摘要
以陆地棉超矮秆突变体超矮1号及其近等基因系M104(正常株高)为材料提取棉花总RNA,以M104为促减物(Dirver),超矮1号为被测物(Tester),利用SSH法建立差异表达cDNA文库;分别以超矮1号和M104的cDNA为探针进行反向Northern杂交,筛选差异表达克隆,进行序列相似性分析。结果表明,通过建立差异表达cD-NA文库共获得2 530个克隆,通过反向Northern杂交共筛选到668个差异表达克隆,测序后得到539个质量合格的EST序列,共得到122个Unigene,有36个重叠群,86个单拷贝序列,平均长度1 319.26 bp。这些EST所涉及的基因,主要是与代谢和次生代谢调节、转录调控、生物合成、细胞凋亡、信号传导有关联。SSH文库的构建为陆地棉超矮秆突变形成的相关基因的克隆和结构功能研究奠定了基础。
The total cotton RNA of upland cotton ultra-dwarf mutant（tester）and its wild-type near-isogenic line M104（driver） was isolated using SSH（suppression subtractive hybridization） to establish differential expression cDNA library.The reverse Northern blot was conducted with cDNA of ultra-dwarf mutant or M104 alone as probes,to screen differential expression clones and analyze the sequence similarity.2 530 clones were obtained from the cDNA library.Totally 668 differential expression clones out of 2 530 were screened through the reverse Northern blot.After sequencing,539 EST sequences were got,including 122 unigenes,36 contigs and 86 singletons,with the average length of 1 319.26 bp.These genes were related to regulation of metabolism and secondary metabolism,transcriptional regulation,biosynthesis,cell apoptosis and signal transduction.So,the construction and analysis of SSH library might provide a foundation for further research on structure and function of gene related to ultra-dwarf mutation in upland cotton.
出处
《江苏农业学报》
CSCD
北大核心
2012年第5期948-956,共9页
Jiangsu Journal of Agricultural Sciences
基金
江苏省农业科技自主创新基金项目(CX09-115)
转基因生物新品种培育科技重大专项(2011ZX08005-001)
关键词
陆地棉
超矮杆突变体
抑制消减杂交
CDNA文库
upland cotton（Gossypium hirsutum L.）
ultra-dwarf mutant
suppression subtractive hybridization
cDNA library
