
Call Number:	QK898.S49 C486 2015
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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