苹果全基因组PAL基因家族成员的鉴定及表达分析
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摘要
根据金冠苹果参考基因组,利用BLAST在线网站鉴定得到苹果基因组中共8个PAL基因,所有MdPAL蛋白都含有MIO保守结构域(Ala-Gly-Ser)。分组鉴定和进化树分析结果显示,MdPAL蛋白可以被分为3组,分子量在47. 713～81. 748 ku,等电点在5. 44～6. 39,进化关系较近的成员拥有相似的基因结构和蛋白保守基序分布。利用GEO数据库和实时荧光定量PCR的方法检测苹果长富2号品种中PAL基因在不同组织器官及5个不同果实发育阶段的表达情况。不同组织器官表达结果表明,MdPAL2、MdPAL3/4/6/7、MdPAL8在叶中表达最高,而MdPAL1和MdPAL5在花中表达最高,表达模式存在多样性。不同果实发育阶段表达结果发现,MdPAL表达规律不同,所有MdPAL在幼果发育期或果实成熟期显著升高(除MdPAL1外),暗示其在果实发育和成熟过程具有重要作用。
Based on apple genome and the genomic database of Golden Delicious,8 apple PAL genes were identified through BLAST tool. All members shared conserved MIO active site including Ala-Gly-Ser. The analysis of classification and evolutionary relationship showed that the 8 MdPAL genes were divided into three groups,the molecular weight was between 47. 713 and 81. 748 ku,the isoelectric point was between 5. 44 and 6. 39,and the members of one subgroup shared similar gene structure and conserved protein motifs. Based on GEO database and q RT-PCR,expression patterns of MdPAL genes in different organs and different stages of fruits in Nagafu No. 2 were determined.The analysis of expression patterns of MdPAL genes in different organs showed that MdPAL2,MdPAL3/4/6/7,MdPAL8 had the highest expression levels in leaves,while MdPAL1 and MdPAL5 expressions were the highest in flowers. The expression patterns were diverse. In 5 stages of the fruit development in Nagafu No. 2,the expression patterns of MdPAL genes were significantly increased in the young stage or the ripening stage except MdPAL1,indicating that they might play important roles in different stages during the fruit development.
Based on apple genome and the genomic database of Golden Delicious,8 apple PAL genes were identified through BLAST tool. All members shared conserved MIO active site including Ala-Gly-Ser. The analysis of classification and evolutionary relationship showed that the 8 MdPAL genes were divided into three groups,the molecular weight was between 47. 713 and 81. 748 ku,the isoelectric point was between 5. 44 and 6. 39,and the members of one subgroup shared similar gene structure and conserved protein motifs. Based on GEO database and q RT-PCR,expression patterns of MdPAL genes in different organs and different stages of fruits in Nagafu No. 2 were determined.The analysis of expression patterns of MdPAL genes in different organs showed that MdPAL2,MdPAL3/4/6/7,MdPAL8 had the highest expression levels in leaves,while MdPAL1 and MdPAL5 expressions were the highest in flowers. The expression patterns were diverse. In 5 stages of the fruit development in Nagafu No. 2,the expression patterns of MdPAL genes were significantly increased in the young stage or the ripening stage except MdPAL1,indicating that they might play important roles in different stages during the fruit development.
引文
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[25]FAN S,ZHANG D,XING L B,et al.Phylogenetic analysis of IDD gene family and characterization of its expression in response to flower induction in Malus[J].Molecular Genetics&Genomics,2017,292(4):755-771.
[26]郭梦月,党美乐,杨亚州,等.苹果新品种‘瑞阳’果实主要特性研究[J].西北植物学报,2017,37(12):2460-2466.GUO M Y,DANG M L,YANG Y Z,et al.Study of the main fruit characteristics of new apple variety‘Ruiyang'[J].Acta Botanica Boreali-Occidentalia Sinica,2017,37(12):2460-2466.(in Chinese with English abstract)
[27]CHU Z,WANG X,LI Y,et al.Genomic organization,phylogenetic and expression analysis of the B-BOX gene family in tomato[J].Frontiers in Plant Science,2016,7:1552.
