草坪草高羊茅高温诱导抑制差减杂交文库的构建及其表达

为了深入研究草坪草对抗高温逆境的分子遗传机制,构建高羊茅在高温胁迫下相关的基因文库。研究以冷季型草坪草高羊茅为研究对象,在长期生理试验的基础上,通过构建高羊茅在高温胁迫下的SSH文库,挑选部分阳性克隆进行测序,并对所获得的差异基因序列进行分析,研究了在38℃/30℃(昼/夜)培养箱中进行高温胁迫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的基因已经被有效去除,使某些特有的差异基因得到了富集;两个文库的基因片段的长度分布在200~900 bp,平均片段长度约500 bp左右;在构建的高羊茅耐热cDNA文库中随机挑选了123个大小约500 bp左右的EST测序,共得到100个有效序列,其中38个是未知序列,其余62个为已报道序列,但是序列功能全部未知,这些EST中有25个与叶绿体染色体有关,其中9个与已提交的羊茅属植物叶绿体内的基因同源。本试验最终得到了合格的差减文库并对部分基因进行了测序,可为高羊茅耐热基因的研究提供依据,同时也为提高草坪草水肥调控措施提供了理论基础。

Construction of SSH Library Induced by High Temperature in Tall Fescue and Analysis of its Expression

In order to study the molecular genetical mechanisms about how turfgrass against heat stress, suppression subtractive hy- bridization was used to construct cDNA library of tall fescue stress-related genes at a high temperature. Experimental material was cool-season turfgrass tall fescue. On the basis of long-term physiological experiments, by building SSH library of tall fescue under high temperature stress, some positive clones to be sequenced were selected, and the differences in gene sequences were analyzed. Thus, the differences between the control group and treatment group of tall fescue leaf RNA were studied, the treatment group was 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; Length of the two gene libraries distributed in 200-900 bp, the average fragment length about 500 bp; in the library, 123 of about 500 bp in size EST to be sequenced were selected randomly, and a total of 100 valid sequences had been got, of which 38 were unknown sequence, and the remaining 62 had been reported, but the entire sequence function were unknown. There were 25 of these EST were related about chloroplast genome, of which 9 were homologous with a submitted fescue plant chloroplast gene. The trial finally got qualified subtracted library, and some genes were sequenced, it may provide the basis for the study of tall fescue resistant genes, as well as provide a theoretical basis of the measures to improve the regulation of turfgrass fertilizer.