黑麦1R染色体微克隆文库的构建与分析

通过玻璃针分离法 ,从黑麦 (SecalecerealeL .)根尖细胞中期分裂相中显微分离出 2条及 5条 1R染色体。经Sau3A接头介导的PCR(LA_PCR)方法对其进行体外扩增 ,得到了 0 .3～ 2 .5kb之间的DNA片段。以DIG标记的探针进行多次Southern杂交 ,证明显微分离出的染色体的体外扩增产物与黑麦基因组DNA同源 ,并且来自 1R染色体。然后利用 5条 1R染色体的第二轮PCR产物构建质粒文库 ,可得到 2 2 0 0 0 0个重组子。随机挑选 172个重组子进行分析 ,发现插入片段主要介于 30 0～ 180 0bp之间。此外 ,根据基因组点杂交结果推算出该文库包含约 42 %的中、高重复序列和 5 8%的单、低拷贝序列 ,而且文库的冗余度较低。研究构建的黑麦 1R染色体微克隆文库为 1R染色体高密度遗传图谱的建立以及位于其上的重要基因的定位与分离提供了便利。

Construction and Characterization of the Microclone Library from Chromosome 1R in Rye

Twoandfive 1Rchromosomesweremicrodissectedfromthemetaphasespreadsofrye (Secalece realeL .)root_ripcellswiththeaidsofglassneedles .Thedissectedchromosomeswereamplifiedinvitroby theSau3AlinkeradaptormediatedPCRtechnique ,bywhich 0 .3to 2 .5kbsmearDNAfragmentswereob tained .AfterhybridizedwithDIGlabeledprobes ,itwasconfirmedthatthePCRproductsofthemicrodissected chromosomeswerehomologouswiththeryegenomicDNA ,andderivedfromthe 1Rchromosomeaswell. Then ,thesecondroundPCRproductsfromfivechromosomesof 1Rweremicroclonedtoconstructtheplasmid library ,including 2 2 0 0 0 0clones .172randomlyselectedcloneswereevaluatedrangedinsizefrom 30 0to 1 80 0bp .Furthermore ,thegenomicdothybridizationresultsindicatedthatthelibrarycontainednearly 4 2 % medium/highrepetitivesequencesand 58%low/singlecopysequences ,anditsredundancywasverylow .In thisresearch ,manyaspectsofthe 1Rchromosomemicroclonelibraryexceededorapproachedthoseofthepre viousreportsintheliteratures .Thosearepotentialforconstructionofahighdensitygeneticmapofchromo some 1R ,fromwhichsomeimportantgenescanbetaggedandisolated .