Cloning Of A Gene Encoding Glutamate Decarboxylase From Banana (Musa Spp.) And Its Roles In Postharvest Ripening

Banana is one of the most important fruit in the world,and is ranked as the world's fourth most important food crop.There are a lot of ethylene production and physiological changes in the postharvest ripening of banana.Banana postharvest ripening plays an important role in fruit quality and shelf life,which influences the development of banana industry directly.So it is worthy of studying banana postharvest ripening process and regulation.In order to gain a better understanding of the mechanism about banana postharvest ripening,we isolate differentially cDNAs at the early stage of banana ripening by suppression subtractive hybridization.A total of 289 clones in the SSH library are sequenced.Blastx results reveal that two cDNAs share high identities with glutamate decarboxylase from other species.The cDNAs are found to be extremely up-regulated by use of cDNA microarray of 10 days and 14 days postharvest banana.So it is possible for the cDNA to be related to banana ethylene biosynthesis and fruit ripening.The full-length cDNA is isolated from banana fruit cDNA library by RT-PCR approach and designated MaGAD1.The full length of MaGAD1 cDNA is 1500bp with an open reading frame encoding 499 amino acids that share high identities with the GAD from Oryza sativa, Citrus sinensis,Populus alba and Solanum lycopersicum(81%,80%,80%and 78%).Amino acid aligmnent indicates MaGAD1 has a CaM-binding domain at the carboxy terminus and a putative PLP-binding motif.Southern hybridization reveals that the gene is really from banana and has low copies in banana.RT-PCR analysis shows that MaGAD1 expresses in roots,stems,leaves,flowers and fruits.The expression lever is higher in fruits than other organs.So MaGAD1 may play some roles in different physiological course and be related to fruit ripening.In order to investigate the relation between MaGAD1 and banana ripening,we measure the ethylene production and analyse the expression level of MaGAD1at different stages with the treatment of ethylene and 1-MCP.The results indicate that MaGAD1 is induced and reach the highest expression level when ethylene biosynthesis start,which heavy induce ethylene biosynthesis with the treatment of ethylene and naturally ripening.Both ethylene production and MaGAD1 expression are at low level at different postharvest ripening stages with the treatment of 1-MCP.So MaGAD1 expression is not only induced by ethylene but also induce ethylene biosynthesis,which indicates that MaGAD1 is really related to ethylene biosynthesis and fruit ripening.In order to further understand the role of MaGAD1 in banana postharvest ripening,we explore the mechanism of MaGAD1 in the ethylene biosynthesis in banana postharvest ripening.we filter the inducement(GABA) and inhibitor(AOAA) of MaGAD1 with the standard of the time from ripening degree one to seven and indicates that GABA induces while AOAA restrains banana fruit ripening.GABA induces while AOAA restrains the abrupt change time of sugar,starch,rigidity in the banana postharvest ripening course.GABA induces while AOAA restrains the expression of MaGAD1 and MaACS1.GABA and AOAA don't affect the qualities of banana.MaACS1 is really the crucial gene in banana ethylene biosynthesis.MaGAD1 can regulate the changes of ripening,sugar,starch,rigidity in postharvest ripening banana fruit.The expression trend of MaGAD1 and MaACS1 are coherent.On the base of pCAMBIA1302,MaGAD1 is inserted downstream of 35s promoter, upstream of GFP gene.The constructed vector is named pCAMBIA1302- MaGAD1.