长白落叶松PAL基因SNPs筛选及木质素含量关联分析
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摘要
为探究长白落叶松PAL基因的多态性以及与木质素含量之间的关系,本研究以吉林省四平市林木种子园中120个长白落叶松无性系为材料,测量各无性系的木质素含量并对不同无性系进行PAL基因克隆及分析,结果显示:不同无性系间木质素含量差异达极显著水平,表明无性系间木质素含量差异较大,对其进行遗传改良意义重大;对3个落叶松无性系不同部位进行PAL基因克隆,发现不同组织均有该基因表达;对120个无性系PAL基因进行克隆、测序及分析结果共筛选出11个SNP位点,均位于基因编码区,其中有7个属于转换类型位点,4个属颠换类型位点;不同无性系间遗传距离范围是0.000～0.035 cM之间;关联分析结果发现4个与木质素含量显著相关的SNP位点(SNP116, SNP922, SNP1150和SNP1186),4个位点均是非同义突变位点,且除SNP116外,纯合突变型无性系木质素含量更高。本研究结果可以为长白落叶松分子遗传改良提供基础,也可以为其他树种分子育种提供借鉴。
In order to study the polymorphism of gene PAL and its relationship with lignin content, this study took120 clones of Larix olgensis in forest seed orchard(Siping city of Jilin Province) as material. The lignin content were measured and the polymorphism in gene PAL was identified using DNA sequencing. The results showed that the lignin content had extremely significant difference among different clones which indicated that there was a great difference in lignin content among clones, so it was of great significance to carry out genetic improvement..Cloning of PAL gene from different organization of 3 L. gmelinii clones showed that the gene was expressed in all the text tissues. After cloning and sequencing of PAL gene form 120 L. olgensis clones, 11 SNP sites(including 7 transformational sites and 4 transversion sites) which were located in the coding region were selected. The range of genetic distance between different clones was 0.000～0.035 cM. The results of correlation analysis showed that four SNP loci(SNP116, SNP922, SNP1150 and SNP1186) were significantly related to lignin content. 4 SNPs were all nonsynonymous mutation sites and the lignin content of mutational clones was higher except SNP 116. The results of this study could provide the basis for molecular genetic improvement of L. olgensis, and it could also provide reference for molecular breeding of other tree species.
In order to study the polymorphism of gene PAL and its relationship with lignin content, this study took120 clones of Larix olgensis in forest seed orchard(Siping city of Jilin Province) as material. The lignin content were measured and the polymorphism in gene PAL was identified using DNA sequencing. The results showed that the lignin content had extremely significant difference among different clones which indicated that there was a great difference in lignin content among clones, so it was of great significance to carry out genetic improvement..Cloning of PAL gene from different organization of 3 L. gmelinii clones showed that the gene was expressed in all the text tissues. After cloning and sequencing of PAL gene form 120 L. olgensis clones, 11 SNP sites(including 7 transformational sites and 4 transversion sites) which were located in the coding region were selected. The range of genetic distance between different clones was 0.000～0.035 cM. The results of correlation analysis showed that four SNP loci(SNP116, SNP922, SNP1150 and SNP1186) were significantly related to lignin content. 4 SNPs were all nonsynonymous mutation sites and the lignin content of mutational clones was higher except SNP 116. The results of this study could provide the basis for molecular genetic improvement of L. olgensis, and it could also provide reference for molecular breeding of other tree species.
