木薯苯丙氨酸解氨酶基因的克隆及其对低温胁迫的响应
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摘要
本研究从全基因组水平对木薯(Manihot esculenta Crantz)苯丙氨酸解氨酶编码基因(Phenylalanine ammonia-lyase,PAL)进行鉴定,并以木薯品种KU50为材料克隆了6个PAL基因,分析了低温胁迫(7℃)对叶片MePAL表达模式、PAL酶活性、总酚含量、类黄酮含量和总抗氧化能力的影响。结果表明:低温处理使木薯叶片迅速萎蔫卷曲,并伴随叶片PAL酶活性、总酚含量、类黄酮含量和总抗氧化能力的显著提高。Real-time PCR分析表明,在低温处理前,MePAL1和MePAL2的相对表达量分别为0.83和1.19,显著高于其他4个MePAL基因,低温处理后6个MePAL基因均不同程度受低温胁迫诱导增强表达,其中MePAL5上调最高,达50.5～142.5倍。本研究结果初步显示低温胁迫上调了木薯叶片MePAL的表达,促进了总酚和类黄酮的合成,提高了抗氧化损伤的能力。
Cassava phenylalanine ammonia-lyase encoding genes(MePALs) were identified from the whole genome sequence data. Six MePALs genes, MePAL1-6, were subsequently cloned by RT-PCR from the leaves of the cultivar KU50 and their expression patterns were analyzed, together with PAL activity, total phenolic content, flavonoid content and total antioxidant capacity, in the leaves under low temperature stress treatment(7 ℃). It was found that low temperature treatment resulted in rapid leaf wilting and curling and correspondingly a significant increase in PAL activity, total phenol content, flavonoid content and total antioxidant capacity in the leaves. Real-time PCR analysis showed that the relative expression level of MePAL1 and MePAL2 was 0.83 and 1.19 respectively before low temperature treatment, which was significantly higher than the other 4 MePALs. Low temperature treatment increased the expression of all the six MePAL genes and the up-regulation of MePAL5 was the highest(increased from 50.5 to 142.5 folds). Our preliminary results showed that low temperature treatment increased the expression of MePALs, promoted the synthesis of total phenols and flavonoids, and enhanced anti-oxidative capacities in cassava leaves.
Cassava phenylalanine ammonia-lyase encoding genes(MePALs) were identified from the whole genome sequence data. Six MePALs genes, MePAL1-6, were subsequently cloned by RT-PCR from the leaves of the cultivar KU50 and their expression patterns were analyzed, together with PAL activity, total phenolic content, flavonoid content and total antioxidant capacity, in the leaves under low temperature stress treatment(7 ℃). It was found that low temperature treatment resulted in rapid leaf wilting and curling and correspondingly a significant increase in PAL activity, total phenol content, flavonoid content and total antioxidant capacity in the leaves. Real-time PCR analysis showed that the relative expression level of MePAL1 and MePAL2 was 0.83 and 1.19 respectively before low temperature treatment, which was significantly higher than the other 4 MePALs. Low temperature treatment increased the expression of all the six MePAL genes and the up-regulation of MePAL5 was the highest(increased from 50.5 to 142.5 folds). Our preliminary results showed that low temperature treatment increased the expression of MePALs, promoted the synthesis of total phenols and flavonoids, and enhanced anti-oxidative capacities in cassava leaves.
引文
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[22]盖江涛,陈振玺,王鹏.茄科植物PAL基因家族的鉴定和序列分析[J].热带作物学报,2015,36(3):474-479.
[23]Raes J,Rohde A,Christensen J H,et al.Genome-wide characterization of the lignification toolbox in Arabidopsis[J].Plant Physiology,2003,133(3):1051-1071.
[24]Dong C J,Shang Q M.Genome-wide characterization of phenylalanine ammonia-lyase gene family in watermelon(Citrullus lanatus)[J].Planta,2013,238(1):35-49.
[25]Joos H J,Hahlbrock K.Phenylalanine ammonia-lyase in potato(Solanum tuberosum L.)Genomic complexity,structural comparison of two selected genes and modes of expression[J].European Journal of Biochemistry,1992,204(2):621-629.
[26]孙梓健,汤青林,宋明,等.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[J].西南大学学报(自然科学版),2010,32(2):90-94.
[27]孙润泽,张雪,成果,等.葡萄苯丙氨酸解氨酶基因家族的全基因组鉴定及表达分析[J].植物生理学报,2016,52(2):195-208.
[28]Chang A,Lim M H,Lee S W,et al.Tomato phenylalanine ammonia-lyase gene family,highly redundant but strongly underutilized[J].Journal of Biological Chemistry,2008,283(48):33591-33601.
[2]Dong An,Jun Yang,Peng Zhang.Transcriptome profiling of low temperature treated cassava apical shoots showed dynamic responses of tropical plant to cold stress[J].BMCGenomics,2012,13(1):64.
[3]Pellegrini L,Rohfritsch O,Fritig B,et al.Phenylalanine ammonia-lyase in tobacco.Molecular cloning and gene expression during the hypersensitive reaction to tobacco mosaic virus and the response to a fungal elicitor[J].Plant Physiol,1994,106:877-886.
