Function And Regulation Mechanism Of MaGBSSI-3 Involved In The Amylose Biosynthesis Of Banana Fruit

Banana(Musa Musaceae)is monocotyledon of large perennial evergreen herbs,which is one of the most important foods in tropical and subtropical developing countries.Starch is the main component of banana fruit,which is closely related to the fruit yield and quality.Especially amylose has a direct impact in banana fruit sweet and waxy quality.Granule-Bound Starch Synthase(GBSS)is the key enzyme in amylose biosynthesis,so it is very significant to improve the banana fruit quality and meet the growing demand for sweet and waxy by a comprehensive,systematic and in-depth study in MaGBSSI-3 of banana.Based on cloning and bioinformatics analysis,genetic transformation in tomato and Pichia pastoris expression of MaGBSSI-3 was studied.In order to get a new systematic understanding of the MaGBSSI-3 regulation mechanism,the MaGBSSI-3 promoter cloning and bioinformatics analysis was future finished.The main results were as follows:(1)Based on banana A genome,MaGBSSI-3 was cloned and bioinformatics analysis.The ORF of MaGBSSI-3 was 675 bp(accession No.KF512022).Bioinformatics analysis showed that the gene is located on 1 chromosomes,included 45 endonuclease enzyme loci and encoded 224 amino acids.Its molecular weight was 55.12 kDa,isoelectric point was 5.38,and the chemical formula was C723H1189N223O231s16.Its protein secondary structure contains 33 alpha helixes,57 stretches and 69 random curly structures,serine and threonine and tyrosine phosphorylated amino acid,and 15 across the membrane structure.(2)Subcellular localization result showed that MaGBSSI-3 was located on the cell membrane by PEG mediated tobacco leaf protoplast.Expression analysis showed that MaGBSSI-3 is specially expressed in banana fruit.(3)MaGBSSI-3 was over-expressed in tomato by agrobacterium tumefaciens mediated methods.The results showed that the MaGBSSI-3 is the highest express in fruit,follow leaves and flowers;With the development of tomato fruit,GBSS enzyme activity showed a trend of gradual decline,and GBSS enzyme activity in transgenic tomato fruit was slightly higher than the wild type tomato,the starch granules number,amylose,amylopectin and total starch content showed a trend of gradual decline;Starch content was the highest in the fruits and leaves of transgenic tomato,followed stems and roots,compared to control.(4)The yeast expression vector(pPICZaA-MaGBSSI-3)was builded and analyzed by GS115 yeast cells transformation,multi-copy screening,methanol induction expression,protein purification,enzyme activity detection.Molecular weight of MaGBSSI-3 protein was about 50 kDa,the result was consistent with theoretical predictions.GBSS protein has physiological and catalytic activity by yeast expression system.(5)MaGBSSI-3 promoter was cloned and analyzed from banana based on bioinformatics and 5' end missing technology,the result showed that the full length of MaGBSSI-3 promoter was 1119 bp and contained TATA-box,GC-box,and CAAT box core elements,which was induced by ABA,GA and drought treatment.Under dark/light condition,-113 bp is critical response components.