A full-length normalized cDNA library for the flower development stages of short-season cotton (Gossypium hirsutum L.) (CCRI36) was constructed. A total of 3 421 clones were randomly selected for sequencing, with a total of 3 175 effective sequences obtained after removal of empty-carriers and low-quality sequences. Clustering the 3 175 high-quality expressed sequence tags (ESTs) resulted in a set of 2 906 non-redundant sequences comprised of 233 contigs and 2 673 singletons. Comparative analyses indicated that 913 (43.6%) of the unigenes had homologues with function-known genes or function-assumed genes in the National Center for Biotechnology Information. In addition, 763 (36.4%) of the unigenes were functionally classified using Gene Ontology hierarchy. Through EST alignment and the screening method, the full-length cDNA of two MADS-box genes viz., GhMADS11 and GhMADS12 were acquired. These genes may play a role in flower development. Phylogenetic analysis indicated that GhMADS11 and GhMADS12 had high homology and close evolutionary relationship with AGL2/SEP-type and PI-type genes, respectively. The expression of both GhMADS11 and GhMADS12, genes was high in reproductive organs. In floral organs, GhMADS11 expression was high in petals (whorl2) and ovules, while GhMADS12 expression was high in petals (whorl2) and stamens (whorl3). Results show that the EST strategy based on a normalized cDNA library is an effective method for gene identification. The study provides more insights for future molecular research on the regulation mechanism of cotton flower development.
WANG Li-na WU Dong YU Shu-xun FAN Shu-li SONG Mei-zhen PANG Chao-you LIU Jun-jie
以短季棉中棉所36(CCRI 36)顶端分生组织和花蕾为材料,以DSN(duplex-specific nuclease)均一化技术与SMART(switching mechanism at 5′end of RNAtranscript)建库技术相结合,构建CCRI 36花发育期均一化全长cDNA文库。经检测原始文库滴度为1.7×106cfumL-1。随机挑取100个克隆,利用PCR方法测得文库重组率达100%,插入片段平均长度为1.2kb。以两个高丰度表达基因Histon3和UBQ7为探针,进行虚拟Northern blot检测显示,其丰度在均一化cDNA中均明显降低,说明均一化效果显著,为节约筛库成本和EST有效测序奠定了基础;同时,利用常规PCR扩增技术从cDNA文库中筛选阳性信号,获得了花发育相关蛋白基因。初步证实CCRI36花发育期均一化全长cDNA文库构建成功,为深入研究棉花花发育机理及发掘与早熟相关的功能基因奠定了基础。