[4]Dixon R A,Paiva N L.Stress-Induced Phenylpropanoid Metabolism[J].Plant Cell,1995,7:1085-1097.
[5]Doudet AM.Evolution and current status of research in phenolic compounds[J].Phytochemistry,2007,68:2722-2735.
[6]Hahlbrock K,Scheel D.Physiology and molecular biology of phenylpropanoid metabolism[J].Plant Biology,1989,40:347-369.
[7]Kim SG,Kim ST,Wang Y,et al.Overexpression of rice isoflavone reductase-like gene(OsIRL)confers tolerance to reactive oxygen species[J].Physiol Plant,2010,138:1-9.
[8]Christie,Peter J,Mark R,et al.Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways:enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings[J].Planta,1994,194(4):541-549.
[9]Janas KM,CvikrováM,Pa??giewicz A,et al.Alterations in phenylpropanoid content in soybean roots during low temperature acclimation[J].Plant Physiology and Biochemistry,2000,38(7-8):587-593.
[10]宋静武,殷德松,赵弟广,等.核桃叶片内多酚黄酮类成分对低温胁迫的响应[J].河北林果研究,2017,32(1):34-41.
[11]董春娟,李亮,曹宁,等.苯丙氨酸解氨酶在诱导黄瓜幼苗抗寒性中的作用[J].应用生态学报,2015,26(7):2041-2049.
[12]Navarre DA,Payyavula RS,Shakya R,et al.Changes in potato phenylpropanoid metabolism during tuber development[J].Plant Physiology and Biochemistry,2013,65:89-101.
[13]Hui Xu,Nam Il Park,Xiaohua Li.Molecular cloning and characterization of phenylalanine ammonia-lyase,cinnamate4-hydroxylase and genes involved in flavone biosynthesis in Scutellaria baicalensis[J].Bioresource Technology,2010,101(24):9715-9722.
[14]Huang J,Gu M,Lai Z,et al.Functional analysis of the Arabidopsis PAL gene family in plant growth,development and response to environmental stress[J].Plant Physiology,2010,153(4):1526-1538.
[15]Campos-Vargas R,Saltveit M E.Involvement of putative chemical wound signals in the induction of phenolic metabolism in wounded lettuce[J].Physiologia Plantarum,2002,114(1):73-84.
[16]Bae H,Kim SH,Kim MS,et al.The drought response of Theobroma cacao(cacao)and the regulation of genes involved in polyamine biosynthesis by drought and other stresses[J].Plant Physiology and Biochemistry,2008,46(2):174-188.
[17]Campos-Vargas R,Nonogaki H,Suslow T,et al.Heat shock treatments delay the increase in wound-induced phenylalanine ammonia-lyase activity by altering its expression,not its induction in Romaine lettuce(Lactuca sativa)tissue[J].Physiologia Plantarum,2005,123(1):82-91.
[18]Sojikul P,Saithong T,Kalapanulak S,et al.Genome-wide analysis reveals phytohormone action during cassava storage root initiation[J].Plant Molecular Biology,2015,88(6):531-543.
[19]Pennycooke JC,Cox S,Stushnoff C.Relationship of cold acclimation,total phenolic content and antioxidant capacity with chilling tolerance in petunia(Petunia hybrida)[J].Environmental and Experimental Botany,2005,53(2):225-232.
[20]Kreps JA,Wu Y,Chang H-S,et al.Transcriptome changes for Arabidopsis in response to salt,osmotic,and cold stress[J].Plant Physiology,2002,130(4):2129-2141.
[21]陈贵林,李建文,何洪巨.蔬菜类黄酮研究进展[J].中国食物与营养,2007,1:57-59.
[22]盖江涛,陈振玺,王鹏.茄科植物PAL基因家族的鉴定和序列分析[J].热带作物学报,2015,36(3):474-479.
[23]Raes J,Rohde A,Christensen J H,et al.Genome-wide characterization of the lignification toolbox in Arabidopsis[J].Plant Physiology,2003,133(3):1051-1071.
[24]Dong C J,Shang Q M.Genome-wide characterization of phenylalanine ammonia-lyase gene family in watermelon(Citrullus lanatus)[J].Planta,2013,238(1):35-49.
[25]Joos H J,Hahlbrock K.Phenylalanine ammonia-lyase in potato(Solanum tuberosum L.)Genomic complexity,structural comparison of two selected genes and modes of expression[J].European Journal of Biochemistry,1992,204(2):621-629.
[26]孙梓健,汤青林,宋明,等.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[J].西南大学学报(自然科学版),2010,32(2):90-94.
[27]孙润泽,张雪,成果,等.葡萄苯丙氨酸解氨酶基因家族的全基因组鉴定及表达分析[J].植物生理学报,2016,52(2):195-208.
[28]Chang A,Lim M H,Lee S W,et al.Tomato phenylalanine ammonia-lyase gene family,highly redundant but strongly underutilized[J].Journal of Biological Chemistry,2008,283(48):33591-33601.