文心兰cDNA-AFLP体系的建立及TDFs分析

ESTABLISHMENT OF A cDNA-AFLP SYSTEM IN Oncidium Orchid AND TDFs ANALYSIS

通过总RNA提取、cDNA合成等试验,建立cDNA-AFLP分析体系。结果表明,采用MseⅠ和EcoRⅠ酶切3h,预扩增产物稀释15倍作为选择性扩增模板,利用筛选的引物进行扩增,可获得丰度和多态性较高的指纹图谱。利用59对引物获得约1160条TDFs(Transcription-derived fragments)。回收、克隆其中128条进行测序,获得102条可读序列,GenBank登陆号为JG014357～JG014458。BLASTN/X同源比对发现,87%的条带在NCBI数据库中能找到功能已知的同源序列;其中2条TDFs分别可能与文心兰子房发育(TDF0073a)和花器官挥发性香味物质合成(TDF0139a)相关。序列分析表明TDFs对应基因的功能涉及植物生命活动的诸多领域。cDNA-AFLP分析体系的建立及TDFs序列分析为进一步克隆花发育相关基因,研究文心兰花发育相关分子调控机制奠定了基础。

The cDNA-AFLP analysis system was established in Oncidium Milliongolds with young leaves and flower buds as materials after a series of work,including total RNA extraction,mRNA isolation,single-and double-strand cDNA synthesis,MseⅠand EcoRⅠ digestion,etc..The results indicated that the ds-cDNA was digested completely by MseⅠand EcoRⅠin 3h.The fingerprint map with high abundance ratio and polymorphism was obtained when the ligation products were used as templates for pre-amplification directly and 15 times-diluted pre-amplification products for selective amplification,using the 114 primer pairs that were gained by screening all 576 primer pairs,as selective amplification primers.Approximately a total of 1160 transcription-derived fragments（TDFs） were generated with 59 primer pairs.Among these TDFs,128 were sub-cloned and sequenced,and 102 readable sequences were produced.The sequences were submitted to the dbEST in NCBI,and the GenBank accession numbers are JG014357~JG014458.BLASTX/N analysis showed that 87% of them had identity genes with functions already known.The TDF0073a is probably involved in ovary development,and TDF0139a is likely to play a role in floral fragrance diffusing.Sequence analysis revealed that the floral differentially expressed genes had putative functions involved in many different fields of plant metabolic activity.The establishment of the cDNA-AFLP system and sequence analysis the TDFs in Oncidium can provide a platform for cloning the genes that involved in flower development and further understanding of the molecular mechanisms underlying flower development.