【摘要】 以未经诱导的棉花下胚轴、经愈伤诱导10d的下胚轴和胚性愈伤组织为材料,利用差异显示反转录 PCR(DDRT PCR)技术对陆地棉体细胞胚胎发生过程中的cDNA差异表达进行了初步研究。反转录获得cDNA第一链,经3个锚定引物和随机引物的DDRT PCR,产物经测序胶电泳分离,共得到约200个差异表达cDNA片段,其中51个为陆地棉胚性愈伤组织所特有,从中还获得相关基因上游或下游的调控系列,从其中一个样品中可成功分离、克隆24个cDNA片段。经Northern杂交验证,从获得的差异片段中找到了一个能稳定出现的阳性cDNA片段,该片段仅出现在胚性愈伤组织阶段。对该片段进行克隆、测序和序列同源性分析,发现该片段是一个功能未知的cDNA片段。更多还原

【Abstract】 In order to investigate the different expression of the genes related to somatic embryogenesis, the hypocotyls section (explant), explant after ten days induction on medium of MS with IBA and kinetin, and embryogenic callus were sampled for total RNA isolation. After reverse transcription with three anchor primers, the first strand cDNA was released. Then DDRTPCR was performed with various pairs of anchor and 10mer random primers. Through separating these PCR products on a sequence gel, about 200 differentially expressed cDNA fragments were obtained, of which 51 cDNA fragments could be seen only in the embryogenic callus. Besides the band difference among the three kinds of samples, we also observed the down and upregulated gene expression among them. We have isolated 24 cDNA fragments that expressed specially in one of the samples. To identify the truth of the differentially expressed cDNA fragments, Northern blotting was performed. Among differential fragments acquired, one was found to be a positive fragment. This fragment appeared only in embryogenic callus, but not in the other samples. Sequence analysis showed that it was a new cDNA fragment, and its function had not been known yet.更多还原