Molecular Identification of Mutant Candidate of Gα Gene from Soybean cv. Slamet Based on mRNA
Identifikasi Secara Molekular Kandidat Mutan Gen Gα dari Kedelai Kultivar Slamet berdasarkan mRNA.
Abstract
Heterotrimeric G proteins composed of a, ß, and . subunits, are bound to the inside surface of the cell membrane. Ga proteins will be activated by extracellular stimuli (biotic or abiotic stress). It is presumably Ga proteins playing an important role in aluminum (Al)-stress tolerance. Soybean cv. Slamet was considered as a tolerant plant to Al and has a single copy of Ga gene. Gamma irradiation to soybean cv. Slamet seeds caused a random chromosomal mutation of second generation plants (designated as M2). The Ga mutant candidates were selected based on stomatal closure in the daylight and dwarf. Ga mutant candidates M4, number 338 and 103 have severe root cell tissues damage compared to non mutant ones. Plant number 338/3S1.174/12/14 (M4) and 103/3S3.103/8/5/1/4 (M5) have a Ga gene mutation at the U3-L4 sequences (exon 5 – exon 11). Whereas, plant number 344/3S1.196/14/1/8 (M4) is assumed as Ga gene mutant. All of those plants were replanted until sixth generation. The candidates of Ga mutant selected have more than fifty percent of closed stomata and dwarf. Four number of M5 plants and three number of M6 plants were selected for Ga mutant identification based on mRNA analysis. The fifth generation plant number 344/3S1.196/14/1/8/7 were decreased its Ga gene expression at L5-L4 sequences (exon 7 – exon 11). The M6 generation plant number 344/3S1.196/14/1/8/7/5 have mutation in Ga gene at L1-L3 sequences (exon 2 – exon 6). A major contributing factor to decreased expression levels of Ga gene may be due to mutation of Ga gene promotor. Three other of M5 plants were assumed as Ga mutant. M6 plant number 103/3S3.103/8/5/1/4/8 has mutation at L3-L2 sequences (exon 6 – exon 15) and number 103/3S3.103/8/5/1/4/9 has mutation at U3-L4 sequences (exon 5 – exon 11). The decreasing of gene expression caused by mutation in those plants was indicated by the less pronounced DNA bands on agarose gel than that of non mutant plants. Protein heterotrimerik G terdiri atas tiga subunit yaitu a, ß, dan . (Ga, Gß dan G.) yang terletak di membran internal sel. Protein Ga teraktivasi ketika adanya cekaman biotik atau abiotik dari luar sel. Protein Ga diduga berperan terhadap ketahanan cekaman Aluminium (Al). Kedelai kultivar Slamet merupakan kedelai toleran Al yang memiliki satu kopi gen Ga. Irradiasi sinar gamma pada biji kedelai kultivar Slamet menyebabkan mutasi random pada kedelai kultivar Slamet generasi M2. Kandidat mutan Ga M2 diseleksi berdasarkan keadaan stomata menutup pada siang hari dan tinggi tanaman. Mutan Ga M4 nomor 338 dan 103 mengalami kerusakan jaringan sel akar lebih besar dibandingkan tanaman non mutan. Tanaman nomor 338/3S1.174/12/14 (M4) dan 103/3S3.103/8/5/1/4 (M5) mengalami mutasi gen Ga pada ruas U3-L4 (ekson 5 – ekson 11). Sedangkan tanaman nomor 344/3S1.196/14/1/8 (M4) diduga mutan gen Ga. Ketiga nomor tersebut ditanam kembali sampai generasi ke 6 dan diseleksi kandidat mutan Ga berdasarkan fenotipe lebih pendek dari pada Slamet non mutan dan stomata menutup >50%. Dipilih empat nomor dari M5 dan tiga nomor dari M6 untuk diidentifikasi mutan Ga bedasarkan mRNA. Tanaman M5 nomor 344/3S1.196/14/1/8/7 mengalami penurunan ekspresi gen Ga pada ruas L5-L4 (ekson 7- ekson 11). Generasi selanjutnya (M6) yaitu nomor 344/3S1.196/14/1/8/7/5 mengalami mutasi gen Ga pada ruas L1 sampai L3 (ekson 2 – ekson 6). Penurunan ekspresi gen Ga dapat terjadi karena adanya mutasi pada promotor gen Ga. Tiga tanaman M5 lain diduga mengalami mutasi gen Ga. Dua tanaman M6 yaitu nomor 103/3S3.103/8/5/1/4/8 mengalami mutasi pada ruas L3-L2 (ekson 6 – ekson 15) dan nomor 103/3S3.103/8/5/1/4/9 pada ruas U3-L4 (ekson 5 – ekson 11). Mutasi yang terjadi pada dua nomor ini adalah penurunan ekspresi gen yang ditandai dengan lebih tipisnya pita gen Ga dibandingan tanaman Slamet non mutan.
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