亚洲棉(Gossypium arboreum L.)全生育期均一化全长cDNA文库的构建和鉴定

Construction and Characterization of Normalized Full-Length cDNA Library of Asian Cotton (Gossypium arboreum L.) in the Whole-Life Cycle

【目的】构建二倍体亚洲棉石系亚1号全生育期均一化全长cDNA文库,建立亚洲棉功能基因组学研究基础平台,发掘亚洲棉发育、抗逆等相关基因。【方法】取石系亚1号子叶期、苗期、蕾期、花铃期不同组织器官,提取总RNA并分离纯化mRNA,采用SMART技术反转录全长双链cDNA。用DSN(duplex-specificnuclease)法对全长双链cDNA进行均一化,均一化的cDNA连接到λZAPⅡ载体,包装蛋白包装后构建石系亚1号全生育期均一化全长cDNA文库。【结果】初级文库滴度4×106pfu·ml-1,库容2×106个独立克隆,重组率大于95%,插入片段平均长度大于1.5kb。随机挑取192个克隆进行EST(expressedsequencetags)测序,冗余度仅为3.49%。BLASTN比对结果显示:78.31%的EST为新EST。【结论】文库均一化效果良好,质量符合要求,可能包含大量新基因,是进行EST测序、发掘新基因等棉花功能基因组学的研究平台。

【Objective】 In order to develop a functional genomics platform for diploid Asia cotton （Gossypium arboreum L.） and discover novel genes related to resistance and development, a normalized full-length cDNA library of Shixiya I （Gossypium arboreum L.） in the whole-life cycle was constructed. 【Method】 Individual plant of Shixiya I at the stages of cotyledon and seedling, leaves at the stage of seedling, flowers, buds and bolls at various stages were used for library construction. Total RNA was extracted from them with a modified CTAB method. Purified from total RNA, mRNA was reverse transcripted to double strands full length cDNA with the SMART technique. The cDNA from different tissues was normalized with the DSN method, then the normalized cDNA was linked with λZAP Ⅱ vector. The normalized full length cDNA library was constructed when the ligation was packed with Gigapack Ⅲ XL packaging extract. 【Result】 The titer of primary library was 4×10^6 pfu·ml^-1, the capacity was 2×10^6 clones, and the recombination ratio was more than 95%. The average insertion size was above 1.5 kb. Random selected 192 clones were sequenced, the redundancy was 3.49%. With BLASTN, sequences were compared against online nucleotide databases, 78.31% of the sequences were not found in GenBank, and 21.69% were found in existing nucleotide databases. 【Conclusion】 It is suggested that the cDNA library was well normalized with high quality. Most likely containing many novel genes, the library can be well used for cotton functional genomics, novel gene screening and high-throughput EST sequencing.