Functional Analysis Of Microtubule Associated Protein Gene SBgLR 5’UTR And Application Of AtMAP18 In Maize Quality Improvement

The microtubule associated protein gene SBgLR is a pollen-specific gene in Solanum tuberosum.Previous work demonstrated that the region from-85 to+184 was critical for gene specefic expression in pollen grains.Transcription start site was determined by 5’RACE,the A of translational start site as+1 and the region from+1 to+184 wasthe 5’UTR of SBgLR.Sequence analysis indicated six putative pollen-specific elements,as well as the Trihelix,DOF and ARR binding sites were located in the 5’UTR.Additionally,a stable triple-hairpin structure was predicted by analysing the secondary structure of the 5’UTR.Quantitative assay ofthe GUS expression and GUS activity showed that the 5’UTR could enhance gene expression in transgenic tobacco.Mutation analysis of the+5 to+17 region,which contained the putative binding site of Trihelix and DOF,revealed that this region was not necessary for the pollen-specific expression.In order to find the crucial elements that specific to pollen,we progressively deleted the pollen elements AGAAA and TTTCT from 3’of 5’UTR.Stable expression results showed that the secondary structure of the 5’UTR had no effect on pollen-specific expression.However,the region from+31 to+60 was crucial for gene expression.Further investigations showed that the mRNA expression level was slightly decreased when the+31 to+60 region was deleted,but the decay rate of mRNA was not changed.Furthermore,mutation analysis also confirmed the pollen-specific element,which located at+37,had an important role in pollen-specific expression.Using yeast one-hybrid screening,we isolated a DOF protein(StDof23)and a AT-hook protein(StAHL).Further investigation indicated that the StDof23 could interact with the+31 to+60 region and have a positive effect.The StAHL could interact with the-49 to+60 region and have a negative effect.All these results demonstrated that the 5’UTR could enhance gene expression,but also change the tissue specificity by regulating the transcription as well as translation.These results provide a further understanding of the regulation mechanism in eukaryotic gene expression.Lysine is the first limiting essential amino acid for animal nutrition in maize grains.Expression of naturally lysine-rich protein genes can increase the lysine contents in maize seeds.AtMAP18 fran Arabidopsis thaliana encoding a microtubule-associated protein with high-lysine content(20.3%)was introduced into the maize genome with the seed-specific promoter F128,and the T4 transgenic maize with AtMAP18 was obtained in the previous work.We cultured offspring of the transgenic maize continuously until the T7 generation.Both the protein and lysine contents were showed more than 20%improvement in the following generations and that the high-lysine and high-protein characters were heritable.Expression of AtMAP18 increased both zein and non-zein protein in the transgenic endosperm.Compared with the wild type,more protein bodies were observed in the endosperm of transgenic maize.These results implied that,as a cytoskeleton binding protein,AtMAP18 facilitated the formation of protein bodies,whichled to accumulation of both zein and non-zein proteins in the transgenic maize grains.Furthermore,F1 hybrid lines with high lysine,high protein and excellent agronomic traits were obtained by hybridizing T6 transgenic offspring with other wild type inbred lines.These results provide evidence supporting the use of cytoskeleton associated proteins to improve the nutritional value of maize.