RESUMEN
With high nutritional value in its fruits, Dangshan Su pear has been widely cultivated in China. The stone cell content in fruits is a key factor affecting fruit quality in pear, and the formation of stone cells has been associated with lignin biosynthesis. O-Methyltransferase (OMT) is a key enzyme involved in lignin metabolism within the phenylpropanoid pathway. Here, we screened 26 OMT genes from the Pyrus bretschneideri cv. Dangshan Su genome using the DNATOOLs software. To characterize the OMT gene family in pear, gene structure, chromosomal localization, and conserved motifs of PbOMTs were analyzed. PbOMTs were divided into two categories, type I (designated PbCCOMTs) and type II (designated PbCOMTs), indicating the differentiation of function during evolution. Based on the analysis of multiple sequence alignment, cis-element prediction, and phylogenetic relationships, two candidate genes, PbCCOMT1 and PbCCOMT3, were selected for the analysis of temporal and spatial gene expression in pear. The promoter regions of both PbCCOMT1 and PbCCOMT3 contain regulatory motifs for lignin synthesis. Moreover, the two genes show high similarity and close phylogenetic relationships with CCOMTs in other species. Expression analysis showed that transcript levels of two PbCCOMTs were positively associated with the contents of both stone cells and lignin during the development of pear fruit. These results suggest that PbCCOMT1 and PbCCOMT3 are closely associated with lignin biosynthesis. These findings will help clarify the function of PbOMTs in lignin metabolism and to elucidate the mechanisms underlying stone cell formation in pear.
Asunto(s)
Biología Computacional , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Lignina/biosíntesis , Metiltransferasas/genética , Proteínas de Plantas/genética , Pyrus/genética , Secuencia de Aminoácidos , Evolución Molecular , Frutas/enzimología , Frutas/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Metiltransferasas/metabolismo , Familia de Multigenes , Filogenia , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Pyrus/clasificación , Pyrus/enzimología , Alineación de Secuencia , Transducción de Señal , Programas InformáticosRESUMEN
The Arabidopsis thaliana genome encodes 56 subtilisin-like serine proteases (subtilases). In order to evaluate the protease activity of a previously uncharacterized subtilase, designated as AtSBT1.9, we cloned its full-length cDNA from A. thaliana seedlings. An AtSBT1.9 mature peptide coding sequence was inserted into the bacterial expression vector, pMAL-c2x, and the recombinant vector was transformed into Escherichia coli BL21 (DE3). The recombinant AtSBT1.9 tagged by maltose binding protein (MBP) was induced as a 117.5-kDa protein in the soluble form in E. coli BL21 (DE3). MBP-AtSBT1.9 was expressed at a level of 11% (w/w) of the bacterial total protein. Protein purification using Amylose Resin revealed a recombinant AtSBT1.9 protease activity of 9.23 U/mg protein at pH 7 and 25°C. Maximal activity occurred over a broad pH (7-8) and temperature (25°-42°C) optimal range. Validation of AtSBT1.9 protease activity would help in characterizing its in vivo function in A. thaliana.