引文
Baucher M.,Halp in C.,Petitconil M.,and Boerjan W.,2003,Lignin:genetic engineering and impact on pulping,Crit.Rev.Biochem.Mol.Biol.,38(4):305-350
Dillon S.K.,Nolan M.,Li W.,Bell C.,Wu H.X.,and Southerton S.G.,2010,Allelic variation in cell wall candidate genes affecting solid wood properties in natural populations and land races of Pinus radiata,Genetics,185(4):1477-1487
Geng S.,Xu C.S.,and Li Y.C.,2003,Advance in biosynthesis of lignin and its regulation,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),23(1):171-181(耿飒,徐存拴,李玉昌,2003,木质素的生物合成及其调控研究进展,西北植物学报,23(1):171-181)
Krutovsky K.V.,and Neale D.B.,2005,Nucleotide diversity and linkage disequilibrium in cold-hardiness and wood quality-related candidate genes in Douglas fir,Genetics,171(4):2029-2041
Lewis N.G.,and Yamamoto E.,1990,Lignin:occurrence,biogenesis and biodegredation,Annu.Rev.Plant Physiol.Plant Mol.Biol.,41:455-496
Li M.D.,and Ji W.L.,1994,Chemical composition of China's larch wood(Larix gmelini Rupr.),Zhongguo Zaozhi(Papermaking in China),(1):58-60(李民栋,纪文兰,1994,兴安落叶松化学组成的研究,中国造纸,(1):58-60)
Li Y.X.,Zhang H.G.,Zhang L.,Zhu H.Y.,and Zhou X.C.,2012,Research on multi-trait selection of the superior families of Larix olgensis pulpwood,Linye Kexue Yanjiu(Forest Research),25(6):712-718(李艳霞,张含国,张磊,朱航勇,周显昌,2012,长白落叶松纸浆材优良家系多性状联合选择研究,林业科学研究,25(6):712-718)
Liu J.,Xu B.L.,Xue Y.Y.,Zhang S.W.,and Chen R.X.,2014,Cloning and expression analysis of PAL gene in seed coat of Cucurbita pepo,Zhongguo Nongye Kexue(Scientia Agricultural Sinica),47(6):1216-1226(刘佳,徐秉良,薛应钰,张树武,陈荣贤,2014,美洲南瓜(Cucurbita pepo)种皮苯丙氨酸解氨酶基因克隆与表达分析,中国农业科学,47(6):1216-1226)
Ning K.,Liu X.P.,Lin Y.H.,Zhao X.Y.,Liu G.F.,Yang Y.,Song X.,and Li H.Y.,2015,Germplasm selection of the progeny genetic variation and superior pulpwood of Betula platyphylla,Zhiwu Yanjiu(Bulletin of Botanical Research),35(1):39-46(宁坤,刘笑平,林永红,赵曦阳,刘桂丰,杨洋,宋鑫,李慧玉,2015,白桦子代遗传变异与纸浆材优良种质选择,植物研究,35(1):39-46)
Polezhaeva M.A.,Semerikov V.L.,and Pimenova E.A.,2013,Genetic diversity of larch at the north of Primorskii krai and limits of Larix olgensis A.Henry distribution,Russ.J.Genet.,5:497-502
Sassner P.,Galbe M.,and Zacchi G.,2005,Steam pretreatment of Salix with and without SO2impregnation for production of bioethanol,Appl.Biochem.Biotech.,124(1-3):1101-1117
Song X.P.,Huang X.,Mo F.L.,Tian D.D.,Yang L.T.,Li Y.R.,and Chen B.S.,2013,Cloning and expression analysis of sugarcane phenylalanine ammonia-lyase(PAL)gene,Zhong guo Nongye Kexue(Scientia Agricultural Sinica),46(14):2856-2868(宋修鹏,黄杏,莫凤连,田丹丹,杨丽涛,李杨瑞,陈保善,2013,甘蔗苯丙氨酸解氨酶基因(PAL)的克隆和表达分析,中国农业科学,46(14):2856-2868)
Thumma B.R.,Matheson B.A.,Zhang D.Q.,Christian M.,and Roger M.,2009,Identification of a cis-acting regulatory polymorphism in a eucalypt COBRA-like gene affecting cellulose content,Genetics,183(3):1153-1164
