Functional and expression analysis of genes involving sugar metabolism in forage grasses

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The objectives of the present research are as follows: 1) Production of gene-silencing plants of forage grass, Lolium perenne with respect to genes involving fructan biosynthesis using RNA interference (RNAi) procedure to clarify the mechanism of functions of fructan biosynthesis in grasses, 2) Isolation of transcriptional factors for cool-inducible genes and elucidation of their function in grasses, 3) Development of available DNA markers for breeding programs of grasses based on genomic information of genes of sugar metabolism.We obtained some results from two years research. For RNAi experiment, we made dsRNA expression vectors for three genes involving fructan biosynthesis which were isolated from L. perenne. The vectors were introduced into embryogenic calli derived from seeds of L. perenne using particle bombardment or Agrobacterium methods. Then calli with antibiotic resistance were obtained. Analysis of gene expression confirmed RNAi in calli, however such calli were not regenerated into plants. Ten novel putative CBFcDNAs of L. perenne (LpCBF genes) were isolated from cold-treated leaf tissue. Some CBF homolog genes were mapped on LG5 forming a cluster. Based on comparative genetic studies, conserved synteny for CBF gene family was observed between the Triticeae cereals and perennial ryegrass. RT-PCR analysis revealed that the expression of LpCBF genes was rapidly induced in response to low temperature and that the expression pattern under the low temperature conditions for a long period was different between the various LpCBF genes. Genes involving sucrose synthase and sucrose phosphate synthase as well as some cold inducible genes were isolated and sequence analysis made DNA markers distinguishable genotypes.