摘要
以香蕉幼苗根系为材料,利用DSN(duplex-specific nuclease)均一化技术与SMART(switching mechanismat5′end of RNA transcript)技术相结合,构建香蕉根系均一化全长cDNA文库。经检测,初级文库滴度1.1×106cfu·mL-1,库容5×106个独立克隆,重组率大于95%,插入片段平均长度大于1kb,表明文库质量较好。随机挑取192个克隆进行EST(expressed sequencetags)测序,成功获得179个高质量EST,含5个conting和174个singlet,冗余度仅为2.35%。小规模测序拼接后获得145个unigenes,对unigenes分析结果表明文库的可靠性好。从序列比对结果中挑选3个与其它植物同源的抗逆基因,利用RT-PCR方法进行了干旱、低温及盐胁迫条件下基因表达分析,结果显示这3个基因在不同的逆境下差异表达。
A normalized full-length cDNA library was constructed with the banana roots,by DSN（duplex-specific nuclease）normalization method combined with SMART（switching mechanism at 5＇end of RNA transcript）technique.The titer of unamplified cDNA library was about 1.1 × 106 cfu · mL-1,the capacity was 5 × 106 clones,and the recombination ratio was more than 95%.The average insertion size was above 1 kb which suggested that the quality of the cDNA was better.Random selected 192 clones were sequenced and 179 high quality EST were obtained,which included 5 contings and 174 singlets.The redundancy was 2.35%.With small scale sequencing,145 unigenes were obtained.The analysis result indicated the library was efficient and reliable.Selecting 3 genes which is homological with other plants＇ anti-abiotic stress genes from above sequences,they are expressed under drought,low temperature and saltstress using RT-PCR method.The result suggested that the 3 genes had different expression under different stresses.
出处
《园艺学报》
CAS
CSCD
北大核心
2011年第9期1667-1674,共8页
Acta Horticulturae Sinica
基金
中央级公益性科研院所基本科研业务费专项(2009hzs1J026)
国家现代农业产业体系项目(CARS-32)
