mRNA差异显示条件的优化

运用优化的mRNA差异显示技术分离受内生真菌诱导的差异基因。优化差异显示条件表现在增如指定引物和随机引物的长度、改变PCR参数和再扩增程序、运用银染显色等。应用这些条件共获得7个阳性差异片段。用未优化的PCR程序1筛选35条差异带，得到3个两端均为随机引物的差示片段。而用优化的PCR程序2，52条差异带中得到9条只能用锚定引物和随机引物才能扩增出的片段。地高辛标记的反向-Northern鉴定为阳性后进行克隆和测序。PCR方法1所得的3个差示片段均无开放的阅读框。PCR程序2得到7个差异表达的基因中，2个为已知基因，5个为未知基因。因此可运用优化的差显技术分离差异表达的基因。

Optimization of mRNA differential display

Optimized mRNAs differential dis play technique(DD)was used to identify genes regulated by endophytic fungi.By increasing the lengths of anchored primer and random primer,modifying the procedures of PCR and re-am plification,and silver staining,seven positive differential fragments were obtained.In35PCR experiments(PCR method1),3differential bands were identified,which were amplified with arbitrary primers on both ends.While in PCR method2,9of52differential bands were re-amplified only with arbitrary primer and random primer.Sequence analysis followed by reverse-Northern blot with DIG probed confirmed positive differential genes.The three differen-tial genes had no ORF(open reading frame)in PCR method1.Among seven differentially expressed genes,two were known genes and five were unknown genes.So the optimized DD protocol can be applied to identify differentially expressed genes.