摘要： 为了挖掘抗病基因,探寻甘蓝抗黑腐病机制,选取抗性材料C7,在幼苗 4~5片真叶期,喷施菌液浓度为1.0×108 cfu/mL的细菌悬浮液,喷施蒸馏水作对照,提取接种后1,3,5 d的总RNA进行等量混合,获得2种不同处理的样品池。采用SRAP分子标记技术进行基因差异表达分析,结果显示:60对SRAP引物组合共扩增出大小在100~750 bp的685条带,平均每对引物扩增11条带,共检测到24个多克隆位点。回收测序后,将得到的9条差异表达片段TDFs(Transcript derived fragments)进行克隆和序列分析,其中3条差异基因片段未搜索到任何同源蛋白,其余6个基因均按功能分类,可分为涉及能量代谢、植物细胞壁蛋白相关基因、液泡加工酶基因和未知功能基因。表明cDNA-SRAP方法简单、重复性好、试验成本低,完全适用于差异表达分析,从中获得一些与甘蓝抗黑腐病相关的差异基因片段,可用于甘蓝抗黑腐病的分子机理研究。
关键词:
甘蓝,
黑腐病,
cDNA-SRAP
Abstract: In order to explore the resistant disease gene and find out relative mechanisms,the suspensions with 1.0×108 colony forming units/mL was sprayed to the resistant variety C7 of seedlings at four to five leaves,while the other group was sprayed distilled water as usual(Control,CK).Leaves were collected in 1,3,5 d time points after XCC and distilled water.A mixture of an equal amount total RNA from the above two groups as two gene pools.Different expression of the genes was analyzed by SRAP technique.The results showed:using 60 pairs of primers,about 685 cDNA fragments in size between 100~750 bp were amplified,averagely 11 bands per primer combination,there were 24 polymorphic loci in these two pool.They were sequenced and the Blast program was used to find out homology with reported genes.Sequence alignment indicated that 6 out of these 9 TDFS(Transcript derived fragments)showed homologies to certain genes from 43% to 59% in NCBI,while the other three showed no significant homology with reported gene.Based on their homologies,these genes were assumed to NADH dehydrogenase,vegetative cell wall protein,DELTA-VPE and unknown functional proteins.The results indicated that cDNA-SRAP method had several advantages over other systems:simplicity,reasonable throughput rate,highly reproducibility and inexpensiveness,and that the cDNA-SRAP technique was applicable to the analysis of gene different expression.Some fragments of differentially expressed genes associated with cabbage black rot were obtained,and may be used for investigating the molecular mechanism of cabbage black rot.
Key words:
Cabbage(Brassica oleracea L.var.capitata),
Black rot(Xanthomonas Campestris pv.Campestris),
cDNA-SRAP
中图分类号:
张云霞, 宋立晓, 曾爱松, 高兵, 严继勇. 利用cDNA-SRAP分离结球甘蓝抗黑腐病相关基因的研究[J]. 华北农学报, 2014, 29(5): 29-32. doi: 10.7668/hbnxb.2014.05.006.
ZHANG Yun-xia, SONG Li-xiao, ZENG Ai-song, GAO Bing, YAN Ji-yong. Studies on Isolation of Resistance Related-genes to Black Rot by cDNA-SRAP in Cabbage[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2014, 29(5): 29-32. doi: 10.7668/hbnxb.2014.05.006.