甘蔗苯丙氨酸解氨酶基因（PAL）的克隆和表达分析

doi:10.3864/j.issn.0578-1752.2013.14.002

Abstract

Abstract: 【Objective】The aim of this study was to clone full-length cDNA of sugarcane phenylalanin ammonia-lyase (PAL) gene (ScPAL), a key enzyme gene related to phenylpropanoid metabolism in sugarcane, investigate its sequence characteristics, analyze its expressions in different organs and under five different stress conditions, thus providing a theoretical support for using this gene in sugarcane stress tolerance breeding. 【Method】 The differentially expressed protein was identified by MALDI TOF/TOF and analyzed using Applied Biosystems GPS Explorer software with Mascot analysis against the NCBI and Uniprot database using a combined peptide mass fingerprint and MS/MS search, then the full sequence of ScPAL was cloned from sugarcane variety ROC22 using RT-PCR and RACE techniques. The bioinformatics method was used to analyze the putative amino acid sequence, and real-time PCR method was used to analyze the expression of ScPAL in different tissues and under different stresses. 【Result】 The full-length cDNA of ScPAL (GenBank accession number: KC172559) in sugarcane was cloned. The sequence consists of 2590 bp with an intact open reading frame of 2115 bp, encoding a polypeptide of 704 amino acids. Sequence analysis showed that it contains the typical PAL enzyme active site sequence (GTITASGDLVPLSYIA). Homology analysis showed that the deduced ScPAL protein was highly homologous to other PAL proteins from different species. Phylogenetic tree analysis indicated that ScPAL was very closely related to PAL of sorghum. Real-time PCR results showed that the ScPAL expressed in root, stalk and leaf, respectively, and its expression was different among three organs. The mRNA of ScPAL in root was the highest among three organs and was about sixty-six times higher than that in leaf. Furthermore, ScPAL transcription level was induced under the treatment of low temperature, PEG, NaCl and H2O2 stresses, but the expression patterns were different. 【Conclusion】 The gene ScPAL which was firstly cloned and characterized from sugarcane (ROC22) is a member of PAL family typically. The results of study indicated that it participated in sugarcane resistance to smut, also played a role in the sugarcane resistance to chilling, drought and salt stress processes.

Key words: sugarcane , PAL , gene cloning , expression analysis

SONG Xiu-Peng-1, HUANG Xing-2, MO Feng-Lian-1, TIAN Dan-Dan-3, YANG Li-Tao-12, LI Yang-Rui-12, CHEN Bao-Shan-1. Cloning and Expression Analysis of Sugarcane Phenylalanin Ammonia-lyase (PAL) Gene[J].Scientia Agricultura Sinica, 2013, 46(14): 2856-2868.

References

[1]李杨瑞, 杨丽涛. 20世纪90年代以来我国甘蔗产业和科技的新发展. 西南农业学报, 2009, 22(5): 1469-1476.

Li Y R, Yang L T. New developments of sugarcane industry and technology in China since 1990s. Southwest China Journal of Agricultural Sciences, 2009, 22(5): 1469-1476. (in Chinese)

[2]Martinez M, Medina I, Naranjo S, Rodriguez C, Armas R, Pinon D, Vicente C, Legaz M E. Changes of some chemical parameters, involved in sucrose recovery from sugarcane juices, related to the susceptibility or resistance of sugarcane plants to smut (Ustilago scitaminea). Indian Sugar, 2000, 50(6): 341-346.

[3]Dixon R A, Paiva N L. Stress-induced phenylpropanoid metabolism. The Plant Cell, 1995, 7(7): 1085.

[4]MacDonald M J, D′Cunha G B. A modern view of phenylalanine ammonia lyase. Biochemistry and Cell Biology-Biochimie Et Biologie Cellulaire, 2007, 85(3): 273-282.

[5]Hahlbrock K, Scheel D. Physiology and molecular biology of phenylpropanoid metabolism. Annual Review of Plant Biology, 1989, 40(1): 347-369.

[6]Liu R, Xu S, Li J, Hu Y, Lin Z. Expression profile of a PAL gene from Astragalus membranaceus var. Mongholicus and its crucial role in flux into flavonoid biosynthesis. Plant Cell Reports, 2006, 25(7): 705-710.

[7]江汉民, 王楠, 赵换, 孙德岭, 宋文芹. 花椰菜苯丙氨酸解氨酶基因的克隆及黑腐病菌胁迫下的表达分析. 南开大学学报: 自然科学版, 2012, 45(4): 87-92, 98.

