Isolasi dan Pengklonan Fragmen CDNA Gen Penyandi H+-ATPase Membran Plasma dari Melastoma Malabathricum L.

Melastoma malabathricum L. grows well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying acid and aluminum stress is a plasma membrane H+ -ATPase protein encoded by PMA gene. The objective of this research was to isolate and clone the cDNA fragment of MmPMA encoding plasma membrane H+ -ATPase from M. malabathricum L. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of MmPMA cDNA had been successfully isolated by PCR by using total cDNA as template and PMA primer designed from conserved region for corresponding gene. This fragment had been successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5". Nucleotide sequence analysis showed that the length of MmPMA fragment is 806 bp encoding 268 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MmPMA fragment was 81% identical to part of PMA of Sesbania rostrata, Juglans regia, and Prunus persica. Based on deduced amino acid sequence, MmPMA was 94% identical to part of PMA of Juglans regia; 93% to PMA of S. rostrata, and Arabidopsis thaliana. MmPMA fragment has phosphorylation intermediate domain (DKTGT) and ATP binding domain (KGAP, DPPR, MITGD, and GDGVN)

Isolasi dan Pengklonan Gen Penyandi H+-ATPase Membran Plasma dari Melastoma Malabathricum L.

Melastoma malabathricum L. is an Al-accumulating plant that grows well in acidic soils with high level of soluble aluminum in the tropics. One of the important proteins in the detoxifying Al stress is a plasma membrane H+-ATPase, a most abundant protein on the plasma membrane, encoded by PMA gene. The objective of this research was to isolate and characterize the gene encoding plasma membrane H+-ATPase from M. malabathricum L. Full length cDNA of MmPMA had been successfully isolated through a gradual isolation of the gene. The 5' end and middle part of the MmPMA gene had been successfully isolated by PCR by using total cDNA as template and pma primers designed from some plants, while the 3' end of Mmpma had been isolated by 3' RACE. The parts of the gene had been successfully joined by PCR. The joining product was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the length of MmPMA coding sequence was 2,871 bp encoding 956 amino acids with molecular weight of 105.29 kDa and a predicted pI value of 6.84. Local alignment analysis based on nucleotide of mRNA showed that MmPMA is 82% identical to pma Vitis vinifera; 81% to pma Juglans regia, pma Populus trichocarpa, pma Sesbania rostrata, and pma Prunus persica and 80% to pma Lycopersicon esculentum. Based on deduced amino acid sequence, MmPMA is 94% identical to PMA Vitis vinifera and PMA Juglans regia; 93% to PMA Populus trichocarpa; 92% to PMA Vicia faba, Lycopersicon esculentum, and Arabidopsis thaliana, AHA4. MmPMA has 10 transmembrane domains, 4 cytoplasm loops, 6 functional domains and 3 autoregulatory domains

Rekayasa Ekspresi Gen Pembungaan Hd3a Dibawah Kendali Promoter Rol C Pada Jarak Pagar (Jatropha Curcas L.) [Engineering of Expression of Hd3a Flowering Gene Driven by Rol C Promoter on Physic Nut {Jatropha Curcas L.)]

Flowering in jatropha (Jatropha curcas L.) was considered as one of major factors that contribute to its productivity. Small number of female flowers produced in each inflorescence was believed as the main cause of low seed production.Introduction of Hd3a flowering gene driven by rol C promoter was supposed to improve total number of flowers including female flower.The objective of this research was to optimize cell proliferation and regeneration medium in Jatropha transformation method mediated by Agrobacterium, to obtain transgenic Jatropha containing Hd3a flowering gene as well as to understand the effect of this transgene on jatropha flowering character.Callus induction medium containing 0.5 mg/1 IBA added with 3 g/1 PVP produced the highest frequency of shoot formation.We obtained 26.67% to 33.33% putative transgenic plantlets that were able to grow in 40mg/l hygromycin selection medium. PCR analysis revealed that seven out of 10 putative transgenic plantlets were positively transgenic.Extremely early flowering character that was confirmed by histological analysis was also shown by some transgenic plantlets