[28]许园园,蔺经,李晓刚,等.梨CBL基因家族全基因组序列的鉴定及非生物胁迫下的表达分析[J].中国农业科学,2015,48(4):735-747.XU Y Y,LIN J,LI X G,et al.Identification and expression analysis under abiotic stresses of the CBL gene family in pear[J].Scientia Agricultura Sinica,2015,48(4):735-747.(in Chinese with English abstract)
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[31]LI J,HOU H,LI X,et al.Genome-wide identification and analysis of the SBP-BOX family genes in apple(Malus×domestica Borkh)[J].Plant Physiology and Biochemistry,2013,70:100-114.
[32]FAN S,ZHANG D,ZHANG L Z,et al.Comprehensive analysis of GASA family members in the Malus domestica genome:identification,characterization,and their expressions in response to apple flower induction[J].BMC Genomics,2017,18(1):827.
[33]邵红霞,陈鸿飞,张东,等.苹果YABBY基因家族的鉴定、进化及表达分析[J].浙江农业学报,2017,29(7):1129-1138.SHAO H X,CHEN H F,ZHANG D,et al.Identification,evolution and expression analysis of the YABBY gene family in apple(Malus×domestica Borkh.)[J].Acta Agriculturae Zhejiangensis,2017,29(7):1129-1138.(in Chinese with English abstract)
[34]YANG S,ZHANG X,YUE J X,et al.Recent duplications dominate NBS-encoding gene expansion in two woody species[J].Molecular Genetics&Genomics,2008,280(3):187-198.
[35]GEOURJON C,DELAGE G.SOPMA:significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J].Bioinformatics,1995,11(6):681-684.
[36]KELLEY L A,MEZULIS S,YATES C M,et al.The PHYRE2 web portal for protein modeling,prediction and analysis[J].Nature Protocols,2015,10(6),845-858.
[37]TAMURA K,STECHER G,PETERSON D,et al.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Molecular Biology&Evolution,2013,30(12):2725-2729.
[38]HU B,JIN J,GUO A Y,et al.GSDS 2.0:an upgraded gene feature visualization server[J].Bioinformatics,2015,31(8):1296-1297.
[39]BAILEY T L,WILLIAMS N,MISLEH C,et al.MEME:discovering and analyzing DNA and protein sequence motifs[J].Nucleic Acids Research,2006,34(Suppl.):W369-W373.
[40]POSTEL D,VANLEMMENS P,GODE P,et al.PLANT-CARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic Acids Research,2002,30(1):325-327.
[41]GAMBINO G,PERRONE I,GRIBAUDO I.A Rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants[J].Phytochemical Analysis,2008,19(6):520-525.
[42]FAN S,ZHANG D,LEI C,et al.Proteome analyses using i TRAQ labeling reveals critical mechanisms in alternate bearing Malus prunifolia[J].Journal of Proteome Research,2016,15(10):3602-3616.
[43]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408.
[44]BARRETT T,WILHITE S E,LEDOUX P,et al.NCBIGEO:archive for functional genomics data sets:update[J].Nucleic Acids Research,2013,41(D1):D991-D995.
[2]KOUKOL J,CONN E E.Metabolism of aromatic compounds in higher plants.Ⅳ.Purification and properties of phenylalanine deaminase of Horden vulgare[J].Journal of Biological Chemistry,1961,236(10):2692-2698.
[3]JOOS H,HAHLBROCK K.Phenylalanine ammonia-lyase in potato(Solanum tuberosum L.):genomic complexity,structural comparison of 2 selected genes and modes of expression[J].European Journal of Biochemistry,1992,204(2):621-629.
[4]WHETTEN R W,SEDEROFF R R.Phenylalanine ammonialyase from loblolly pine:purification of the enzyme and isolation of complementary DNA clones[J].Plant Physiology,1992,98(1):380-386.
[5]HAMBERGER B,ELLIS M F M,SOUZA C D A,et al.Genome-wide analyses of phenylpropanoid-related genes in Populus trichocarpa,Arabidopsis thaliana,and Oryza sativa:the Populus lignin toolbox and conservation and diversification of angiosperm gene families[J].Canadian Journal of Botany,2007,85(12):1182-1201.
[6]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.
[7]REICHERT A I,HE X Z,DIXON R A.Phenylalanine ammonia-lyase(PAL)from tobacco(Nicotiana tabacum):characterization of the four tobacco PAL genes and active heterotetrameric enzymes[J].Biochemical Journal,2009,424(2):233-242.
