利用cDNA-AFLP技术筛选菊花开花相关基因

Differential Analysis of Flowering Related Genes by cDNA-AFLP in Chrysanthemum

【目的】分析不同光周期诱导下菊花茎尖差异表达的基因,筛选菊花开花相关基因。【方法】将菊花品种‘神马’分别进行长日照(16h/8h,昼/夜)和短日照(8h/16h,昼/夜)处理后,利用cDNA-AFLP技术筛选菊花茎尖差异表达的转录衍生片段(Transcript-derived fragment,TDFs),并对其进行验证、克隆、测序和生物信息学分析。【结果】64对引物组合检测到2 917个cDNA片段,获得差异表达的TDF共835个,其中584个为上调表达,251个为下调表达。对克隆测序成功的50个差异表达TDF进行分析,结果表明,36个TDF可以在NCBI数据库中找到同源序列。其中31个TDF为已知功能的基因(dbEST登录号:JG700127-JG700157),5个TDF未找到同源序列。按照其功能分类,将31个差异表达基因分为7类:信号转导、分化发育、转录调控、蛋白质代谢、能量与代谢、抗病与防御等相关基因、未知或假定蛋白基因及未知基因。对选取的6个与开花相关的TDF进行半定量RT-PCR验证,结果表明,6个基因片段在短日照条件下均特异表达或表达上调,而在长日照下均未见表达或表达下调,这与cDNA-AFLP表达谱结果一致。【结论】利用cDNA-AFLP技术筛选了菊花花芽分化期多个与菊花开花相关的基因片段,这些基因片段可进一步用于功能鉴定和转基因利用。

【Objective】Differentially expressed transcripts of the apical buds in chrysanthemum induced by different photoperiod were analyzed and to isolate the flowering related genes.【Method】cDNA-AFLP technique was used to identify differentially expressed transcript-derived fragments（TDFs） in the apical buds of chrysanthemum induced by different photoperiods of long-day（16h/8h,day/night） and short-day（8h/16h,day/night） treatments,and the TDFs were validated,cloned,sequenced and the bioinformation analyzed.【Result】 A total of 2 917 TDFs were screened by 64 primer combinations,and 835 TDFs were identified differentially expression,including 584 TDFs up regulation and 251 TDFs down regulation.Fifty TDFs were successfully cloned and the sequence was analyzed.The results indicated that 36 TDFs had homology in NCBI database,and 30 of which displayed homology to genes with known functions,5 no had a match.Functional analysis indicated that 31 TDFs mainly participated in 7 kinds of processes of signal transduction,development,transcription regulation,protein degradation and synthesis,metabolism,stress responses etc.Semi-quantitative RT-PCR analysis with selected transcripts of 6 flowering related genes indicated that the 6 flowering related genes all differentially expressed or up regulated under the short-day conditions,while they all did not express or down regulate under the long-day conditions.These results were similar to those of cDNA-AFLP expression patterns in chrysanthemum.【Conclusion】In this study several flowering related gene pigments were selected by cDNA-AFLP techniques.These novel genes could be used in future for functional analysis and strategies of molecular breeding.