Tian J.X.,Chang M.Q.,Du Q.Z.,Xu B.H.,and Zhang D.Q.,2014,Single-nucleotide polymorphisms in PtoCesA7 and their association with growth and wood properties in Populus tomentosa,Mol.Genet.Genomics,289(3):439-455
Vasyutkina E.A.,Reunova G.D.,Tupikin A.E.,and Zhuravlev Y.N.,2014,Mitochondrial DNA variation in olga bay larch(Larix olgensis A.Henry)from Primorsky Krai of Russia,Genetics,50(3):253-260
Wang M.,Cao F.X.,Wang S.L.,and Long J.L.,2010,Molecular cloning and sequence analysis of phenylalanine ammonia-lyase cDNA from Pinus massoniana,Zhongnan Linye Ke ji Daxue Xuebao(Journal of Central South University of Forestry&Technology),30(1):79-83(王猛,曹福祥,王仕利,龙绛雷,2010,马尾松苯丙氨酸解氨酶基因的克隆与序列分析,中南林业科技大学学报,30(1):79-83)
Wang Y.H.,Jia Q.B.,Zhang L.,and Zhang H.G.,2016,Allelic variation in 4-coumarate:CoA ligase(4CL)associated with wood properties of Larix olgensis,Zhiwu Yanjiu(Bulletin of Botanical Research),36(2):242-251(王艳红,贾庆斌,张磊,张含国,2016,长白落叶松4CL基因单核苷酸多态性及其与木材材性的关联分析,植物研究,36(2):242-251)
Xia H.,Zhao G.H.,Zhang L.S.,Sun X.Y.,Yin S.P.,Liang D.Y.,Li Y.,Zheng M.,and Zhao X.Y.,2016,Genetic and variation analyses of growth traits of half-sib Larix olgensis families in northeastern China,Euphytica,212:387-397
Xu F.,Zhu J.,Zhang F.X.,Wang Y.,Cheng S.Y.,Cheng S.H.,2008,Cloning of PAL gene from Sophora japonica,construction of anti-sense gene of SjPAL and its genetic transformation in Arabidopsis,Linye Kexue Yanjiu(Forest Research),21(5):611-618(许峰,朱俊,张风霞,王燕,程水源,程述汉,2008,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,21(5):611-618)
Xu N.,Loflin P.,Chen C.Y.,and Shyu A.B.,1998,A broader role for AU-rich element-mediated m RNA turnover revealed by a new transcriptional pulse strategy,Nucleic Acids Res.,26(2):558-565
Xu Y.,Thammannagowda S.,Thomas T.P.,Azadi P.,Schlarbaum S.,and Liang H.Y.,2013,LtuCAD1 is a cinnamyl alcohol dehydrogenase ortholog involved in lignin biosynthesis in Liriodendron tulipifera L.,a basal angiosperm timber species,Plant Mol.Biol.Rep.,31(5):1089-1099
Xue Y.C.,Li J.H.,Lu M.Z.,and Zhang Q.W.,2004,Cloning and identification of PAL gene amplified by RT-PCR form Populus×euramericana cv."74/76"second xylem m RNA,Linye Kexue(Scientia Silvae Sinicae),40(4):193-197(薛永常,李金花,卢孟柱,张绮纹,2004,107杨次生木质部PAL基因的RT-PCR扩增及其鉴定,林业科学,40(4):193-197)
Yao Y,2013,Genetic diversity of Larix olgensis henry seed orchard using SSR markers,Thesis for M.S.,Northeast Forestry University,Supervisor:Zhang H.G.,pp.30(姚宇,2013,长白落叶松初级无性系种子园SSR遗传多样性分析,硕士学位论文,东北林业大学,导师:张含国,pp.30)
Yin S.P.,Xiao Z.H.,Zhao G.H.,Zhao X.,Sun X.Y.,Zhang Y.,Wang F.W.,Li S.C.,Zhao X.Y.,and Qu G.Z.,2017,Variation analyses of growth and wood properties of Larix olgensis clones in China,J.Forest.Res.,28(4):687-697
