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

本研究的目的如下：1)利用RNA干扰（RNA interference, RNAi）技术在牧草中培育与果聚糖生物合成相关基因的基因沉默植物——麦穗（Lolium perenne），阐明草中果聚糖生物合成的功能机制；2)分离冷诱导基因转录因子并阐明其在草中的功能；3)基于糖代谢基因的基因组信息开发可用于草育种计划的DNA标记。我们通过两年的研究获得了一些结果。在RNAi实验中，我们将从白羊草中分离到的三个与果聚糖生物合成有关的基因制作成dsRNA表达载体。采用颗粒轰击法或农杆菌法将载体导入由二年生小麦种子产生的胚性愈伤组织。获得耐药愈伤组织。基因表达分析证实了RNAi在愈伤组织中的作用，但这些愈伤组织并没有再生成植株。从冷处理叶片组织中分离到10个新的假定的白羊草cbfcdna （LpCBF基因）。一些CBF同源基因被定位在LG5上形成一个簇。通过比较遗传研究，发现小麦科谷物和多年生黑麦草CBF基因家族具有保守的同源性。RT-PCR分析表明，低温对LpCBF基因的表达具有快速诱导作用，且不同LpCBF基因在长时间低温条件下的表达模式存在差异。分离到蔗糖合成酶和蔗糖磷酸合成酶相关基因以及一些冷诱导基因，通过序列分析，利用DNA标记区分基因型。

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.