核桃内果皮苯丙氨酸解氨酶基因克隆及表达分析
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摘要
【目的】苯丙氨酸解氨酶(PAL)为木质素单体生物合成途径中的关键酶。采用RT-PCR技术克隆露仁核桃WJ-PAL基因和硬壳完整核桃ZJ-PAL基因,研究PAL基因在硬壳完整和露仁核桃内果皮中发育过程中的表达特性。【方法】以‘温138’核桃为材料,‘纸皮’核桃作为对照,利用反转录PCR(RT-PCR)方法克隆WJ-PAL、ZJ-PAL基因、通过生物信息学方法分析,采用实时荧光定量PCR(qRT-PCR)分析WJ-PAL、ZJ-PAL在核桃硬核过程8个不同时期的表达特性。【结果】WJ-PAL基因cDNA序列包括600 bp ORF,编码200个氨基酸,分子量21.11 k D,理论等电点8.44; ZJ-PAL基因包含690 bp ORF,编码230个氨基酸,分子量24.10 kD,理论等电点7.44。实时荧光定量PCR分析表明,露仁核桃PAL基因在花后100 d的相对表达量为花后51 d相对表达量的117倍,硬壳完整核桃PAL基因在花后65 d相对表达量为花后72 d相对表达量的5倍,初步证明PAL基因在硬壳完整和露仁核桃的不同发育时期表达量存在明显差异。【结论】结果表明该基因参与核桃内果皮(硬壳)硬化过程,影响内果皮木质素的合成,从而造成核桃内果皮发育不全,出现露仁。
【Objective】The phenylalanine ammonia-lyase gene(PAL) gene is a key enzyme involved in biosynthesis pathway of lignin monolignols.In this study,RT-PCR technology was used to clone WJ-PAL gene in bared-nut walnut and ZJ-PAL gene in hard shell intact walnut,and the expression characteristics of PAL gene in endocarp development of bared-nut walnut and hard shell intact walnut were studied.【Method】Taking walnut germplasm 'wen138' as materials,'zhipi' as contrast,Gene WJ-PAL and ZJ-PAL was amplified by RT-PCR,and the amino acid sequence of its encoded protein(WJ-PAL and ZJ-PAL) was predicted by bioinformatics software.Expressions of gene WJPAL and ZJ-PAL in 8 different stages in endocard of walnut were analyzed by real-time fluorescence quantitative PCR(qPCR).【Result】The cDNA sequence in WJ-PAL gene contained 600 bp ORF,encoding 200 amino acids,with the molecular weight of 21.11 kD and isoelectric point as 8.44.ZJ-PAL gene cDNA sequence contained 690 bp ORF,encoding 230 amino acids,and the molecular weight and isoelectric point were 24.10 kD and 7.44,respectively.Real-time fluorescent quantitative PCR analysis indicated that relative expression of WJ-PAL genes in the 100 d after flowering was 117 times of the relative expression in the 51 d after flowering,and the relative expression of ZJ-PAL genes in the 65 d after flowering was 5 times of the relative expression in the 72 d after flowering,which preliminarily certified that the expression of PAL genes was different in different development stages of two kinds of walnut species.【Conclusion】Research results proved that the PAL gene participated in the development of walnut endocarp harden processing and affected the synthesis of lignin from endocarp,resulting in the hypoplasia of walnut endocarp and bared-nut.
