落叶松扦插生根过程SSH文库构建及部分基因的表达分析

Construction of Suppression Subtractive Hybridization Library and Expression Pattern of a Few Genes Involved in Rooting of Larix Cuttings

为研究落叶松在扦插生根过程中基因的表达情况,以落叶松扦插生根率存在显著差异的2个全同胞无性系为材料构建差异表达cDNA文库,获得正、反2个消减文库。随机挑取正库和反库各500个克隆进行测序。将测序结果整理后,正库得到272个UniEST,反库得到249个UniEST。经生物信息学分析表明,这些UniEST分别属于新陈代谢、信号途径、物质运输、抗性相关、生长发育等类别基因,这些基因可能在扦插生根过程中具有重要作用。选择部分有代表性的基因,运用半定量PCR技术对这些基因在落叶松不同器官、不同扦插阶段的表达情况及对IBA的响应进行检测。检测结果表明激素在落叶松扦插生根过程中发挥重要作用,除激素类基因外,小G蛋白、ABC转运蛋白类、因伤诱导物质等相关基因也参与落叶松扦插生根的过程。

In order to investigate the expression pattern of genes during rooting of Larix cuttings,two differentially expressed cDNA libraries were constructed by using two Larix clones whose rates of rooting are significantly different. Each 500 clones, stochastically selected from positive and negative library,were sequenced. After sequencing and assembling,we obtained 521 UniEST（272 from positive library and 249 from negative library）. The UniEST included the genes related to metabolism,signal pathway,transport,resistance,development and cell structure. The result suggested that these genes might have the function during rooting of Larix cuttings. The representational genes were selected and further detected by using semi-quantitative RTPCR to understand their expression patterns in roots,stems,leaves and at different stages of rooting of Larix cuttings. The result indicated that hormones played pivotal roles in rooting of Larix cuttings. Besides genes related to hormones,small G protein genes,ATP binding cassette transporters genes and WRCs genes were all involved in the rooting procedure.