摘要
【目的】为获得大豆种子发育相关基因,并为大豆基因组资源提供材料。【方法】采用SMART(switching mechanism at 5′end of RNA transcript)与DSN(duplex-specific nulease)均一化相结合的技术,构建了全长均一化cDNA文库,采用涂平板测定和PCR快速鉴定的技术分析文库质量,用3730测序仪对文库克隆进行测序。【结果】构建了大豆种子不同发育时期全长均一化cDNA文库,最大限度地获得了大豆种子不同发育阶段表达的基因序列,原始文库的库容为6.0×105cfu/mL,重组率接近100%,插入片段大小在0.6—2.0kb之间,平均长度超过1.0kb。经过大规模的质粒提取和测序,共得到了36656条高质量的EST序列,序列的平均读长在600bp以上。【结论】经过EST序列拼接分析,整个文库有着很高的非冗余性。文库的代表性和重组片段的完整性均达到了分离筛选目的基因的建库要求。
[Objective] The objective of the study is to obtain full-length genes related to seed development and to provide resources for soybean genomics. [Method] A normalized cDNA library enriched in full-length sequences was constructed using DSN （duplex-specific nulease）-normalization method combined with SMARTrM（switching mechanism at 5＇ end of RNA transcript） technique. [ Result ] The titer of unamplified cDNA library was about 6.0× 10^5 cfu/mL. The average cDNA inserts was more than 1.0 kb with a recombination rate of nearly 100%. After large-scale plasmid extraction and sequencing, 36 656 EST sequences were generated from the soybean seed cDNA library and the average length was more than 600 bp. All the EST sequences were assembled and 27 982 unigenes were gotten, which indicated that the cDNA library was a non-redundant library. [Conclusion] These results indicate that the normalized full-length cDNA library has been established successfully, which is convenient for further studying the molecular mechanism and gene cloning of seed development.
出处
《中国农业科学》
CAS
CSCD
北大核心
2010年第3期462-467,共6页
Scientia Agricultura Sinica
基金
国家自然科学基金面上项目(30671312)
中央级公益性科研院所基本科研业务费专项资金(20065049)
