砀山酥梨萼片发育的SSH文库构建及EST序列分析

SSH library construction and EST sequence analysis on the development of the calyx of ‘Dangshansu’ pear

为了寻找梨幼果期萼片发育中差异表达基因,为梨幼果萼片脱落分子机制研究提供依据,以砀山酥梨萼片脱落开始期幼果为材料,利用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建砀山酥梨幼果期宿萼果与脱萼果差异表达cDNA文库。随机挑取200个阳性克隆,经菌液PCR检测及测序后得到185个EST序列片段,其中小于100 bp、大于300 bp以及介于两者之间的EST片段分别占3%、96%和1%。将这些片段在NCBI上进行BLAST比对,得到调节因子(regulator)、蛋白质(protein)和相关酶类(related enzyme)3类代谢途径相关物质,分别占21%、63%和16%。其中,在Gene Ontology网站上比对到3-异丙基苹果酸脱氢酶(3-isopropylmalate dehydrogenase)和N-酰基-L-氨基酸氨基水解酶(N-acyl-L-amino acid amidohydrolae)2种酶,推测可能是砀山酥梨幼果期萼片发育代谢途径中的重要的相关酶。

The aim of this experiment is to investigate the differentially expressed genes on the development of calyx in the fruitlet period of pear and to provide evidence for the research of molecular mechanisms of its calyx shedding. A subtracted cDNA library about the different expressions between, the calyx-setting and the calyx-shedding fruitlet was constructed by using suppression subtractive hybridization（SSH）, and the fruitlets of ＇Dangshansu＇ pear at the time of calyx shedding were used as materials. A total 200 positive clones were randomly screened out, and 185 expressed sequence tags （ESTs） were identified after PCR and sequencing, in which the EST fragments of more than 100 bp, less than 300 bp and 100-300 bp account for 3%, 96% and 1%, respectively. Regulator, protein and related enzyme, which are related to the metabolic process, were found out after BLAST on the NCBI website, accounting for 21%, 63% and 16%, respectively. 3-isopropylmalate dehydrogenase and N-acyl-L-amino acid amidohydrolase were obtained on the Gene Ontology website and it was presumed that they were important related enzymes in the metabolic process of calyx development in the fruitlet period of ＇Dang- shansu＇ pear.