Jiang H M, Wang N, Zhao H, Sun D L, Song W Q. Cloning of phenylanlanine ammonia-lyase gene from cauliflower and its expression under Xcc stress. Acta Scientiarum Naturalium Universitatis Nankaiensis, 2012, 45(4): 87-92, 98. (in Chinese)

[8]Dixon R A, Achnine L, Kota P, Liu C J, Reddy M, Wang L. The phenylpropanoid pathway and plant defence-a genomics perspective. Molecular Plant Pathology, 2002, 3(5): 371-390.

[9]Koukol J, Conn E E. The metabolism of aromatic compounds in higher plants. Journal of Biological Chemistry, 1961, 236(10): 2692-2698.

[10]Sanchez-Ballesta M, Zacarias L, Granell A, Lafuente M. Accumulation of PAL transcript and PAL activity as affected by heat-conditioning and low-temperature storage and its relation to chilling sensitivity in mandarin fruits. Journal of Agricultural and Food Chemistry, 2000, 48(7): 2726-2731.

[11]Lafuente M T, Zacarias L, Mart??nez-Téllez M A, Sanchez-Ballesta M T, Granell A. Phenylalanine ammonia-lyase and ethylene in relation to chilling injury as affected by fruit age in citrus. Postharvest Biology and Technology, 2003, 29(3): 309-318.

[12]孙梓健, 汤青林, 宋明, 任雪松. 红叶芥低温胁迫下苯丙氨酸解氨酶活性及其基因的克隆表达. 西南大学学报: 自然科学版, 2010, 32(2): 90-94.

Sun Z J, Tang Q L, Song M, Ren X S. Cloning and expression of PAL gene and PAL activity assay in red -leaf mustard (Brassica juncea var. garrhiza Tsenet Lee) under low temperature stress. Journal of Southwest University: Natural Science Edition, 2010, 32(2): 90-94. (in Chinese)

[13]Vogt T, Ibdah M, Schmidt J, Wray V, Nimtz M, Strack D. Light-induced betacyanin and flavonol accumulation in bladder cells of Mesembryanthemum crystallinum. Phytochemistry, 1999, 52(4): 583-592.

[14]Pina A, Errea P. Differential induction of phenylalanine ammonia-lyase gene expression in response to in vitro callus unions of Prunus spp. Journal of Plant Physiology, 2008, 165(7): 705-714.

[15]商闯, 马春红, 李运朝, 翟彩霞, 董文琦, 崔四平, 侯立白, 贾银锁. 玉米小斑病菌C毒素培养滤液对玉米叶片苯丙氨酸解氨酶(PAL)活性的诱导效应. 玉米科学, 2008, 16(2): 131-134.

Shang C, Ma C H, Li Y C, Zhai C X, Dong W Q, Cui S P, Hou L B, Jia Y S. The induced effects of filtration of Bipolaris maydis race C toxin cultivation on PAL activities in maize leaves. Journal of Maize Sciences, 2008, 16(2): 131-134. (in Chinese)

[16]张淑珍, 靳立梅, 徐鹏飞, 陈维元, 吴俊江, 李文滨, 邱丽娟, 常汝镇. 野生大豆接种大豆疫霉根腐病后苯丙氨酸解氨酶(PAL)活性的变化. 大豆科学, 2009, 28(6): 1044-1048.

Zhang S Z, Jin L M, Xu P F, Chen W Y, Wu J J, Li W B, Qiu L J, Chang R Z. Response of PAL activity to Phytophthora sojae inoculation in Glycine soja. Soybean Science, 2009, 28(6): 1044-1048. (in Chinese)

[17]王永琦, 张显, 咸丰, 王军辉. 不同品种西瓜接种辣椒疫霉菌后3种保护酶活性的变化. 西北农业学报, 2011, 20(11): 123-128.

Wang Y Q, Zhang X, Xian F, Wang J H. The changes of the protective enzyme activities in different watermelon cultivars inoculated by Phytophthora capsici. Acta Agriculturae Boreali-Occidentalis Sinica, 2011, 20(11): 123-128. (in Chinese)

[18]Giberti S, Bertea C M, Narayana R, Maffei M E, Forlani G. Two phenylalanine ammonia lyase isoforms are involved in the elicitor- induced response of rice to the fungal pathogen Magnaporthe oryzae. Journal of Plant Physiology, 2012, 169(3): 249-254.

[19]Lois R, Dietrich A, Hahlbrock K, Schulz W. A phenylalanine ammonia-lyase gene from parsley: Structure, regulation and identification of elicitor and light responsive cis-acting elements. The EMBO Journal, 1989, 8(6): 1641.

[20]Cochrane F C, Davin L B, Lewis N G. The Arabidopsis phenylalanine ammonia lyase gene family: Kinetic characterization of the four PAL isoforms. Phytochemistry, 2004, 65(11): 1557-1564.