[8]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.SUN R Z,ZHANG X,CHENG G,et al.Genome-wide characterization and expression analysis of the phenylalanine ammonia-lyase gene family in grapevine(Vitis vinifera L.)[J].Plant Physiology Communications,2016,52(2):195-208.(in Chinese with English abstract)
[9]RITTER H,SCHULZ G E.Structural basis for the entrance into the phenylpropanoid metabolism catalyzed by phenylalanine ammonia-lyase[J].Plant Cell,2004,16(12):3426-3436.
[10]徐晓梅,杨署光.苯丙氨酸解氨酶研究进展[J].安徽农业科学,2009,37(31):15115-15119.XU X M,YANG S G.Advances in the studies of phenylalanine ammonia-lyase[J].Journal of Anhui Agricultural Sciences,2009,37(31):15115-15119.(in Chinese with English abstract)
[11]SANTIAGO R,ARMAS R D,LEGAZ M E,et al.Changes in phenolic acids content,phenylalanine ammonia-lyase and peroxidase activities in sugarcane leaves induced by elicitors isolated from Xanthomonas albilineans[J].Australasian Plant Pathology,2009,38(4):357-365.
[12]GALLO M I,CORTELAZZOL,FEVEREIRO M P S,et al.Biochemical and morphological responses to abiotic elicitor chitin in suspension-cultured sugarcane cells[J].Brazilian Archives of Biology&Technology,53(2):253-260.
[13]樊胜,张晓晓,周会玲,等.紫外线照射对采后苹果抗灰霉病作用的研究[J].西北农林科技大学学报(自然科学版),2016,44(2):172-178.FAN S,ZHANG X X,ZHOU H L,et al.Effect of UV-C irradiation on gray mold of postharvest apples[J].Journal of Northwest A&F University(Natural Science Edition),2016,44(2):172-178.(in Chinese with English abstract)
[14]VILJOEN M M,HUYSAMER M.Biochemical and regulatory aspects of anthocyanin synthesis in apples and pears[J].Journal of Southern African Society for Horticultural Sciences,1995,29(3):307-311.
[15]BLANKENSHIP S M,UNRATH C R.PAL and ethylene content during maturation of red and golden delicious apples[J].Phytochemistry,1988,27(4):1001-1002.
[16]KUBO Y,TAIRA S,ISHIO S,et al.Color development of 4apple cultivars grown in the southwest of japan,with special reference to fruit bagging[J].Engei Gakkai Zasshi,1988,57(2):191-199.
[17]CHENG G,HE Y N,YUE T X,et al.Effects of climatic conditions and soil properties on cabernet sauvignon berry growth and anthocyanin profiles[J].Molecules,2014,19(9):13683-13703.
[18]ADAMS D O.Phenolics and ripening in grape berries[J].American Journal of Enology&Viticulture,2006,57(3):249-256.
[19]CHEN J Y,WEN P F,KONG W F,et al.Changes and subcellular localizations of the enzymes involved in phenylpropanoid metabolism during grape berry development[J].Journal of Plant Physiology,2006,163(2):115-127.
[20]BRAIDOT E,ZANCANI M,PETRUSSA E,et al.Transport and accumulation of flavonoids in grapevine(Vitis vinifera L.)[J].Plant Signaling&Behavior,2008,3(9):626-632.
[21]聂继云,吕德国,李静,等.苹果果实中类黄酮化合物的研究进展[J].园艺学报,2009,36(9):1390-1397.NIE J Y,LYU D G,LI J,et al.Advances in studies on flavonoids in apple fruit[J].Acta Horticulturae Sinica,2009,36(9):1390-1397.(in Chinese with English abstract)
[22]TEIXEIRA A,EIRASDIAS J,CASTELLARIN S D,et al.Berry phenolics of grapevine under challenging environments[J].International Journal of Molecular Sciences,2013,14(9):18711.
[23]LISTER C E,LANCASTER J E,WALKER J R L.Phenylalanine ammonia-lyase(PAL)activity and its relationship to anthocyanin and flavonoid levels in new zealand-grown apple cultivars[J].American Society for Horticultural Science,1996,121(2):281-285.
[24]VELASCO R,ZHARKIKH A,PRUSS D,et al.The genome of the domesticated apple(Malus×domestica Borkh.)[J].Nature Genetics,2010,42(10):833.
[25]FAN S,ZHANG D,XING L B,et al.Phylogenetic analysis of IDD gene family and characterization of its expression in response to flower induction in Malus[J].Molecular Genetics&Genomics,2017,292(4):755-771.
