Cloning And Identification Of Granule-bound Starch Synthase Gene In Banana Fruit

The banana is known as the tropical and sub-tropical fruit and is the fourth most important fruit after apple, pear, citrus, while it is the fourth crop in the developing countries. Banana has the very high economic value and nutritional value. Its fruit contains rich starch which is the source of the energy and carbon skeletons in biological body and is the major component and basic substances of fruits. Starch is closely related to fruit yield and quality.According to the chemical structure, starch can be divided into amylose and amylopectin. Granule-bound starch synthase (GBSS) was responsible for amylose synthesis. The study of GBSS would help to understand the molecular mechanism of banana starch synthesis, more effective regulate the metabolism of starch.Banana (Musa acuminata L. AAA group cv. Brazilian) was used as material in the study. Pulps of develop0d,10d,20d,30d,40d,50d,60d and fruit postharvest0d,2d,6d,10d,14d,16d,18d were collected. The systematic study of banana fruit GBSS was conducted in the physiological and molecular level, the main results as fllow:(1) In the process of banana fruit development, starch granules were nearly elliptic and became larger and the quantity of starch granules increased, while the content of total starch, amylose, amypolectin, resistant starch gradually increased. The maximum were66.93%(50d),233.28mg.g-1(5O d),436.03mg.g-1(50d),402.96mg.g-1(3O d), respectively. During the stage of fruit postharvest, the size and quantity of starch granules decreased, while the content of total starch, amylose, amypolectin, resistant starch gradually decreased. At18d of fruit postharvest, the contents were0.18%,3.88mg.g-1,0mg.g-1,39.52mg.g-1, respectively.(2) By homology cloning method, the full length cDNA of six MaGBSS family genes were cloned from banana pulp, designated as MaGBSSI-1(KF512020). MaGBSSI-2(KF512021)、 MaGBSSI-3(KF512022)、MaGBSSI-4(KF512023)、MaGBSSII-1(KF512024)、MaGBSSII-2(KF512025). ORF were1851bp,1851bp,675bp,1845bp,2265bp,906bp and translated616,616,224,614,754,301amino acids, respectively.(3)Bioinformatics analysis of MaGBSS family genes showed that MaGBSSI-1、 MaGBSSI-2、MaGBSSI-3、MaGBSSI-4、MaGBSSII-1、MaGBSSII-2in the1,1,1,9,4,8chromosomes, contained13,13,6,13,10,1exons. Amino acid alignment analysis showed that MaGBSS family contained3conservative domain, which were Box1(PWSKTGGLGDVLGGLP), Box2(TSPFEPCGL), Box3(ATTGGLVDTV). And amino acid homology with other species shared50%-84%identity. (4) Q-RTPCR revealed significant defferences in the expression of MaGBSS family genes in different banana tissues. The results suggested MaGBSSI-1, MaGBSSI-2, MaGBSSI-4, MaGBSSII-1and MaGBSSII-2were upregulated in vegetative organs such as root, stem, leaf and bract, In contrast, MaGBSSI-3was highly expressed in banana reproductive organs such as flower, peel, and pulp. The expression of MaGBSSI-3in the pulp was326times of the quantity in the leaf. These results suggested that MaGBSSI-3might be involved in amylose synthesis in reproductive organs, while amylose accumulation in banana vegetative organs might be correlated with MaGBSSI-1, MaGBSSI-2, MaGBSSI-4, MaGBSSII-1and MaGBSSII-2.(5) QRT-PCR was used to research the differential expression of MaGBSS in the process of banana fruit development and postharvest. Results suggested that the expression of MaGBSSI-3gradually increased during the banana fruit development, and decreased in banana fruit during postharvest. Correlation analysis showed the expression of MaGBSSI-3had a extremely positive correlation with the change of amylose content. Therefore, MaGBSSI-3might be closely related with amylose metabolism in banana fruit.(6) GBSS activity of banana pulp gradually increased during the process of fruit development and gradually decreased during the stage of postharvest. The analysis of SDS-PAGE suggested that the protein band between25.0kDa and35.0kDa showed a trend of first increased and then decreased, the same with the prediction of MaGBSSI-3protein molecular weight(25.12kDa).(7) After polyclonal antibody preparation of MaGBSSI-3, Western blot analysis suggested that the MaGBSSI-3protein molecular weight was25.12kDa and up-regulated expression during fruit development while it was down-regulated expression during postharvest, the same with the changes of starch granule, amylose content, GBSS activity and the expression of MaGBSSI-3. Therefore, in the6members of MaGBSS family, MaGBSSI-3might be the key gene in banana fruit amylose synthesis.