Construction Of Normalized Full-length CDNA Library Of Shixiya Ⅰ (Gossypium Arboreum L.) In The Whole-life Cycle

As an Asiatic A-genome diploid species, Shixiya I (2n=26) has good genetic stability, resistance to wilt, barrenness tolerance, drought resistance. The bolls of Shixiya I are not easy to shed and perish. It is considered Asiatic cotton genetic standard line. In order to accelerate the development of cotton functional genomics and clone new genes of Shixiya I, a normalized full-length cDNA library of Shixiya I in the whole life cycle has been constructed.The materials used for library included the individual plant of the Shixiya I in the stage of cotyledon and seedling, leaves in the stage of seedling and bud and boll, flowers, buds and bolls in different stages. Total RNA was extracted from those materials with a modified CTAB method. mRNA was purified from total RNA with the Oligotex mRNA Mini Kit. Then mRNA was reverse transcripted to double strands full length cDNA with the SMART technique.cDNA from different tissues was normalized with the DSN method, then the normalized cDNA was ligated with λ ZAP II vector. The normalized full length cDNA library was constructed after packing the ligation with Gigapack III XL packaging extract. The titer of primary library was 2×10~6 pfu/ml;the recombination ratio was 90%. The average insertion size was above 1kb. Results indicated that the number of clones and recombination rates of these libraries all met standards of library construction. The titer of the amplified library is 5xl09 pfu/ml. After normalizing, abundant classes of different genes tended to equalize, which can increase the chance to discover new genes.In order to clone the disease resistance gene, six pairs of degenerate primers were designed from conserved motifs of plant resistant gene products knownly. Six RGAs were obtained from the normalized full length cDNA library with the PCR method. One RGA was sequenced, sequence analysis revealed it had 82% similarity compared to protein carrier AT3G22845 mRNA of Arabidopsis thaliana. The sequence data was submitted to GenBank, the GenBank accession number was DQ668368.Construction of the normalized full length cDNA library was important for the functional genomics of Shixiya I. The library was suitable for cloning the genes associated with the development of Shixiya I. The chance to discover new genes was increased after the normalization. The library was also suitable for EST project.