[26]郭梦月,党美乐,杨亚州,等.苹果新品种‘瑞阳’果实主要特性研究[J].西北植物学报,2017,37(12):2460-2466.GUO M Y,DANG M L,YANG Y Z,et al.Study of the main fruit characteristics of new apple variety‘Ruiyang'[J].Acta Botanica Boreali-Occidentalia Sinica,2017,37(12):2460-2466.(in Chinese with English abstract)
[27]CHU Z,WANG X,LI Y,et al.Genomic organization,phylogenetic and expression analysis of the B-BOX gene family in tomato[J].Frontiers in Plant Science,2016,7:1552.
[28]许园园,蔺经,李晓刚,等.梨CBL基因家族全基因组序列的鉴定及非生物胁迫下的表达分析[J].中国农业科学,2015,48(4):735-747.XU Y Y,LIN J,LI X G,et al.Identification and expression analysis under abiotic stresses of the CBL gene family in pear[J].Scientia Agricultura Sinica,2015,48(4):735-747.(in Chinese with English abstract)
[29]HUNTER S,APWEILER R,BINNS D,et al.ExPASy:SIBbioinformatics resource portal[J].Nucleic Acids Research,2012,40(W1):W597-W603.
[30]刘仁虎,孟金陵.MapDraw:在Excel中绘制遗传连锁图的宏[J].遗传,2003,25(3):317-321.LIU R H,MENG J L.MapDraw:a microsoft excel macro for drawing genetic linkage maps based on given genetic linkage data[J].Hereditas(Beijing),2003,25(3):317-321.(in Chinese with English abstract)
[31]LI J,HOU H,LI X,et al.Genome-wide identification and analysis of the SBP-BOX family genes in apple(Malus×domestica Borkh)[J].Plant Physiology and Biochemistry,2013,70:100-114.
[32]FAN S,ZHANG D,ZHANG L Z,et al.Comprehensive analysis of GASA family members in the Malus domestica genome:identification,characterization,and their expressions in response to apple flower induction[J].BMC Genomics,2017,18(1):827.
[33]邵红霞,陈鸿飞,张东,等.苹果YABBY基因家族的鉴定、进化及表达分析[J].浙江农业学报,2017,29(7):1129-1138.SHAO H X,CHEN H F,ZHANG D,et al.Identification,evolution and expression analysis of the YABBY gene family in apple(Malus×domestica Borkh.)[J].Acta Agriculturae Zhejiangensis,2017,29(7):1129-1138.(in Chinese with English abstract)
[34]YANG S,ZHANG X,YUE J X,et al.Recent duplications dominate NBS-encoding gene expansion in two woody species[J].Molecular Genetics&Genomics,2008,280(3):187-198.
[35]GEOURJON C,DELAGE G.SOPMA:significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J].Bioinformatics,1995,11(6):681-684.
[36]KELLEY L A,MEZULIS S,YATES C M,et al.The PHYRE2 web portal for protein modeling,prediction and analysis[J].Nature Protocols,2015,10(6),845-858.
[37]TAMURA K,STECHER G,PETERSON D,et al.MEGA6:molecular evolutionary genetics analysis version 6.0[J].Molecular Biology&Evolution,2013,30(12):2725-2729.
[38]HU B,JIN J,GUO A Y,et al.GSDS 2.0:an upgraded gene feature visualization server[J].Bioinformatics,2015,31(8):1296-1297.
[39]BAILEY T L,WILLIAMS N,MISLEH C,et al.MEME:discovering and analyzing DNA and protein sequence motifs[J].Nucleic Acids Research,2006,34(Suppl.):W369-W373.
[40]POSTEL D,VANLEMMENS P,GODE P,et al.PLANT-CARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic Acids Research,2002,30(1):325-327.
[41]GAMBINO G,PERRONE I,GRIBAUDO I.A Rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants[J].Phytochemical Analysis,2008,19(6):520-525.
[42]FAN S,ZHANG D,LEI C,et al.Proteome analyses using i TRAQ labeling reveals critical mechanisms in alternate bearing Malus prunifolia[J].Journal of Proteome Research,2016,15(10):3602-3616.
[43]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408.
[44]BARRETT T,WILHITE S E,LEDOUX P,et al.NCBIGEO:archive for functional genomics data sets:update[J].Nucleic Acids Research,2013,41(D1):D991-D995.
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