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Title:	Isolation and functional characterization of mr219sap8, a member of sap gene family from Oryza Sativa Var MR219
Authors:	Poya Hedayati (P46896)
Supervisor:	Zamri Zainal, Prof.Dr.
Keywords:	Salinity ESTs Transgenic Universiti Kebangsaan Malaysia -- Dissertations
Issue Date:	2014
Description:	Salinity stress is one of the most important environmental factors that impose huge reduction in crop growth and yield. Thus, there is a need for continued effort to understand the salt tolerance mechanism towards the development of salt tolerant plants. The present study was undertaken to generate information on salt stress responsive genes in Oryza sativa var. MR219, using suppression subtractive hybridization technique. A SSH library was constructed after exposing the two weeks old rice seedlings to 150 mM NaCl for 0 h as driver and 2, 4, 8, 12 , 24 h as tester. A total of 260 high quality of ESTs were obtained. BLASTX search revealed 125 unique transcripts. The majority of these unique transcripts are homologous to the previously known salt-responsive genes. Based on GO annotation, unique transcripts were divided into three groups in which: 143 ESTs were located under biological process, 107 under cellular component and 76 were annotated under molecular functions. Using RT-PCR, we examined the expression of two candidate genes (MR219SAP8 and SALT ) and confirmed that their expression levels increased at different time points under salinity stress. Further functional study was carried out on MR219SAP8, a member of Stress Associated Protein (SAP) and multiple stress inducible gene. To overexpress MR219SAP8 in Arabidopsis thaliana, pCAMBIA1301 was selected as a transformation vector where its gus gene was replaced with a cDNA of MR219SAP8 under the control of CaMV 35S promoter. Five transgenic Arabidopsis lines were obtained by the floral-dip transformation method using Agrobacterium tumefaciens strain GV3101. Selection was performed on MS medium containing 25 µg/mL hygromycin. The presence of the of the transgene in the transgenic lines was assessed through PCR amplification using two set of primers designed specifically to MR219SAP8 and CaMV35S promoter sequences. The results obtained gave with the size of 513 bp and 700 bp. RT-PCR was done in order to determine the ability of the transgene to be expressed in the transgenic lines. Three T3 transgenic lines were chosen and the result obtained proven that the transgenic lines contain transcripts of the transgene. Evaluation of the survival of T3 transgenic lines under salinity stress was conducted at 100, 150, 200 and 250 mM NaCl concentrations. The result showed that seeds from transgenic line-5 has the highest percentage of germination at 250 mM as compared to the wild type where not a single seed was able to germinate. Our results indicate that MR219SAP8 may play a important role in response to salt stress.,Kepayahan saliniti merupakan salah satu faktor persekitaran sangat penting yang dapat mempengaruhi pertumbuhan dan hasil tanaman. Oleh itu, usaha yang berterusan sangat diperlukan untuk memahami mekanisma toleransi saliniti dalam menghasilkan tanaman yang dapat bertoleransi terhadap kepayahan saliniti. Kajian yang dijalankan adalah untuk menghasilkan maklumat peringkat gen yang bergerakbalas terhadap kepayahan saliniti pada Oryza sativa var. MR219, menggunakan teknik suppression substractive hybrydisation (SSH). Sebuah perpustakaan cDNA SSH telah dibina dari mRNA anak benih padi berumur dua minggu yang telah didedahkan kepada kepekatan 150mM NaCl selama 0, 2, 4, 8, 12 dan 24 jam. Sejumlah 260 EST yang berkualiti tinggi telah diperoleh. Pencarian melalui BLASTX telah mengenalpasti sebanyak 125 unigen.Majoriti jujukan EST ini mempunyai homolog kepada gen-gen yang telah diketahui bergerakbalas terhadap kepayahan saliniti. Berdasarkan anotasi Ontologi Gen (GO),jujukan-jujukan EST boleh dibahagikan kepada tiga kumpulan; sebanyak 143 EST dapat dikelompokkan dibawah proses biologi, 107 di bawah komponen selular dan76 dinotasikan di bawah fungsi molekul. Menggunakan teknik RT-PCR dua gen ( MR219SAP8 dan SALT) telah dipilih untuk mengetahui corak pengekspresannya. Keputusan mengesahkan bahawa transkrip gen tersebut didapati meningkat dalam masa yang berbeza di bawah tekanan saliniti. Kajian kefungsian turut dilakukan keatas gen MR219SAP8, iaitu ahli famili gen bagi protein berkait kepayahan (SAP) dan gen teraruh kepayahan berganda. Untuk mengekspreskan gen MR219SAP8 secara berlebihan dalam Arabidopsis, vektor transformasi pCAMBIA1301 telah dipilih dengan menggantikan jujukan gen gus dengan jujukan cDNA MR219SAP8 dibawah kawalan promoter CaMV35S. Sebanyak 5 titisan Arabidopsis transgenik telah diperoleh dari langkah transformasi menggunakan Agrobacterium tumefaciens strain GV3101 secara floral-dip. Pemilihan titisan transgenik telah dijalankan ke atas medium MS yang mengandungi 25 µg/mL antibiotik higromisin. Kehadiran transgen dalam tumbuhan transgenik telah ditentukan melalui amplifikasi PCR dengan mereka bentuk dua set primer yang khusus terhadap gen SAP8 dan promoter CaMV35S. Hasil dari amplifikasi PCR mendapati dua jalur bersaiz 513 bp dan 700 bp seperti yang dijangkakan. Penentuan keupayaan transgen diekspreskan dalam titisan transgenik telah berjaya dibuktikan secara RT-PCR terhadap 3 titisan transgenik. Penilaian terhadap keupayaan tumbuhan transgenik bermandiri di bawah kepayahan saliniti pada kepekatan 100,150,200 dan 250 mM NaCl dikaji semasa tahap percambahan. Hasil kajian ini menunjukkan MR219SAP8 berkemungkinan memainkan peranan yang sangat signifikan dalam gerakbalas terhadap tekanan saliniti.,Ph.D.
Pages:	147
Call Number:	QK753.S32.H435 2014
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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