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

牧草糖代谢相关基因的功能和表达分析

基本信息

批准号：
17380163
负责人：
YAMADA Toshihiko
金额：
$ 6.53万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17380163/
关键词：
forage grasses sugar metabolism RNAi winter hardiness transcriptional factor transgenic DNA marker 転写遺伝子フルクタン

项目摘要

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.

本研究的目标如下：1）利用RNA干扰（RNAi）方法产生牧草多年生黑麦草（Lolium perenne）中涉及果聚糖生物合成的基因的基因沉默植物，以阐明禾本科植物中果聚糖生物合成的功能机制，2）分离冷诱导基因的转录因子并阐明其在禾本科植物中的功能，（3）利用糖代谢基因的基因组信息，开发禾本科牧草育种的DNA标记。在RNAi实验中，我们将从L.多年生植物将该载体导入由苜蓿种子产生的胚性愈伤组织中。用粒子轰击法或农杆菌法测定多年生植物。获得了具有抗生素抗性的愈伤组织。基因表达分析证实了愈伤组织中的RNAi，然而这样的愈伤组织没有再生成植物。10个新的L.从冷处理的叶组织中分离多年生植物（LpCBF基因）。一些CBF同源基因被定位在LG 5上形成一个簇。通过比较遗传学研究，发现CBF基因家族在小麦族禾本科和多年生黑麦草中具有保守的同线性。RT-PCR分析表明，LpCBF基因的表达在低温条件下被迅速诱导，并且在低温条件下长时间表达的模式在不同的LpCBF基因之间是不同的。分离了蔗糖合成酶和蔗糖磷酸合成酶相关基因以及低温诱导基因，序列分析表明，DNA标记可区分基因型。