Study On The Role Of Over-expression AGpase Cytosolic Large Subunit (LSU?) In Starch Content Of Common Wheat

Starch is the primary factor in yield of wheat,accounting for about65–80% of wheat kernel weight.The starch synthesis is completed by a series of enzyme-catalyzed reactions in endosperm.AGPase is the rate-limiting enzyme of regulating starch biosynthesis in cereal seeds.In plant cells,there are four types of AGPase subunits: cytosolic small subunit(SSU?);plastidial small subunit(SSU?);cytosolic large subunit(LSU?)and plastidial large subunit(LSU?).In this experiment,the rates of starch accumulation,and the activities of AGPase were measured and compared between two wheat cultivars with different seed weights.Then,the transcript levels of genes encoding four AGPase subunits were compared using semi-quantitative RT-PCR during the grain filling periods.c DNA sequence of the open reading frame(ORF)of LSU? was amplified,an over-expression vector of LSU? gene was constructed,and transferred into wheat by pollen tube mediated transformation methods.PCR,southern blotting and real-time RT-PCR analysis indicated that LSU? gene was integrated into the genome of wheat.The starch contents were significantly improved in transgenic plants.The main experimental results were as follows:1.Two different seed weight of wheat cultivars(Yujiao2 and Lankao Aizao8)were selected in this study,the rates of starch accumulation,the activities of AGPase,and the transcript levels of its four subunits were measured and compared.During the whole grain filling periods,there were no differences in the rates of starch accumulation and AGPase activities in the first 10 days,whereas significant difference in subsequent grain filling period.Then,the transcript levels of AGPase subunits were compared between them,LSU? transcripts were higher in Yujiao2 than that in Lankao Aizao8 after 15 days of anthesis,but no difference was found between the two wheat cultivars in the other three subunits.Moreover,only the variation trend of LSU? subunit transcript levels was corresponded with the starch accumulation rates and AGPase activities,implying that LSU? was closely related with the rates of starchaccumulation.2.The ORF sequence(Gen Bank no: DQ839506)of LSU? was cloned,and constructed for an over-expression vector containing LSU? gene.Then it was transferred into wheat by pollen tube mediated transformation method.PCR,Southern blot,and real-time RT-PCR analyses indicated that the transgene was integrated into the genome of transgenic plants,and was overexpressed in their progeny.The overexpression of the LSU? gene remarkably enhanced AGPase activity,endosperm starch weight,and single grain weight.These results suggest that transgenic wheat plants overexpressing the LSU? gene may have potential applications in wheat breeding to develop a high-yield wheat cultivar with high starch weight and kernel weight.