Yin S.P.,Zhao G.H.,Xia H.,Sun X.Y.,PanY.Y.,Wang F.W.,Li S.C.,and Zhao X.Y.,2016,Stacly on progeny test of half-sibs families in Larix olgensis,Xinan Linye Daxue Xuebao(Journal of Southwest Forestry University),36(1):63-68(尹绍鹏,赵国辉,夏辉,孙晓阳,潘艳艳,王福维,李树春,赵曦阳,2016,长白落叶松半同胞子代测定研究,西南林业大学学报,36(1):63-68)
Yu G.H.,Wang G.P.,Wang L.,Yin H.Y.,Mu J.J.,Wu H.Y.,Guo X.X.,Wang H.W.,Kong L.R.,and Li A.F.,2015,Cloning of PAL genes and their response to FHB in wheat,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),15(6):1055-1061(虞光辉,王桂平,王亮,尹华燕,牟晶晶,吴洪燕,郭秀秀,王宏伟,孔令让,李安飞,2015,小麦PAL基因的克隆及赤霉菌诱导下的表达分析,植物遗传资源学报,15(6):1055-1061)
Yu H.Y.,2015,Superior provenances selection of Larix olgensis in different sites based on analytic timber,Thesis for M.S.,Northeast Forestry University,Supervisor:Zhang H.G.,pp.30(于宏影,2015,基于多点解析木的长白落叶松优良种源选择,硕士学位论文,东北林业大学,导师:张含国,pp.30)
Zhang D.Y.,Li W.B.,Li D.M.,and Li Y.G.,2009,Relationship between the contents of soybean isoflavanones and relative expression quantity of PAL gene,Dadou Kexue(Soybean Science),28(4):670-673(张大勇,李文滨,李冬梅,李永光,2009,大豆叶片异黄酮含量与PAL基因相对表达量的关系,大豆科学,28(4):670-673)
Zhou S.M.,Wang L.P.,Xiang X.,Wei B.H.,Li Y.R.,Fang F.X.,Wei W.X.,and Cao J.S.,2008,Cloning,expression and analysis of a full-length cDNA sequence encoding PAL in yam,Henongxue Bao(Journal of Nuclear Agricultural Sciences),22(6):781-788(周生茂,王玲平,向珣,韦本辉,李杨瑞,方锋学,韦威旭,曹家树,2008,山药PAL基因全长cDNA序列的克隆、表达与分析,核农学报,22(6):781-788)
Zhu J.J.,Yang K.,Yan Q.L.,Liu Z.G.,Yu L.Z.,and Wang H.X.,2010,Feasibility of implementing thinning in even-aged Larix olgensis plantations to develop uneven-aged larch-broadleaved mixed forests,Journal of Forest Research,15(1):71-80
Dillon S.K.,Nolan M.,Li W.,Bell C.,Wu H.X.,and Southerton S.G.,2010,Allelic variation in cell wall candidate genes affecting solid wood properties in natural populations and land races of Pinus radiata,Genetics,185(4):1477-1487
Geng S.,Xu C.S.,and Li Y.C.,2003,Advance in biosynthesis of lignin and its regulation,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),23(1):171-181(耿飒,徐存拴,李玉昌,2003,木质素的生物合成及其调控研究进展,西北植物学报,23(1):171-181)
Krutovsky K.V.,and Neale D.B.,2005,Nucleotide diversity and linkage disequilibrium in cold-hardiness and wood quality-related candidate genes in Douglas fir,Genetics,171(4):2029-2041
Lewis N.G.,and Yamamoto E.,1990,Lignin:occurrence,biogenesis and biodegredation,Annu.Rev.Plant Physiol.Plant Mol.Biol.,41:455-496
Li M.D.,and Ji W.L.,1994,Chemical composition of China's larch wood(Larix gmelini Rupr.),Zhongguo Zaozhi(Papermaking in China),(1):58-60(李民栋,纪文兰,1994,兴安落叶松化学组成的研究,中国造纸,(1):58-60)
