美洲南瓜(Cucurbita pepo)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

【目的】克隆有壳美洲南瓜种皮PAL基因(CP-PAL),研究PAL基因在有壳和裸仁美洲南瓜种皮发育过程中的表达特性,为揭示美洲南瓜种皮发育机理及木质素积累在南瓜种皮发育中的作用等方面提供理论依据。【方法】利用RT-PCR,结合RACE技术克隆CP-PAL的全长序列并进行生物信息学分析;利用实时荧光定量PCR技术,采用2-△△Ct方法对种皮发育过程中PAL基因的表达进行分析。【结果】CP-PAL序列全长为1 720 bp,含有一个1 359bp的ORF,114 bp 5′端非翻译区、236 bp 3′端非翻译区及11 bp polyA结构,可编码452个氨基酸,分子量为48.86 kD,等电点为6.55,原子总数为6 885个,分子式为C2158H3449N607O657S14。通过BLASTX比对表明CP-PAL核苷酸序列及其氨基酸序列与黄瓜PAL核苷酸及其氨基酸序列的相似性最高。CP-PAL包含PAL-HAL、PLN02457及phe_am_lyase 3个结构域及酶活性中心序列(GTITASGDLVPLSYIA),属于Lyase_I_Like超家族。CP-PAL不具有导肽及信号肽,为非跨膜蛋白,可能定位于细胞质及内质网上,属可溶性蛋白。CP-PAL蛋白含有4个酪蛋白激酶Ⅱ识别位点、6个蛋白激酶C识别位点、12个豆蔻酰化位点及2个糖基化位点。此外,分析可知CP-PAL有18个丝氨酸磷酸化位点、6个苏氨酸磷酸化位点及5个酪氨酸磷酸化位点。无规则卷曲是CP-PAL蛋白二级结构中最大量的结构元件,α-螺旋和延伸链分散于整个蛋白质中,且N-末端以无规则卷曲形式存在,C-末端以延伸链形式存在。CP-PAL氨基酸序列同挑选的其他14种植物的PAL氨基酸序列进行多重序列比较,发现功能区域的氨基酸序列较为保守,N-端的差异最大。系统进化树分析表明CP-PAL和黄瓜PAL蛋白的亲缘关系最近。CP-PAL蛋白三级结构以α-螺旋为主要结构元件,β-转角和无规则卷曲较少。实时荧光定量PCR分析表明PAL基因在有壳和裸仁美洲南瓜种皮发育中呈现反向对应的变化趋势:有壳美洲南瓜种皮PAL基因在自交授粉20 d后表达量增加,而裸仁美洲南瓜种皮PAL基因在20 d后表达量下降。整个种皮发育过程中,PAL基因在裸仁美洲南瓜中的表达量低于其在有壳美洲南瓜中的表达量。【结论】从有壳美洲南瓜种皮中克隆得到与木质素合成相关的PAL基因,该基因可能通过参与调控种皮木质素的合成从而影响美洲南瓜裸粒品种的种皮发育。

[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 eDNA sequence and deduced amino acid sequence, and the real-time PCR and 2＂zct 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 I1 bp, encoding a polypeptide of 452 amino acids. The molecular weight of CP-PAL was 48.86 kD, plwas 6.5, total number of atoms was 6 885 and the formula was C2158H3449N6070657514. 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 fimctional 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-glyeosylation 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 was -Helix, less in 13-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.