菜心抗虫性的cDNA-AFLP分析

cDNA-AFLP Analysis of Insect Resistance in Flowering Chinese Cabbage(Brassica rapa var. parachinensis)

【目的】探讨菜心抗虫性的分子机制,为抗虫品种选育和抗虫基因挖掘提供理论支持和中间材料。【方法】选用抗小菜蛾品种65和感小菜蛾品种69菜心为亲本构建F2作图群体,利用cDNA-AFLP标记构建转录图谱、分析差异基因表达,筛选和克隆抗虫相关转录衍生片段。【结果】14对引物组合共扩增出330条扩增带,其中238条为多态性带,多态性条带比例为56.00%~100.00%,平均为73.86%,平均每对引物扩增24条带,多态性条带17个。构建的cDNA-AFLP转录图谱由5个连锁群组成,193个标记未能定位在连锁群上,42个位点定位在5个连锁群上,总长度为575.869 c M,平均图距为115.1738 c M。每个连锁群上的标记数为2~23个,平均为8个。5个连锁群的LOD值变化范围为9.923~12.71,平均为13.207。A1连锁群上的分子标记分布不均匀,出现成簇分布现象和出现了5个较大的断裂。共有18个偏分离位点,偏分离比例为42.86%。在330个位点中,异常分离位点103个,占总位点数的34.24%。对克隆测序成功的10个转录衍生片段分析表明,1个TDF5-1-1与防御相关基因GW775571.1具有高度的序列同源性,8个TDF与大白菜花蕾败育相关基因GR308173.1高度同源,1个与功能未知基因EX106546.1高度的序列同源。【结论】cDNA-AFLP标记能可初步从转录组水平上揭示抗虫菜心群体的连锁遗传规律及抗虫分子机制,其抗性相关基因可进一步用于小菜蛾抗性育种。

[ Objective] In order to provide theoretical support and intermediate materials for the breeding of insect-resistant cultivms and find defense-related genes, the linkage genetic law and molecular mechanisms in insect resistance of flowering Chinese cabbage were analyzed. [ Method ] cDNA-AFLP was used to analyze 96 progenies of the F2 segregating population obtained by hybridization of insect-susceptible flow- ering Chinese cabbage 65 and insect-resistance flowering Chinese cabbage 69. The transcription map was constructed and differential expres- sion was detected by cDNA-AFLP and TDFs cloned. [ Result] 330 bands were amplified with 14 primers and 238 were polymorphic bands. The polymorphic ratio varied between 56 % - 100 % and the average was 73.86 %. The average 24 bands were amplified with each primer combination and 17 were polymorphic bands. A total of 42 cDNA-AFLPs marker loci were mapped on 5 linkage groups which covered 575. 869 cM with an average distance of 115. 1738 cM. 2 -23 markers were on each linkage group and average was 8. The value of LOD was 9. 923 - 12.71 and average was 13. 207. The molecular markets on A1 was uneven distribution, clustered together and there were 5 big gaps. The total segregation distortion markers were 18 and segregation ratio was 42.86 %. 10 TDFs （transcripts-derived fragments） were cloned and analyzed. One was homologous sequence of GW775571 which was Brassica rapa PR2 Peronospora parasitica induced defense-re- lated genes. Eight were homologous sequences of GR30817337 aborting bud in Chinese cabbage Brassica rapa eDNA. One was high homo- logous sequence of function unknown gene EX106546. 1. [Conclusion]The linkage genetic law and molecular mechanisms in insect resistance of flowering Chinese cabbage could be re- vealed by cDNA-AFLP. These defense-related genes can fur- ther be used to study resistance to diamondback ninth.