【Objective】The phenylalanine ammonia-lyase gene(PAL) gene is a key enzyme involved in biosynthesis pathway of lignin monolignols.In this study,RT-PCR technology was used to clone WJ-PAL gene in bared-nut walnut and ZJ-PAL gene in hard shell intact walnut,and the expression characteristics of PAL gene in endocarp development of bared-nut walnut and hard shell intact walnut were studied.【Method】Taking walnut germplasm 'wen138' as materials,'zhipi' as contrast,Gene WJ-PAL and ZJ-PAL was amplified by RT-PCR,and the amino acid sequence of its encoded protein(WJ-PAL and ZJ-PAL) was predicted by bioinformatics software.Expressions of gene WJPAL and ZJ-PAL in 8 different stages in endocard of walnut were analyzed by real-time fluorescence quantitative PCR(qPCR).【Result】The cDNA sequence in WJ-PAL gene contained 600 bp ORF,encoding 200 amino acids,with the molecular weight of 21.11 kD and isoelectric point as 8.44.ZJ-PAL gene cDNA sequence contained 690 bp ORF,encoding 230 amino acids,and the molecular weight and isoelectric point were 24.10 kD and 7.44,respectively.Real-time fluorescent quantitative PCR analysis indicated that relative expression of WJ-PAL genes in the 100 d after flowering was 117 times of the relative expression in the 51 d after flowering,and the relative expression of ZJ-PAL genes in the 65 d after flowering was 5 times of the relative expression in the 72 d after flowering,which preliminarily certified that the expression of PAL genes was different in different development stages of two kinds of walnut species.【Conclusion】Research results proved that the PAL gene participated in the development of walnut endocarp harden processing and affected the synthesis of lignin from endocarp,resulting in the hypoplasia of walnut endocarp and bared-nut.
引文
[1]赵鹏,周惠娟,刘占林,等.胡桃属植物分子系统发育和生物地理研究进展[J].林业科学,2014,50(11):147-157.
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[9]文菁,赵书岗,王红霞,等.核桃硬壳发育期内果皮木质素与相关酶活性的变化[J].园艺学报,2015,42(11):2144-2152.
[10]戴超,刘雪梅,周菲.白桦基因表达半定量RT-PCR中内参基因的选择[J].经济林研究,2011,29(1):34-39.
[11]刘佳,徐秉良,薛应钰,等.美洲南瓜(Cucurbita pepo)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,2014,47(6):1216-1226.
[12]Kim D S,Hwang B K. An important role of the pepper phenylalanine ammonia-lyase gene(PAL1)in salicylic acid-dependent signalling of the defence response to microbial pathogens[J]. J. Exp.Bot.,2014,65(9):2295-2306.
[13]雒军,王引权,温随超,等.当归苯丙氨酸解氨酶基因片段克隆和组织特异性表达分析[J].草业学报,2014,23(4):130-137.
[14]王猛,曹福祥,王仕利,等.马尾松苯丙氨酸解氨酶基因的克隆与序列分析[J].中南林业科技大学学报,2010,30(1):79-83.
[15]Shang Q M,Li L,Dong C J. Multiple tandem duplication of the phenylalanine ammonia-lyase genes in Cucumis sativus L.[J]. Planta,2012,236(4):1093-1105.
[16]Bate N J,Orr J,Ni W,et al. Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis[J]. Proc. Natl. Acacl. Sci. USA,1994,91:7608-7612.
[17]Cochrane F C,Davin L B,Lewis N G. The Arabidopsis phenylalanine ammonia lyase gene family:kinetic characterization of the four PAL isoforms[J]. Phytochemistry,2004,65(11):1557-1564.
[18]郑洁红,邱栋梁.龙眼叶片PAL基因的克隆与表达研究[J].热带作物学报,2012,33(1):79-83.
[19]乔枫,罗桂花,耿贵工,等.独一味苯丙氨酸解氨酶基因的克隆与表达分析[J].西北植物学报,2013,33(12):2361-2368.
[20]Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ctmethod[J].Methods,2001,25(4):402-408.
[21]李含芬,徐莹,苏鹏,等.石细胞发育对“黄金梨”果肉硬化症的影响[J].北方园艺,2017(10):20-24.
[22]靳萍,靳同霞,赵青青,等. 11种植物石细胞形成相关基因pal的生物信息学分析[J].河南农业科学,2012,41(4):117-120.
[23]宋福南,邢磊,陈肃,等.白桦苯丙氨酸解氨酶(PAL)基因的分离及其表达[J].东北林业大学学报,2009,37(12):14-17+47.
[24]薛应钰,师桂英,徐秉良,等.裸仁美洲南瓜种皮性状遗传规律研究[J].草业学报,2011,20(3):205-210.
