关键词: 亚洲棉, 全生育期, 均一化, cDNA文库, EST

Abstract:

【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 4×106 pfu?ml-1, the capacity was 2×106 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.

Key words: asian cotton, whole-life cycle, normalization, cDNA library, EST