干旱胁迫下油菜消减文库的构建及分析

Construction and Analysis of SSH Library in Rapeseed(Brassica napus L.) under Drought Stress

以抗旱能力强的油菜Holiday为材料,以干旱处理的样品为Tester,正常水分管理(对照)为Driver构建干旱诱导的甘蓝型油菜叶片正向抑制性消减杂交(SSH)文库。随机挑选24个阳性克隆进行PCR验证,结果表明,其中23个含有插入片段,平均大小在750bp左右。将96个阳性克隆测序,运用CAP3 Sequence Assembly Program聚类、拼接和去除冗余,获得重叠群4条,单拷贝序列82条,平均长度为542bp。经BlastX程序比对蛋白数据库发现,11条EST没有找到同源性序列,75条有同源序列。KOBAS分析发现,28条EST被定位到67条代谢途径中,根据P值推测,光合中的碳固定、有氧呼吸的电子供体、氮代谢、乙醛酸和二羧酸代谢在植物干旱胁迫中发挥着极为重要的作用。这些EST涉及到的功能中所占比例最大的分别是细胞器(58.82%)、结合(30.77%)和新陈代谢过程(43.72%),表明甘蓝型油菜受到干旱胁迫时,细胞器组件上的功能发挥了重要的作用,结合基因和蛋白被激活,加强了新陈代谢。

The positive SSH library in leaves of rapeseed （Brassica napus L.） Holiday （with high drought resistance ability） was constructed using the sample under drought stress as Tester and the sample under normal water management as Driver. Twenty-four positive clones selected randomly were verified by using PCR. The result of electrophoresis showed that 23 clones contained inserts whose average length was 750 bp. Ninety-six positive clones were sequenced and analyzed by CAP3 Sequence Assembly Program, showing that four contigs and 82 singletons were contained and the average length of 86 ESTs was 542 bp. BlastX alignment with Nr database showed 11 ESTs without significant similarity and 75 ESTs with similarity in GenBank data- base. KOBAS suggested that 28 ESTs were mapped to 67 pathways. It could be predicted that the carbon fixation in photosyn- thetic organism, electron donor III of aerobic respiration, nitrogen metabolism, glyoxylate and dicarboxylate metabolism played a key role in drought tolerance of rapeseed. The analysis of ESTs showed that a large share of fimction could be attributed to organelle （58.82%）, binding （30.77%）, and metabolic process （43.72%）, which plays important roles in metabolism of rapeseed under drought stress.