Proteomic Analysis Of Soluble Starch Synthase Binding Proteins In Maize Endosperm

Proteomic Analysis of Soluble Starch Synthase Binding Proteins in Maize EndospermSoluble starch synthase (SSs, soluble starch synthase) presents in the amyloplasts, including SSâ… , SSâ…¡, SSâ…¢and other subtypes.In 1981, Boyer found two kinds of soluble starch synthase from maize matrix, named them zSSI and zSSâ…¡. A recent detection of a maize transposon insertion mutant indicated that dull 1 gene encodes a protein whose amino acid sequence has a high homology with potato SSâ…¢, named zSSâ…¢.Maize soluble starch synthase (zSSs) is mainly responsible for amylopectin synthesis. Each isozyme performs a special role. SSI is mainly responsible for glucose-based degree of polymerization of 10 below (DP<10) of short-chain extension, and SSâ…¡or SSâ…¢are mainly responsible for further extensions of short-chain catalysis.Although individual enzymatic steps in stach metabolic pathways have been adequately revealed over the last decades, detailed mechanisms of starch synthesis remain unknown. Recently, some experimental data support that starch metabolism is under the control of a complex metabolic network, which operates through functional interactions among multiple starch metabolizing enzymes. Identified of new starch metabolizing enzymes binding proteins was thus important for further explored starch metabolic network.In this study, maize soluble starch synthase familys were choosed as baits to screen their binding proteins. Therefore, zssâ… and zssâ…¡b clon were acquired from maize grains cDNA by nested PCR method. The conserved region of zssâ…¡clon was got from the existing zssâ…¢. A cDNA library was constructed from the high amylopectin maize kernel. Using zSSâ… , zSSâ…¡b and zSSâ…¢HD as bait proteins, some binding proteins were identified by a series assays including yeast two-hybrid screening, GST pull-down and western blot. The detailed results were showed as follows:(1) A cDNA library was constructed from the high amylopectin maize kernel. Then using zSSI, zSSâ…¡b and zSSâ…¢HD as bait proteins,14,10 and 18 proteins were identified as new SSs-binding protein candidates respectively.(2) To further validate the interaction, a pair of proteins (SSâ…¡b versus cyanate hydratase) was selected randomly from the interations detected by yeast two-hybrid, and their interaction were verified by GST pull-down analysis in vitro. SSâ…¡b was fusion with Myc tag, and expressed in yeast. Cyanate hydratase was fusion with GST tag, and further expressed in E.coli. Our primary data validated that SSIIb associated with cyanate hydratase in vitro.In conclusion, our study supplemented the maize starch synthesis regulatory networks. This is the first report of new zSSs binding protein, and provide a basic experiment data for futher dissection of starch synthesis regulatory network in maize.