高温胁迫下草坪草高羊茅差异表达基因的分子研究  

Molecular Study about Differential Expressed Genes in Tall Fescue Induced by High Temperature

作　　者：李卉[1] 王艳[2] 王海宏[3] 梁珂珂 李建龙[1] 

机构地区：[1]南京大学生命科学学院,江苏南京210046 [2]中国科学院成都生物研究所,四川成都610041 [3]南京交通职业技术学院,江苏南京211188 [4]南京农业大学草业学院,江苏南京210095

出　　处：《天津农业科学》2015年第1期1-5,共5页Tianjin Agricultural Sciences

基　　金：苏州市科技计划项目(科技支撑计划-农业项目(SNG201447));江苏省农业三新工程项目(SXGC[2014]287);APN项目(Pro-ject:ARCP2013-16NMY-Li);张家港市凤凰水蜜桃营养生理;腐烂机理及保鲜综合技术研究(ZKN1002)

摘　　要：通过构建高羊茅高温胁迫下的消减文库,并使用斑点印迹技术从中筛选出差异表达基因,以期深入研究高羊茅适应高温的分子机制。以冷季型草坪草高羊茅为研究对象,在两年生理指标测定的基础上通过构建高羊茅在高温胁迫下的SSH文库,分别以正、反向SSH探针与尼龙膜上的消减c DNA质粒进行杂交,并经相应的显色反应得到杂交信号,比较了38/30°C(昼/夜)的培养箱中高温胁迫6 h的高羊茅叶片和对照组高羊茅叶片的RNA差异。结果表明:试验得到的差减文库中大量组成型表达基因已经被有效去除,使某些特有的差异基因得到了富集;利用斑点杂交共得到252个差异片段,库中阳性克隆约占50%以上;阳性克隆中91个为上调基因,161个为下调基因,这些与高羊茅的耐热性密切相关的基因即为试验所寻的差异表达基因。本试验为找出高羊茅耐热相关基因奠定了基础,为草坪草的转基因育种工作提供了相关依据,结合恰当的水肥调控措施可有效提高草坪草的抗逆性。In order to study the molecular genetical mechanisms about how turfgrass adjust heat stress, suppression subtractive hybridization was used to contract cDNA library of tall fescue stress-related genes at a high temperature and the method of dot blot hybridization was used to find differentially expressed genes. Experimental material is cool-season turfgrass tall fescue. After two years of physiological experiments, first, SSH library of tall fescue under high temperature stress was built. Second, the subtracted cDNA plasmid in the nylon membrane was used to two-way hybridized with SSH probes and show hybridization signal by the corresponding color reaction. Thus, the differences between the control group and treatment group of tall fescue leaf RNA was studied, the treatment group is the plant on the temperature stress of 38/30 ℃ （day / night） incubator for 6 h. The results showed that a large constitutively expressed genes of the subtractive library had been effectively removed, so that some of the specific enrichment of differentially expressed genes obtained; 384 clones were selected from the forward and reverse SSH cDNA library, respectively. 252 different fragments were then obtained by dot-blot screening, and the positive clones in the library were about 50%, 91 of which are up-regulated genes and 161 down-regulated genes. The study provides the basis for the study of tall fescue resistant genes, as well as the relevant evidence for transgenic turfgrass breeding. Adding appropriate water and fertilizer measures, the resistance of turfgrass can be effectively improved.

关 键 词：高温胁迫 逆境生理 差异表达基因 抑制差减杂交 斑点印迹技术 阳性克隆 

分 类 号：S688.4[农业科学—观赏园艺]