中国农业科学Issue(6)：1216-1226,11.
美洲南瓜（Cucurbitapepo）种皮苯丙氨酸解氨酶基因克隆与表达分析
Cloning and Expression Analysis of PAL Gene in Seed Coat of Cucurbita pepo
摘要
Abstract
Objective]The aim of this study was to clone full-length cDNA of phenylalanine ammonia-lyase (PAL) gene (CP-PAL) in seed coat of the hulled Cucurbita pepo, analyze its expressions in seed coat development of the hulled and hull-less C. pepo, thus providing a theoretical support for revealing the mechanism of seed development and function of accumulation of lignin in seed coat development.[Method]The full sequence of CP-PAL was cloned by RT-PCR and RACE techniques. The bioinformatics method was used to analyze cDNA sequence and deduced amino acid sequence, and the real-time PCR and 2-△△Ct method were used to analyze the expression profile of PAL gene in the whole period of seed coat development. [Result] The full-length sequence of CP-PAL consists of 1 720 bp with an intact open reading frame of 1 359 bp, 5′UTR of 114 bp, 3′UTR of 236 bp, polyA of 11 bp, encoding a polypeptide of 452 amino acids. The molecular weight of CP-PAL was 48.86 kD, pI was 6.5, total number of atoms was 6 885 and the formula was C2158H3449N607O657S14. Homology analysis showed that the CP-PAL nucleotide sequences and deduced amino acids were highly homologous to that of PAL gene in Cucumis sativus. CP-PAL contained three functional domains of PAL-HAL, PLN02457, phe_am_lyase and the typical PAL enzyme active site sequence (GTITASGDLVPLSYIA), and it was one member of Lyase_I_Like superfamily. CP-PAL was most likely to be located in the cytoplasm and endoplasmic reticulum, without signal peptide and leader peptide. And it was non-transmembrane and soluble protein. Besides, CP-PAL included casein kinase II phosphorylation site for four, protein kinase C phosphorylation site for six, N-myristoylation site for twelve and N-glycosylation site for two. In addition, CP-PAL had eighteen serine phosphorylation sites, six threonine phosphorylation sites and five tyrosine phosphorylation sites. Random coil was the maximum structural part in the protein secondary structure of CP-PAL, alpha helix and extended strand dispersed in whole protein, N-terminal region was presented mainly in the form of random coil, and C-terminal domain was existed in the form of extended strand. The multiple sequence alignment based on the deduced amino acid sequences of CP-PAL and other PALs of 14 plants showed the functional region was conserved, differences in the N-end. Phylogenetic tree analysis indicated that CP-PAL was very closely related to PAL of C. sativus. The main structural element in CP-PAL protein tertiary structure wasα-Helix, less inβ-Turn and random coil. Real-time PCR analysis revealed that PAL gene in hull-less C. pepo showed an opposite tendency to hulled C. pepo. The expression of PAL gene in hulled C. pepo was increased, but the expression in hull-less C. pepo was decreased after 20d of self-pollination. The expression of PAL gene in hull-less C. pepo was lower than that in hulled C. pepo.[Conclusion]The gene CP-PAL which related to lignin synthesis was firstly cloned and characterized in seed coat of hulled C. pepo. The results of study indicated that it may affect seed coat development of the naked kernel variety from C. pepo by regulating the synthesis of lignin.关键词
美洲南瓜/种皮/苯丙氨酸解氨酶/基因克隆/表达分析Key words
Cucurbita pepo/seed coat/phenylalanine ammonia-lyase (PAL)/gene cloning/expression analysis引用本文复制引用
刘佳,徐秉良,薛应钰,张树武,陈荣贤..美洲南瓜（Cucurbitapepo）种皮苯丙氨酸解氨酶基因克隆与表达分析[J].中国农业科学,2014,(6):1216-1226,11.基金项目
国家自然科学基金项目（31060261）、甘肃省科技支撑计划（1104NKCA074）、甘肃省自然科学基金项目 （）
