甘蓝型油菜矮化突变体抑制消减cDNA文库的构建及初步分析

Construction and primary analysis of suppression subtractive library of dwarf mutant 'NDF-1' in Brassica napus

采用抑制性消减杂交（suppressionsubtraetivehybridization，SSH）技术，以甘蓝型油菜矮化突变体“NDF—1”为实验方（tester），野生型“3529”为驱动方（driver）构建植株差异表达的抑制消减cDNA文库。所建eDNA文库的插入片段大小主要集中在100-1000bp之间。斑点杂交筛选得到了32个阳性克隆，序列测定和同源性对比分析表明，其中的12个克隆功能未知，其余涉及到激素信号转导、第二信使、光合作用、脂肪酸代谢及衰老等多个方面。还对部分差异表达基因的功能进行了分析。为进一步认识油菜植株矮化的机理奠定了基础。

cDNA library of differential expressed genes of a dwarf mutant ＇ NDF - 1 ＇ and its wild type ＇ 3529＇ was constructed by suppression subtractive hybridization （ SSH ） , with dwarf mutant ＇ NDF - 1 ＇ as tester and ＇3529＇ as driver, respectively. These clones were verified by colony PCR. The recombined efficiencies of the reverse subtractive library were 90%. The inserted fragment size ranged from 100bp to 1 000bp. One hundred clones in the reverse subtractive library were screened by dot blot and thirty - two positive clones were obtained. They were sequenced and analyzed for homology by BLAST search. Seventeen of these clones were annotated into several functional categories including signaling, secondary messenger, photosynthesis, fatty acid metabolism, transport and accumulation, and senescence. Others remain functionally unknown. Some of these putative genes with high homology to known genes in Genbank were also functionally analyzed. The successful construction of subtractive cDNA library provided a basis for further study of mechanism for plant dwarfness.