休眠解除过程中牡丹花芽均一化酵母杂交文库的构建

Construction of a Normalized Yeast Hybridization cDNA Library during Dormancy Release in Tree Peony

以不同低温处理的牡丹花芽为试材,采用DSN(duplex-specific nuclease)均一化技术结合SMARTTM(switching mecha-nism at 5'end of RNA transcript)建库技术合成cDNA,cDNA与pGADT7-Rec重组并转化酵母AH109,构建了冬季牡丹低温累积进程中的花芽均一化cDNA文库。经检测,原始文库滴度为1.77×106cfu/ml,库容量为2.3×108。对管家基因18S rRNA均一化前后丰度检测表明,均一化程度>26,随机挑取了188个克隆,PCR方法测得文库重组率大于95%,插入片段平均长度1200bp,构建的文库质量较高。该文库为进一步通过酵母杂交技术筛选低温解除牡丹休眠进程中受低温调控的转录因子和互作蛋白,进而为构建低温解除休眠的基因调控网络奠定了基础。

To obtain full-length genes related to dormancy release and provide resources for tree peony genomic study,flower buds of different chilling duration were used to isolate mRNA of tree peony.A normalized full-length yeast hybridization cDNA library was then constructed by DSN（duplex-specific nuclease） normalization method in combination with SMART（switching mechanism at 5′end of RNA transcript） technique.The amplified cDNA was recombined with pGADT7-Rec and transformed to the yeast strain AH109.The titer of un-amplified cDNA library was about 1.77×106cfu/ml,which contained 2.3×108independent clones.The results of PCR and Tanon GIS analysis indicated that the abundance of transcripts 18S rRNA decreased by 26in normalized cDNA library compared with that in non-normalized samples.The average size of cDNA inserts was 1200 bp with a recombination rate of over 95%.These results indicated that the normalized yeast hybridization cDNA library has been successfully established with high quality,which is convenient to screen transcription factors and interaction protein regulated by chilling temperature,and finally to develop gene regulation network during dormancy release in tree peony.