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核农学报  2015 

砀山酥梨果实COMT基因的克隆和表达分析

DOI: 10.11869/j.issn.100-8551.2015.03.0454, PP. 454-461

Keywords: 砀山酥梨,PbCOMT,生物信息学分析,原核表达,荧光定量表达

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Abstract:

梨果实石细胞的发育和木质素的合成、转运及沉积密切相关。咖啡酸氧甲基转移酶(COMT)是木质素合成途径中的1种关键酶,主要参与紫丁香基木质素(syringyllignin,S-木质素)的生物合成。本研究以砀山酥梨果实为材料,利用RT-PCR技术并结合RACE方法,得到砀山酥梨COMT基因的全长cDNA,命名为PbCOMT(GenBank登录号为KC905086)。该基因全长1371bp,开放阅读框为1098bp,编码365个氨基酸。进化树分析发现砀山酥梨COMT蛋白具有很高同源性,与苹果的相似性达到94%。将该基因重组到表达载体pET-32a(+)中进行原核表达,电泳检测到1条大约60.0kD的外源蛋白,与预测的融合蛋白分子量相符。荧光定量表达分析得知,梨果实PbCOMT基因表达量变化与木质素的含量变化趋势基本一致,并且和石细胞发育规律有很大的相关性。本研究为阐明梨石细胞发育分子机理奠定基础,为分子调控石细胞含量、改善梨果实口感提供了科学依据。

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