打顶烟草根尖组织抑制性消减cDNA文库的构建及其序列分析

Construction and Analysis of Roots Suppression Subtraction Hybridization cDNA Library After Tobacco Topping

【目的】构建烟草打顶后根尖组织的消减文库,寻找调控烟碱生物合成的相关候选基因。【方法】以打顶株为测验子(tester)、以非打顶株为驱赶子(driver),利用抑制性消减杂交(suppression subtractive hybridization,SSH)和反向Northern杂交技术构建和筛选烟草打顶前后根尖组织消减文库,并对差异表达克隆进行生物信息学分析。【结果】构建了具有高消减效率的cDNA文库。PCR验证消减文库阳性克隆的插入片段为200—1 000 bp。经反向Northern杂交,从850个克隆中筛选到560个杂交信号差异明显的克隆,测序后得到273条有效序列。对273个已知功能基因的ESTs进行分类,生物碱合成类占4%、植物激素代谢类占3%、信号转导和转录因子类占18%、胁迫和防御类占32%、蛋白质代谢占9%、碳代谢占6%、其它代谢占15%、功能未知类占13%。RT-PCR结果显示,NTAT84和NTAT71序列在烟株打顶后转录活性均升高。【结论】应用SSH技术构建了烟草打顶前后根尖组织消减cDNA文库。

【Objective】 This study was carried out to construct suppression subtractive hybridization（SSH） library of tobacco roots after topping and to search some candidate genes involved in regulation of nicotine biosynthesis.【Method】 An sSSH library was constructed using cDNA from control tobacco plants as driver and those from topped tobacco plants as tester.The positive clones were selected and further screened by reverse-northern blotting,and significantly differently expressed clones were sequenced and analyzed by bioinformatics.【Result】 The SSH library was constructed successfully.The insert size of positive clones was 200-1 000 bp confirmed by PCR.After reverse-northern blotting further screening,560 significantly differently expressed clones among 850 positive clones were acquired,sequenced and 273 high quality expressed sequence tags（ESTs） were acquired.The results of nucleotide blast homological analysis indicated that these ESTs mainly involved in alkaloid biosynthesis（4%）,plant hormone metabolism（3%）,signaling/transcription（18%）,stress/defense（32%）,protein metabolism（9%）,carbon metabolism（6%）,other metabolism（15%） and function unknown（13%）.The RT-PCR result of NTAT84 and NTAT71 indicated that their transcription amount increased after tobacco topping.【Conclusion】 The SSH library of tobacco roots after topping were well constructed,and thus a basis for screening candidate genes involved in regulation of nicotine biosynthesis has been established by the information generated in this study.