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Rapid EST isolation from chromosome 1R of rye

Rapid EST isolation from chromosome 1R of rye

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中国遗传学会第八次代表大会暨学术讨论会

作者： Ruo-nan Zhou~(1,4) Rui Shi~(1,5) Shu-Mei Jiang~(1,2) Wei-Bo yin~1 Huang-Huang Wang~1 yu-Hong Chen~1 Jun Hu~1 Richard RC Wang~3 Xiang-Qi Zhang~1 Zan-Min Hu~1 1 Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,P.R.China 2 South China Sea Institute of Oceanology,Chinese Academy of Sciences,Guangzhou 510301,P.R.China 3 USDA-ARS,FRRL,Utah State University,Logan,UT 84322-6300,Usa 4 Graduate University of Chinese Academy of Sciences,Beijing 100049,P.R.China 5 Forest Biotechnoiogy Group,n.C.State University,Campus Box 7247,Raleigh,nC 27695-7247,Usa

Background To obtain important expressed sequence tags(ESTs)located on specific chromosomes is currently *** of single-chromosome EST library could be an efficient strategy to isolate important ESTs located on specific *** this research we developed a method to rapidly isolate ESTs from chromosome 1R of rye by combining the techniques of chromosome microdissection with hybrid specific amplification(Hsa).Results Chromosome 1R was isolated by a glass needle and digested with proteinase K(PK).The DnA of chromosome 1R was amplified by two rounds of PCR using a degenerated oligonucleotide 6-MW sequence with a sau3AI digestion site as the *** PCR product was digested with sau3AI and linked with adaptor Hsa1,then hybridized with the sau3AI digested cDnA with adaptor Hsa2 of rye leaves with and without salicylic acid(sa)treatment,*** hybridized DnA fragments were recovered by the Hsa method and cloned into pMD18-T *** cloned inserts were released by PCR using the partial sequences in Hsa1 and Hsa2 as the primers and then *** the 94 ESTs obtained and analyzed,6 were known sequences located on rye chromosome 1R or on homologous group 1 chromosomes of wheat;all of them were highly homologous with ESTs of wheat,barley and/or other plants in Gramineae,some of which were induced by abiotic or biotic *** in this research were 22 ESTs with unknown functions, probably representing some new genes on rye chromosome 1R,Conclusion We developed a new method to rapidly clone chromosome-specific ESTs from chromosome 1R of *** information reported here should be useful for cloning and investigating the new genes found on chromosome 1R.

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