[21]Kumar A, Ellis B E. The phenylalanine ammonia-lyase gene family in raspberry. Structure, expression, and evolution. Plant Physiology, 2001, 127(1): 230-239.

[22]Song J, Wang Z. Molecular cloning, expression and characterization of a phenylalanine ammonia-lyase gene (SmPAL1) from Salvia miltiorrhiza. Molecular Biology Reports, 2009, 36(5): 939-952.

[23]Hsieh L S, Hsieh Y L, Yeh C S, Cheng C Y, Yang C C, Lee P D. Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii. Molecular Biology Reports, 2011, 38(1): 283-290.

[24]Hu G S, Jia J M, Hur Y J, Chung Y S, Lee J H, Yun D J, Chung W S, Yi G H, Kim T H, Kim D H. Molecular characterization of phenylalanine ammonia lyase gene from Cistanche deserticola. Molecular Biology Reports, 2011, 38(6): 3741-3750.

[25]Gallao M I, Cortelazzo A L, Fevereiro M P S, de Brito E S. Biochemical and morphological responses to abiotc elicitor chitin in suspension-cultured sugarcane cells. Brazilian Archives of Biology and Technology, 2010, 53(2): 253-260.

[26]Santiago R, de Armas R, Legaz M E, Vicente C. Changes in phenolic acids content, phenylalanine ammonia-lyase and peroxidase activities in sugarcane leaves induced by elicitors isolated from Xanthomonas albilineans. Australasian Plant Pathology, 2009, 38(4): 357-365.

[27]de Armas R, Santiago R, Legaz M E, Vicente C. Levels of phenolic compounds and enzyme activity can be used to screen for resistance of sugarcane to smut (Ustilago scitaminea). Australasian Plant Pathology, 2007, 36(1): 32-38.

[28]龚得明, 陈如凯, 林彦铨. 甘蔗受黑穗病菌侵染后苯丙烷类代谢变化及与其抗性的关系. 福建农业大学学报, 1995, 24(4): 394-398.

Gong D M, Chen R K, Lin Y Q, Relation between phenylpropanoid metabolism in sugarcane and resistance to smut. Journal of Fujian Agricultural University, 1995, 24(4): 394-398. (in Chinese)

[29]周桂, 靳晓芸, 邓光辉, 丘立杭, 邹成林, 李杨瑞. 壳寡糖诱导甘蔗叶多酚与防御酶活性的变化. 南方农业学报, 2011, 42(8): 874-877.

Zhou G, Jin X Y, Deng G H, Qiu L H, Zou C L, Li Y R. Chitosan oligosaccharide induced changes in polyphenols and defense enzymes activities in sugarcane leaves. Journal of Southern Argriculture, 2011, 42(8): 874-877. (in Chinese)

[30]周桂, 李杨瑞, 杨丽涛, 韦雪雪, 丘佩玲. 干旱胁迫下甘蔗叶类黄酮及相关酶活性的变化. 干旱地区农业研究, 2009, 27(6): 185-188, 203.

Zhou G, Li Y R, Yang L T, Wei X X, Qiu P L. Changes in flavonoids content and related enzyme activity of sugarcane leaves under PEG stress. Agricultural Research in the Arid Areas, 2009, 27(6): 185-188, 203. (in Chinese)

[31]罗聪, 何新华, 陈虎, 韦泳丽, 李明娟. 一种高效获取基因5′末端的RACE方法. 植物生理学报, 2011, 47(4): 409-414.

Luo C, He X H, Chen H, Wei Y L, Li M J. A high-efficient method of RACE technique for obtaining the gene 5′ end. Plant Phsiology Journal, 2011, 47(4): 409-414. (in Chinese)

[32]Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 2011, 28(10): 2731-2739.

[33]程水源, 陈昆松, 刘卫红, 杜何为. 植物苯丙氨酸解氨酶基因的表达调控与研究展望. 果树学报, 2003, 20(5): 351-357.

Cheng S Y, Chen K S, Liu W H, Du H W. Regulation and expression of the PAL in plant and its outlook. Journal of Fruit Science, 2003, 20(5): 351-357. (in Chinese)

[34]Minami E I, Ozeki Y, Matsuoka M, Koizuka N, Tanaka Y. Structure and some characterization of the gene for phenylalanine ammonia-lyase from rice plants. European Journal of Biochemistry, 2005, 185(1): 19-25.

[35]Rosler J, Krekel F, Amrhein N, Schmid J. Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity. Plant Physiology, 1997, 113(1): 175-179.

