Study of MA-ACS Gene Family and MA-ACO Gene Expression During Vegetative Development and Fruit Ripening Stages of Pisang Ambon (Musa sp. AAA group) Using REALTIME PCR (qPCR) Method Student
: Resnanti Utami Handayani Thesis (2010), Master’s program in Biotechnology, School of Life Sciences and Technology – ITB, email :
[email protected]
Advisor
: Dr. Fenny M. Dwivany1 1 School of Life Sciences and Technology ITB, email:
[email protected] atau
[email protected]
Degree
: Magister Sains (M.Si), Conferred July 2010
Abstract Pisang Ambon (Musa sp. AAA group) is a banana cultivar exclusive from Indonesia. Banana fruit is one of climacteric fruit, which is the ripening is regulated by a phytohormone, ethylene. Ethylene plays important roles in plant growth and development, flower senescence, abscission and fruit ripening. Ethylene biosynthesis is regulated by two genes, gene encoding ACC synthase (ACS) and ACC oxidase (ACO). Pisang Ambon has nine ACS genes (MA-ACS 1-9) and one ACO gene (MAACO). In this research, we investigated five ACS genes member of multigenes ACS ACS (MA-ACS1, MA-ACS2, MA-ACS4, MA-ACS5 and MA-ACS6) and one ACO gene (MA-ACO) during vegetative and fruit ripening stages of pisang Ambon. Vegetative tissues (roots and leaves) were obtained from tissue cultures, while fruit samples (peels and pulps) were taken from pre-climacteric, climacteric and postclimacteric stages at day 1, 3, 5 and 7 after harvested. Fruit’s physical characteristic were done by observing the changes of peels colour, ratio of peels and pulps and starch conversion into sugars. Gene expression levels were analyzed using qPCR (quantitative PCR) method which normalized to MA-GADPH and MA-18srRNA as gene references. Results showed that MA-ACS1 and MA-ACS5 were expressed specifically in fruit samples, either in peels and pulps tissues. MA-ACS1 increased significantly in pulps and peels tissues at day 5 and decreased at day 7 after harvested. MA-ACS5 in peels tissue were tended to decrease from day 1 to day 7 after harvested while in pulps tissue, MA-ACS5 expressions were increased significantly at day 3 and reached maximum level at day 5 and decreased significantly at day 7 after harvested. In the mean time, MA-ACS6 and MA-ACO were expressed since vegetative stages until fruit ripening process happened. The expression of MA-ACO gene in fruit ripening stage reached maximum level at pulps iii
samples day 5 and decreased significantly at day 7 after harvested. MA-ACS2 gene which induced by wounding and stress, were expressed very high in vegetative tissue infected by fungi, especially in roots tissue. From this research, we conclude that MAACS1 and MA-ACS5 were fruit ripening specific genes and the promoters can applied in various transgenic plants, especially edibble vaccine. Key words : Pisang Ambon, MA-ACS, MA-ACO, gene expression, qPCR
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Studi Pola Ekspresi Keluarga Multigen MA-ACS dan Gen MA-ACO pada Tahap Perkembangan Vegetatif dan Proses Pematangan Buah Pisang Ambon (Musa sp. AAA group) Menggunakan Metode REALTIME PCR (qPCR) Mahasiswa
: Resnanti Utami Handayani Tesis (2010), Program Studi Magister Bioteknologi, Sekolah Ilmu dan Teknologi Hayati – ITB, email :
[email protected]
Pembimbing : Dr. Fenny M. Dwivany1 1 SITH-ITB, email :
[email protected] atau
[email protected] Gelar
: Magister Sains (M.Si), Wisuda Juli 2010
Abstrak Pisang Ambon (Musa sp. AAA group) merupakan pisang kultivar khas Indonesia. Buah pisang termasuk ke dalam buah klimaterik, yang pematangannya diregulasi oleh suatu fitohormon, yaitu etilen. Etilen berperan penting dalam pertumbuhan dan perkembangan tanaman, senesens bunga, absisi dan pematangan buah. Biosintesis etilen diregulasi oleh dua gen, yaitu gen pengkode ACC sintase (ACS) dan ACC oksidase (ACO). Pisang ambon terdiri dari sembilan gen ACS (MA-ACS 1-9) dan satu gen ACO (MA-ACO). Pada penelitian ini dilakukan studi ekspresi terhadap lima multigen ACS (MA-ACS1, MA-ACS2, MA-ACS4, MA-ACS5 dan MA-ACS6) dan satu gen ACO (MA-ACO) pada tahap vegetatif dan pematangan pisang ambon. Jaringan vegetatif (akar dan daun) diperoleh dari hasil kultur jaringan, sedangkan sampel buah pisang diambil mulai dari tahap pre-klimaterik, klimaterik sampai post-klimaterik setelah panen. Pada sampel buah pisang dilakukan analisis secara fisik, meliputi pengamatan warna kulit buah pisang, rasio berat kulit dan daging, serta uji kandungan pati dan gula. Analisis level ekspresi dilakukan dengan menggunakan metode qPCR (quantitative PCR) yang dinormalisasi terhadap MA-GADPH dan MA-18srRNA sebagai gene references. Hasil menunjukkan bahwa MA-ACS1 dan MA-ACS5 diekspresikan secara spesifik pada buah, baik pada jaringan kulit dan daging buah. MA-ACS1 meningkat secara cukup signifikan pada jaringan kulit dan daging buah pada hari ke-5 dan menurun pada hari ke-7 setelah panen. Sedangkan level ekspresi MA-ACS5 pada jaringan kulit cenderung menurun dari hari ke-1 sampai hari ke-7, pada jaringan daging buah level ekspresi MA-ACS5 meningkat signifikan pada hari ke-3, mencapai maksimum pada hari ke-5 dan menurun secara signifikan pada hari ke-7 setelah panen. Sementara itu, MA-ACS6 dan MA-ACO diekspresikan sejak tahap vegetatif sampai proses pematangan buah. Ekspresi MA-ACO pada proses pematangan buah mulai mencapai maksimum pada jaringan daging buah hari ke-5 i
setelah panen. Sedangkan MA-ACS2, yang diinduksi oleh adanya pelukaan dan stres diekspresikan jauh lebih tinggi pada jaringan vegetatif yang terinfeksi jamur, terutama pada jaringan akar. Dari hasil tersebut dapat diambil kesimpulan bahwa MA-ACS1 dan MA-ACS5 merupakan gen yang spesifik pada proses pematangan buah dan promoternya dapat diaplikasikan dalam berbagai tanaman transgenik, terutama edibble vaccine. Kata Kunci : Pisang Ambon, MA-ACS, MA-ACO, ekspresi gen, qPCR
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