| Chinese chestnut(Castanea mollissima Blume)is in the fagaceae family and is known as woody food,which act as one of the important commercial nut tree.Starch is the major metabolite in chestnuts,accounting for 46 ~ 64 % of the chestnut dry weight.Starch consists of amylose and amylopectin,amylopectin is the main constituents of total starch and account for around 70 %.There are large aggregation of studies shows that the structure of amylopectin can affect the properties of chestnut starch and thus the nut processing quality.Samples were collected from the Chinese chestnut cultivar‘Yanshanhongli’ in the Chestnut Experiment Station in the Huairou district of Beijing.Chestnuts were collected from three 15-year-old trees at 60,64,68,71,74,77,80,and 83 days after pollination(DAP).The cotyledons were frozen in liquid nitrogen and stored at-80 °C for further studies.1.The properties of chestnut starch granule.Light-microscope,scanning electron microscope and polarizing microscope were used to observe the morphological characteristics.The results indicated that the increase of total starch result from amylopectin synthesis.Amylopectin contributed up to 76 % of the total starch content at 80 days after pollination(DAP),which is stage before ripening in chestnut.Starch granules have various shapes and irregular starch granule appeared mostly in the earlier period.The sizes of starch granules are among 1-30 μm~2,maltese cross can be observed in all the granule.The pasting temperature declined during the development.2.The expression patterns of main genes(AGPase、GBSS、SSS、PUL、SBE)involved in starch synthesis were evaluated in order to found out the key gene which affected chestnut starch physico-chemical properties.SBE showed the most significant change compared to other genes.The pasting temperature is negatively correlated to the SBE expression,while the sizes of starch granule are positively correlated to the SBE expression.Those results indicated that SBE is the key gene affected the starch characteristics during development.3.We identified and cloned the full length sequence of two starch branching enzymes,SBE I and SBE II,from developing chestnut cotyledons.The open reading frame(ORF)of SBE I was predicted to be 2,460 bp in length(Gen Bank accession number: KY429029).The full length ORF of SBE II was predicted to be 2,595 bp in length(Gen Bank accession number: KY429028.The expressions of two SBE genes reached the highest levels at 60 DAP and 80 DAP.SBE I and SBE II both have a N-conservative domain and a C conservative domain,with a barrel amylase catalytic domain. |
