砀山酥梨果实COMT基因的克隆和表达分析

Cloning and Expression Analysis of COMT Gene in Pyrus bretschneideri cv. Dangshan Su Fruit

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(syringyl lignin,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c DNA,命名为Pb COMT(Gen Bank登录号为KC905086)。该基因全长1 371 bp,开放阅读框为1 098 bp,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p ET-32a(+)中进行原核表达,电泳检测到1条大约60.0 k D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Pb COMT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。

The development of pear stone cell is closely related to the synthesis,transfer and deposition of lignin. Caffeic acid o-methyltransferase（ COMT） is a key enzyme in the lignin biosynthesis pathway that mainly catalyzes Syringyl lignin. In this study,a COMT gene named Pb COMT（ Gen Bank Accession No. KC905086） from Pyrus bretschneideri cv. Dangshan Su fruit using RT-PCR and RACE methods was cloned. The Pb COMT c DNA comprises 1371 bp,with an ORF of 1098 bp,which encodes a putative protein containing 365 amino acid residues. Phylogenetic analysis indicated that Pb COMT shared high similarity with other plant COMTs,and showed 94% homology with apples. Furthermore,a prokaryotic expression vector p ET-32a-Pb COMT was constructed and transformed into E. coli BL21（ DE3）. Then a fusion protein with molecular mass 60. 0 k D was produced. Fluorescence quantitative expression analysis showed that the change of Pb COMT expression level was similar to that of lignin content,and also had great relevance with stone cell development rules. The present study is to elucidate the molecular mechanism of stone cell development in pear,which will provide a basis for further researches on the regulation of stone cell content and the improvement of pear taste.