[2]潘学军,张文娥,李琴琴,等.核桃感官和营养品质的主成分及聚类分析[J].食品科学,2013,34(8):195-198.
[3]孙树杰,王兆华,宋康,等.核桃营养价值及功能活性研究进展[J].中国食物与营养,2013,19(5):72-74.
[4]冀爱青.华北山地核桃遗传多样性和矿质营养特性研究[D].晋中:山西农业大学,2014.
[5]王国安,张强,黄闽敏,等.新疆核桃产业现状与分析[A].中国园艺学会干果分会.第八届全国干果生产、科研进展学术研讨会论文集[C].中国园艺学会干果分会,2013:4.
[6]徐凯,夏玉芳,李允祥,等.贞丰核桃主要经济性状评价[J].种子,2016,35(4):118-121.
[7]田贝贝,高疆生,赵明,等.南疆核桃品种脂肪酸及氨基酸的营养评价[J].新疆农业科学,2009,46(5):970-974.
[8]武际,郭熙盛,鲁剑巍,等.不同水稻栽培模式下小麦秸秆腐解特征及对土壤生物学特性和养分状况的影响[J].生态学报,2013,33(2):565-575.
[9]文菁,赵书岗,王红霞,等.核桃硬壳发育期内果皮木质素与相关酶活性的变化[J].园艺学报,2015,42(11):2144-2152.
[10]戴超,刘雪梅,周菲.白桦基因表达半定量RT-PCR中内参基因的选择[J].经济林研究,2011,29(1):34-39.
[11]刘佳,徐秉良,薛应钰,等.美洲南瓜(Cucurbita pepo)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,2014,47(6):1216-1226.
[12]Kim D S,Hwang B K. An important role of the pepper phenylalanine ammonia-lyase gene(PAL1)in salicylic acid-dependent signalling of the defence response to microbial pathogens[J]. J. Exp.Bot.,2014,65(9):2295-2306.
[13]雒军,王引权,温随超,等.当归苯丙氨酸解氨酶基因片段克隆和组织特异性表达分析[J].草业学报,2014,23(4):130-137.
[14]王猛,曹福祥,王仕利,等.马尾松苯丙氨酸解氨酶基因的克隆与序列分析[J].中南林业科技大学学报,2010,30(1):79-83.
[15]Shang Q M,Li L,Dong C J. Multiple tandem duplication of the phenylalanine ammonia-lyase genes in Cucumis sativus L.[J]. Planta,2012,236(4):1093-1105.
[16]Bate N J,Orr J,Ni W,et al. Quantitative relationship between phenylalanine ammonia-lyase levels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis[J]. Proc. Natl. Acacl. Sci. USA,1994,91:7608-7612.
[17]Cochrane F C,Davin L B,Lewis N G. The Arabidopsis phenylalanine ammonia lyase gene family:kinetic characterization of the four PAL isoforms[J]. Phytochemistry,2004,65(11):1557-1564.
[18]郑洁红,邱栋梁.龙眼叶片PAL基因的克隆与表达研究[J].热带作物学报,2012,33(1):79-83.
[19]乔枫,罗桂花,耿贵工,等.独一味苯丙氨酸解氨酶基因的克隆与表达分析[J].西北植物学报,2013,33(12):2361-2368.
[20]Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ctmethod[J].Methods,2001,25(4):402-408.
[21]李含芬,徐莹,苏鹏,等.石细胞发育对“黄金梨”果肉硬化症的影响[J].北方园艺,2017(10):20-24.
[22]靳萍,靳同霞,赵青青,等. 11种植物石细胞形成相关基因pal的生物信息学分析[J].河南农业科学,2012,41(4):117-120.
[23]宋福南,邢磊,陈肃,等.白桦苯丙氨酸解氨酶(PAL)基因的分离及其表达[J].东北林业大学学报,2009,37(12):14-17+47.
[24]薛应钰,师桂英,徐秉良,等.裸仁美洲南瓜种皮性状遗传规律研究[J].草业学报,2011,20(3):205-210.