[36]康晓慧, 雷桅, 张梅. 水稻苯丙氨酸解氨酶的生物信息学分析. 湖南师范大学自然科学学报, 2010, 33(4): 89-94.

Kang X H, Lei W, Zhang M. Bioinformatic analysis of phenylalanine ammonia-lyase gene in Oryza sativa. Journal of Natural Science of Hunan Normal University, 2010, 33(4): 89-94. (in Chinese)

[37]Fukasawa-Akada T, Kung S D, Watson J C. Phenylalanine ammonia-lyase gene structure, expression, and evolution in Nicotiana. Plant Molecular Biology, 1996, 30(4): 711-722.

[38]许锋, 曹腾, 宁迎晶, 蒋丽阳, 张威威, 程水源. 夏枯草苯丙氨酸解氨酶基因的克隆与表达分析. 华北农学报, 2012, 27(1): 39-44.

Xu F, Cao T, Ning Y J, Jiang L Y, Zhang W W, Cheng S Y. Molecular cloning and expression analysis of a phenylalanne ammonial-lyase gene from Prunella vulgaris. Acta Agriculturae Boreali-Sinica, 2012, 27(1): 39-44. (in Chinese)

[39]许锋, 朱俊, 张风霞, 王燕, 程水源, 程述汉. 国槐苯丙氨酸解氨酶基因的克隆、反义表达载体构建及遗传转化. 林业科学研究, 2008, 21(5): 611-618.

Xu F, Zhu J, Zhang F X, Wang Y, Cheng S Y, Cheng S H. Cloning of PAL gene from Sophora japonica, construction of anti-sense gene of SjPAL and its genetic transformation in Arabidopsis. Forest Research, 2008, 21(5): 611-618. (in Chinese)

[40]Bolwell G P, Bell J N, Cramer C L, Schuch W, Lamb C J, Dixon R A. L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. European Journal of Biochemistry, 2005, 149(2): 411-419.

[41]Sequeira L. Mechanisms of induced resistance in plants. Annual Reviews in Microbiology, 1983, 37(1): 51-79.

[42]范树国, 杨跃齐, 邱璐, 杨海艳, 李国树, 梁晓华, 李易洲, 谢美华. 低温对玉米幼苗生长和苯丙氨酸解氨酶活性的影响. 江苏农业科学, 2009, (3): 70-71.

Fan S G, Yang Y Q, Qiu L, Yang H Y, Li G S, Liang X H, Li Y Z, Xie M H. Effects of low temperature on phenylalanine ammonia enzyme activity and seedling growth in maize. Jiangsu Agricultural Sciences, 2009, (3): 70-71. (in Chinese)

[43]秦鑫, 吕忠恕. 低温对植物幼苗L-苯丙氨酸解氨酶活性的影响. 兰州大学学报, 1985, 21(3): 89-93.

Qin X, Lv Z S. Effects of low temperature on L-phenylalanine ammonia-lyase activity in plant seedlings. Journal of Lanzhou University Natural Sciences, 1985, 21(3): 89-93. (in Chinese)

[44]王改利, 魏忠, 贺少轩, 周雪洁, 梁宗锁. 土壤干旱胁迫对酸枣叶片黄酮类代谢及某些生长和生理指标的影响. 植物资源与环境学报, 2011, 20(3): 1-8.

Wang G L, Wei Z, He S X, Zhou X J, Liang Z S. Effects of drought stress in soil on flavonoids metabolism in leaf and some growth and physiological indexes of Ziziphus jujuba var. spinosa. Journal of Plant Resources and Environment, 2011, 20(3): 1-8. (in Chinese)

[45]刘丽萍, 臧小云, 袁巧云, 蔡庆生. 外源蔗糖对盐胁迫荞麦幼苗根系生长的缓解效应. 植物生理学通讯, 2006, 42(5): 847-850.

Liu L P, Zang X Y, Yuan Q Y, Cai Q S. Mitigating effect of exogenous sucrose on root growth of buckwheat (Fagopyrum esculentum Moench) seedlings under salt stress. Plant Physiology Communications, 2006, 42(5): 847-850. (in Chinese)

[46]周宝利, 吕娜, 王子晗, 叶雪凌. NaCl胁迫下嫁接对茄子生长及抗性生理指标的影响. 中国蔬菜, 2010, 20: 42-46.

Zhou B L, Lü N, Wang Z H, Ye X L. Effect of grafting to eggplant growth and resistance physiology under NaCl. China Vegetables, 2010, 20: 42-46. (in Chinese)

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Cloning and Expression Analysis of Sugarcane Phenylalanin Ammonia-lyase (PAL) Gene

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