The Study Of Starch Formation And The EfSBE1 Expression In The Development Of Gordon Euryale Seed(Euryale Ferox Salisb)

Gordon euryale(Euryale ferox Salisb),an annual aquatic herbaceous plant,belongs to genus Euryale of family Nymphaeaceae,which is origined from East Asia.The gordon euryale seeds are rich in nutrients and various active ingredients.Among which,starch accounts for more than 70%of the dry matter weight in the seeds,so it is the main storage.The composition of starch and grain structure decide the edible quality of gordon euryale.At present,there are few studies on the starch formation in the developmental seed of gordon euryale in literature,and the molecular mechanism of starch synthesis has not been reported.In this study,the change of seed starch content and developmental process of starch grain in two cultivars('Zi Hua Su Qian ' and' Zi Hua Ci Qian ')of gordon euryale were analyzed.Some proteins relevant to starch metabolism were explored by proteomics analysis,and important gene was cloned with aim to study the molecular mechanism of starch synthesis in the development of gordon euryale seed.1.The study on starch content and starch grain morphology in the development of gordon euryale seed:The results showed that starch accumulation was rapidly increased more than 30%from 10 to 20 d after anthesis,and only 15%was accumulated from 30 to 40 d after anthesis.The amylose content in 'Zi Hua Ci Qian ' was higher than that of 'Zi Hua Su Qian '.However,starch content,amylopectin content and the ratio between amylopectin and amylose were significant higher in ' Zi Hua Su Qian ' than in ' Zi Hua Ci Qian '.Microstructure analysis revealed that starch grain in ' Zi Hua Su Qian ' was polyhedron with distinct edges and comers,and the corners of starch grain in 'Zi Hua Ci Qian' was blunt and partly round.The starch grain in 'Zi Hua Su Qian ' showed more close in arrangement and higher plumpness than that of 'Zi Hua Ci Qian'.2.The proteomics analysis in the development of gordon euryale seed:969 changed proteins in expression were identified from four stages of gordon euryale seed development by iTRAQ technique based on genome of Arabidopsis thaliana.These change proteins were mainly involved in carbohydrate metabolism,biosynthesis and degradation,protein folding and synthesis,transcriptional translation,nucleotide metabolism and so on.Twenty proteins related with starch and sucrose metabolism were found from these changed proteins.Among which,four proteins such as SBE2.2(Starch branching enzyme 2.2),APL2(ADP-glucose pyrophosphorylase large subunit 2),APS 1(ADP-glucose pyrophosphorylase small subunit 1)and SS2(Starch synthase 2)showed high correlation with starch synthesis.SBE was selected to further study due to its important role on the formation of starch branch,the results elucidated that SBE decreased the expression during gordon euryale seed development.The abundance of protein 20,30 and 40 d after postanthesis was only 0.68-,0.16-and 0.15-fold compared with that of 10 d after anthesis.3.The cloning of EfSBE1:A cDNA sequence of SBE was isolated from 'Zi Hua Su Qian 'by PCR method.The full length of this gene was 2 796 bp.More than 76%similarity was found when compared with SBEs derived from other species by blastx tool in NCBI database,so this gene was designated as EfSBE1.EfSBE1 composing of 2 782 bp of open reading frame encoded 821 amino acid residues with a 94.4 kDa molecular weight of protein.Because this gene showed 78%similarity with SBEI of Nelumbo nucifera Gaertn after comparison,so we believed that EfSBE1 belonged to SBEI type.In addition,DNA sequence of EfSBE1 was cloned from the same sample as above,and the sequence was 11 106 bp consisting of 13 exons and 12 introns.4.The analysis of EfSBE1 expression:The expression of EfSBE1 was significantly different in 'Zi Hua Ci Qian ' and ' Zi Hua Su Qian ' after anthesis for 10 d,15 d,20 d,25 d,30 d,35 d and 40 d,and the expression showed a down regulated tendency.The mRNA level of EfSBE1 at 40 d after anthesis was only 0.13-and 0.25-fold of 10 d after anthesis in ' Zi Hua Ci Qian ' and ' Zi Hua Su Qian ' respectively.The transcriptional level EfSBE1 at early stage of 'Zi Hua Ci Qian ' seed development was lower than that of 'Zi Hua Su Qian ',and while,this gene expression in the middle and later development of ' Zi Hua Ci Qian ' seed was higher than that of' Zi Hua Su Qian '.