利用抑制消减杂交技术构建苹果同源四倍体差异表达基因文库研究

Construction of Expression cDNA Libirary of Autotetraploid Apple by Suppression Subtractive Hybridization

寒富苹果是沈阳农业大学育成的二倍体、抗寒大苹果品种,通过秋水仙素诱导获得寒富苹果同源四倍体在田间表现明显矮化等特性。为了探寻苹果同源四倍化后表型差异的分子机理,采用抑制消减杂交(suppression subtractive hybridization,SSH)技术,分别建立寒富四倍体上调和下调表达cDNA文库。分别从这两个cDNA文库中各随机挑取200个阳性克隆测序,发现其中有211条序列与已知植物基因同源性大于80%,定量RT-PCR验证了部分uni ESTs的表达趋势和表达水平。初步筛选获得了与植株矮化相关的BKI1和DWF4基因,与植物光合特性相关的光系统II中的关键基因Psb Z;发现了四倍体中表达显著上调的Chitinase基因,推测四倍体可能在真菌病害的胁迫中表现更为优良的抗性。本研究为探究同源多倍体差异表型的分子机理奠定基础。

Hanfu is an apple cultivar in diploid with high tolerance of cold stress, bred by Shenyang Agriculture University. The autotetraploid induced by colchicine treatment, apple cultivar Hanfu have a dwarf character. In order to find the molecular mechanism of the autotetraploid and diploid, both up-regulate expression and down-regulate expression cDNA librarys of autotetraploid were constructed by suppression subtractive hybridization. 200 positive clones and 200 negative clones were selected for sequencing and 211 sequences had high identity with known proteins. Some of the uni ESTs were verified by fluorescence quantitative RT-PCR. Height related gene and a photosynthetic system II key gene BKI1, DWF4 and Psb Z were founded. The chitinase gene was also founded in autotetraploid library. It suggested the autotetraploid may have high resistance to stress of the fungal diseases. This study supplied a foundation to explore the molecular mechanism of the performance of autopolyploid.