东乡野生稻及其渐渗系应答低磷胁迫的基因差异表达分析

Analysis of Differential Expression Genes in Dongxiang Wild Rice and Its Introgression Lines under Phosphorus-deficiency Stress

以东乡野生稻耐低磷渐渗系IL171及其双亲（栽培稻‘协青早B’和东乡野生稻）为试材,采用cDNA-AFLP技术分析其幼苗期应答低磷胁迫的差异表达谱特征,并采用实时荧光定量PCR分析验证差异表达基因的表达特性,为探究东乡野生稻耐低磷胁迫的分子机制、发掘耐低磷相关的基因奠定基础。结果显示：（1）基于17对扩增效果较好的cDNA-AFLP引物分析发现,不同胁迫时间的参试材料与其对照组（正常磷水平）相比,具有很多（20～159个）上调或下调表达的差异片段。（2）与‘协青早B’相比,IL171中特异性上调的差异带有36条,下调表达61条,而东乡野生稻中分别有79条和136条;IL171与东乡野生稻共有的上调差异带为13条,下调差异带有15条。（3）回收纯化其中60条特异性差异表达条带,最终克隆测序获得50个差异表达基因片段TDFs;通过Blast比对和功能分析,可将TDFs分为8类,包括能量与代谢、基因表达调控、信号转导和转录因子等。（4）实时荧光定量PCR验证其中6个TDFs发现,各差异片段的荧光定量PCR表达模式与cDNA-AFLP结果一致,表明该实验的cDNAAFLP差异表达结果可靠,东乡野生稻的部分耐低磷相关基因已成功导入到渐渗系中,是发掘利用东乡野生稻耐低磷相关基因、探究其耐低磷分子机制的重要资源。

In the present study,the phosphorus-deficiency tolerance introgression line IL171 and its parents（Oryza sativa cv.‘Xieqingzao B＇and Dongxiang wild rice）were used to investigate the differential expression genes at early seedling stage by cDNA-AFLP technique under the phosphorus-deficiency stress condition.Real-time fluorescent quantitative PCR was used to verify the differentially expressed genes.（1）cDNA-AFLP analysis revealed that numerous cDNA fragments（20-159）were obtained in each material under the phosphorus-deficiency condition compared to the control（normal phosphorus level）.（2）Compared with‘Xieqingzao B＇,36 and 61cDNA fragments were differentially up-and down-regulated in IL171,respectively.In Dongxiang wild rice line,79 and 136cDNA fragments were differentially up-and down-regu-lated,respectively.Further analysis showed that IL171 and Dongxiang wild rice shared the same expression pattern at 13up-regulated loci and 15down-regulated loci.（3）Among the 60 recovered and sequenced TDFs（transcript-derived fragments）,50 of them were obtained and their functions were determined through Blast search against the RAP-DB database.The functions of them were grouped into eight classes,including energy and metabolism,regulating genes,signal transduction and transcription factors et al.（4）Six above functional genes were subjected to real-time fluorescentquantitative PCR（qRT-PCR）analysis which was all in agreement with those of the cDNA-AFLP analysis,comfirming that cDNA-AFLP was reliable in detecting differentially expressed gens involved in responding to the phosphorus-deficiency stress.The results in this study suggested that partial alien DNA fragments of Dongxiang wild rice related to phosphorus-deficiency tolerance has been transferred into the introgression lines and they could be acted as excellent bridging germplasm in exploring and utilizing the phosphorus-deficiency tolerant genes in rice.In additon,the introgression linses derived from Dongxiang wild rice can be used a good experimental system for understanding the molecular mechanism to tolerate phosphorus-deficiency stress in wild rice.