Molecular Identification Of Starch Branching Enzyme-Ⅰ (SBE Ⅰ) In Triticum Monococcum

In this study, we have isolated the starch branching enzyme Ⅰ(SBE I) gene, predicted the molecular structure, analysed the expression of SBE I gene and researched the evolution of the relationship among diploid wheat by using homologous cloning, overlap extension PCR (SOE-PCR) and quantitative real-time PCR (RT-qPCR). In addition, from 20 accessions of T. monococcum with various total starch content, we previously identified two accessions (Y59 and Y63) with significant differences in total starch content between each other. Then we measured starch content, determined SBE activity within endosperms, analyzed expression patterns of SBE I by real-time quantitative PCR during grain filing period of two accessions. The main results in this study are as follow:1. The sequences of SBE I gene in diploid wheat were about 5313-5526 bp, which had 14 exons and 13 introns. Based on the homology comparison of corresponding exons and introns in the materials, we found that the first exon varied obviously in different species. The homology comparison of corresponding exons showed that the sequence similarity ranged from 70.50% to 98.02% and their average similarity was 92.32%. While in introns, they were 15.50% to 83.63% and with 92.32% in average similarity. This phenomenon demonstrated that the open reading frame ware conserved and the first varied exon maybe the reason that the expression of SBE I were different in different plants. Based on the deduced amino acid sequences from diploid wheat, the results of phylogenetic tree indicated that SBE I in 71 monococcum is more closely related to T. boeoticum and T. urartu.2. According to the GT-AG composition rules between exons and introns and the cDNA sequences of other species from the NCBI, we speculated the composition structure of introns and exons and gained the ORF. Through the alignment of amino acid, we founded the protein struture in diploid wheat have 4 conserved domains:DWHSHA, GFRFDGVTS, EYFS and YAESHDQ. The D of GFRFDGVTS, E of EYFS and D of YAESHDQ were regarded as the nucleophile base and catalytic site.3. From 20 accessions of T. monococcum with various total starch content, we previously identified two accessions (Y59 and Y63) with significant differences in total starch content between each other. Y59 had higher total starch content and more plump endosperm than Y63. By exploring the starch accumulation during grain filling period, we founed that accumulating rates of starch and amylopectin in Y59 were significantly higher than that in Y63. Meanwhile the SBE enzyme activities in Y59 was higher than those in Y63. The analysis that the correlation of SBE enzyme activities to starch filling rate suggests that, as reported in wheat endosperm and rice grains previously, SBE activity in T. monococcum grains plays an important role in starch accumulation process. On the basis of predicted amino acids and amino acids from other species which contain of A, B, D group of wheat, rice and maize, we found that sites of Tyr303, Asp338, His343, Arg410, Asp412, Glu467 and His535 were conserved in these species. This result indicated that:these sites are equally necessary for SBE activity in endosperm of T. monococcum:Different expression of Y59 and Y63 may resulted from the differences on non-conservative sites. The function of these varied sites in influencing the significant changes of SBE I enzyme activity need to be studied in the future.4. We have determined the differential expression of SBE I by RT-qPCR. Here, SBE I transcripts of both Y59 and Y63 increased about 50 times from 5 DAP to 17 DAP. SBE I of both accessions were highly expressed from 14 DAP to 23 DAP, however the SBE activity was mainly present at early and middle period of grain development. These results demonstrated the following conclusions. Firstly, in T. monococcum, SBE I mainly plays roles at middle stage of grain development while in wheat it mainly functioned at later stage. The difference may be caused by the different genetic background. Secondly, the possibly existing of other SBE isoforms which may be SBE Ⅱa or SBE Ⅱb in T. monococcum endosperms play an important role for SBE activity at early grain development according to previous study from other plants. Lastly, SBE I transcripts were positively correlated to SBE activity at middle grain development and then may influence the accumulation of amylopectin.