Li Y.X.,Zhang H.G.,Zhang L.,Zhu H.Y.,and Zhou X.C.,2012,Research on multi-trait selection of the superior families of Larix olgensis pulpwood,Linye Kexue Yanjiu(Forest Research),25(6):712-718(李艳霞,张含国,张磊,朱航勇,周显昌,2012,长白落叶松纸浆材优良家系多性状联合选择研究,林业科学研究,25(6):712-718)
Liu J.,Xu B.L.,Xue Y.Y.,Zhang S.W.,and Chen R.X.,2014,Cloning and expression analysis of PAL gene in seed coat of Cucurbita pepo,Zhongguo Nongye Kexue(Scientia Agricultural Sinica),47(6):1216-1226(刘佳,徐秉良,薛应钰,张树武,陈荣贤,2014,美洲南瓜(Cucurbita pepo)种皮苯丙氨酸解氨酶基因克隆与表达分析,中国农业科学,47(6):1216-1226)
Ning K.,Liu X.P.,Lin Y.H.,Zhao X.Y.,Liu G.F.,Yang Y.,Song X.,and Li H.Y.,2015,Germplasm selection of the progeny genetic variation and superior pulpwood of Betula platyphylla,Zhiwu Yanjiu(Bulletin of Botanical Research),35(1):39-46(宁坤,刘笑平,林永红,赵曦阳,刘桂丰,杨洋,宋鑫,李慧玉,2015,白桦子代遗传变异与纸浆材优良种质选择,植物研究,35(1):39-46)
Polezhaeva M.A.,Semerikov V.L.,and Pimenova E.A.,2013,Genetic diversity of larch at the north of Primorskii krai and limits of Larix olgensis A.Henry distribution,Russ.J.Genet.,5:497-502
Sassner P.,Galbe M.,and Zacchi G.,2005,Steam pretreatment of Salix with and without SO2impregnation for production of bioethanol,Appl.Biochem.Biotech.,124(1-3):1101-1117
Song X.P.,Huang X.,Mo F.L.,Tian D.D.,Yang L.T.,Li Y.R.,and Chen B.S.,2013,Cloning and expression analysis of sugarcane phenylalanine ammonia-lyase(PAL)gene,Zhong guo Nongye Kexue(Scientia Agricultural Sinica),46(14):2856-2868(宋修鹏,黄杏,莫凤连,田丹丹,杨丽涛,李杨瑞,陈保善,2013,甘蔗苯丙氨酸解氨酶基因(PAL)的克隆和表达分析,中国农业科学,46(14):2856-2868)
Thumma B.R.,Matheson B.A.,Zhang D.Q.,Christian M.,and Roger M.,2009,Identification of a cis-acting regulatory polymorphism in a eucalypt COBRA-like gene affecting cellulose content,Genetics,183(3):1153-1164
Tian J.X.,Chang M.Q.,Du Q.Z.,Xu B.H.,and Zhang D.Q.,2014,Single-nucleotide polymorphisms in PtoCesA7 and their association with growth and wood properties in Populus tomentosa,Mol.Genet.Genomics,289(3):439-455
Vasyutkina E.A.,Reunova G.D.,Tupikin A.E.,and Zhuravlev Y.N.,2014,Mitochondrial DNA variation in olga bay larch(Larix olgensis A.Henry)from Primorsky Krai of Russia,Genetics,50(3):253-260
Wang M.,Cao F.X.,Wang S.L.,and Long J.L.,2010,Molecular cloning and sequence analysis of phenylalanine ammonia-lyase cDNA from Pinus massoniana,Zhongnan Linye Ke ji Daxue Xuebao(Journal of Central South University of Forestry&Technology),30(1):79-83(王猛,曹福祥,王仕利,龙绛雷,2010,马尾松苯丙氨酸解氨酶基因的克隆与序列分析,中南林业科技大学学报,30(1):79-83)
Wang Y.H.,Jia Q.B.,Zhang L.,and Zhang H.G.,2016,Allelic variation in 4-coumarate:CoA ligase(4CL)associated with wood properties of Larix olgensis,Zhiwu Yanjiu(Bulletin of Botanical Research),36(2):242-251(王艳红,贾庆斌,张磊,张含国,2016,长白落叶松4CL基因单核苷酸多态性及其与木材材性的关联分析,植物研究,36(2):242-251)
Xia H.,Zhao G.H.,Zhang L.S.,Sun X.Y.,Yin S.P.,Liang D.Y.,Li Y.,Zheng M.,and Zhao X.Y.,2016,Genetic and variation analyses of growth traits of half-sib Larix olgensis families in northeastern China,Euphytica,212:387-397
Xu F.,Zhu J.,Zhang F.X.,Wang Y.,Cheng S.Y.,Cheng S.H.,2008,Cloning of PAL gene from Sophora japonica,construction of anti-sense gene of SjPAL and its genetic transformation in Arabidopsis,Linye Kexue Yanjiu(Forest Research),21(5):611-618(许峰,朱俊,张风霞,王燕,程水源,程述汉,2008,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,21(5):611-618)
Xu N.,Loflin P.,Chen C.Y.,and Shyu A.B.,1998,A broader role for AU-rich element-mediated m RNA turnover revealed by a new transcriptional pulse strategy,Nucleic Acids Res.,26(2):558-565
Xu Y.,Thammannagowda S.,Thomas T.P.,Azadi P.,Schlarbaum S.,and Liang H.Y.,2013,LtuCAD1 is a cinnamyl alcohol dehydrogenase ortholog involved in lignin biosynthesis in Liriodendron tulipifera L.,a basal angiosperm timber species,Plant Mol.Biol.Rep.,31(5):1089-1099
Xue Y.C.,Li J.H.,Lu M.Z.,and Zhang Q.W.,2004,Cloning and identification of PAL gene amplified by RT-PCR form Populus×euramericana cv."74/76"second xylem m RNA,Linye Kexue(Scientia Silvae Sinicae),40(4):193-197(薛永常,李金花,卢孟柱,张绮纹,2004,107杨次生木质部PAL基因的RT-PCR扩增及其鉴定,林业科学,40(4):193-197)
Yao Y,2013,Genetic diversity of Larix olgensis henry seed orchard using SSR markers,Thesis for M.S.,Northeast Forestry University,Supervisor:Zhang H.G.,pp.30(姚宇,2013,长白落叶松初级无性系种子园SSR遗传多样性分析,硕士学位论文,东北林业大学,导师:张含国,pp.30)
Yin S.P.,Xiao Z.H.,Zhao G.H.,Zhao X.,Sun X.Y.,Zhang Y.,Wang F.W.,Li S.C.,Zhao X.Y.,and Qu G.Z.,2017,Variation analyses of growth and wood properties of Larix olgensis clones in China,J.Forest.Res.,28(4):687-697
Yin S.P.,Zhao G.H.,Xia H.,Sun X.Y.,PanY.Y.,Wang F.W.,Li S.C.,and Zhao X.Y.,2016,Stacly on progeny test of half-sibs families in Larix olgensis,Xinan Linye Daxue Xuebao(Journal of Southwest Forestry University),36(1):63-68(尹绍鹏,赵国辉,夏辉,孙晓阳,潘艳艳,王福维,李树春,赵曦阳,2016,长白落叶松半同胞子代测定研究,西南林业大学学报,36(1):63-68)
Yu G.H.,Wang G.P.,Wang L.,Yin H.Y.,Mu J.J.,Wu H.Y.,Guo X.X.,Wang H.W.,Kong L.R.,and Li A.F.,2015,Cloning of PAL genes and their response to FHB in wheat,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),15(6):1055-1061(虞光辉,王桂平,王亮,尹华燕,牟晶晶,吴洪燕,郭秀秀,王宏伟,孔令让,李安飞,2015,小麦PAL基因的克隆及赤霉菌诱导下的表达分析,植物遗传资源学报,15(6):1055-1061)
Yu H.Y.,2015,Superior provenances selection of Larix olgensis in different sites based on analytic timber,Thesis for M.S.,Northeast Forestry University,Supervisor:Zhang H.G.,pp.30(于宏影,2015,基于多点解析木的长白落叶松优良种源选择,硕士学位论文,东北林业大学,导师:张含国,pp.30)
Zhang D.Y.,Li W.B.,Li D.M.,and Li Y.G.,2009,Relationship between the contents of soybean isoflavanones and relative expression quantity of PAL gene,Dadou Kexue(Soybean Science),28(4):670-673(张大勇,李文滨,李冬梅,李永光,2009,大豆叶片异黄酮含量与PAL基因相对表达量的关系,大豆科学,28(4):670-673)
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