杉木NAC转录因子基因ClNAC1的克隆、表达及单核苷酸多态性分析
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摘要
【目的】克隆与杉木次生壁形成相关的Cl NAC1基因,在组织表达特异性分析基础上开展该基因的单核苷酸多态性(SNP)及其连锁不平衡(LD)分析,以期为深入解析该基因功能和开展LD作图提供重要依据。【方法】基于杉木根、茎、叶等混合样本转录组测序获得的相关数据,分离杉木Cl NAC1的c DNA序列,进行同源比对和进化树构建分析。利用实时荧光定量PCR技术(q PCR)检测Cl NAC1的表达模式。通过MEGA 6.0和Dna SP 5.0分析Cl NAC1在40个杉木无性系的SNP变异,以及LD衰减程度。【结果】分离到Cl NAC1基因c DNA序列1 286 bp,开放阅读框(ORF)长度为1 092 bp,编码蛋白质在N端含有1个由128个氨基酸残基组成的NAC结构域。相应基因组序列长2 546 bp,有3个外显子和3个内含子,且第1个内含子位于5'非翻译区(UTR)。系统进化树分析表明,Cl NAC1蛋白位于B分支,与拟南芥的NST1/2/3、毛果杨的Ptr WND2A/B等聚在一起,属于次生生长相关的NAC转录因子类别。Cl NAC1在雄球花中的表达量最大,在当年生成熟叶中最小;该基因在当年生半木质化茎中的表达量是未木质化茎的2.8倍,且在去年生茎木质部中的表达量比皮层大3倍左右。利用来自6个地理种源的40个无性系发现Cl NAC1内存在104个常见SNP位点,平均发生频率为1/24 bp,多样性水平达到0.012 53。编码区域有32个SNP位点,其中25个属于同义突变,7个属于错义突变。SNP多样性指数πtot、πsil、πs、πn在6个群体间的差异不显著,且不同群体的非同义突变多样性πn皆小于同义突变多样性πs。LD分析显示,当r2>0.1时,在6个群体中LD衰退序列长度在1 025~2 460 bp间变化,在基因内部LD水平已衰退至不显著。【结论】杉木Cl NAC1基因可能参与次生壁的发育。Cl NAC1在不同无性系间存在丰富的SNP变异,且在进化中主要受纯化选择作用。不同杉木群体中Cl NAC1基因SNP位点间的LD皆在较短序列长度内迅速消失,表明基于候选基因的LD作图策略在杉木关联作图研究中是可行的。
【Objective】 A NAC transcription factor gene Cl NAC1 was cloned,which was related to formation of the secondary wall of Cunninghamia lanceolata. Based on tissue differential expression detection, single nucleotide polymorphism( SNP) analysis and linkage disequilibrium( LD) test of Cl NAC1 were conducted to provide an important foundation for further functional dissection and LD mapping.【Method】The c DNA sequence of Cl NAC1 was isolated based on transcriptome sequencing of C. lanceolata mixed samples, and bioinformatics characteristics were analyzed by homologous alignment and phylogenetic tree construction. The expression patterns in different organs and tissues were detected by real-time quantitative polymerase chain reaction( q PCR). The SNP variations and the patterns of LD decay among 40 clones of C. lanceolata were analyzed using MEGA 6. 0 and Dna SP 5. 0.【Result】 The isolated Cl NAC1 c DNA was 1 286 bp long with an open reading frame( ORF) of 1 092 bp,and the encoding protein possessed a NAC domain of128 amino acids residues at the N-terminus. The corresponding genomic sequence of 2 546 bp length contained three exons and three introns,and the first intron was located in the 5'untranslated region( UTR). Phylogenetic tree analysis showed that the Cl NAC1 protein was grouped with Arabidopsis thaliana NST1/2/3 and Populus trichocarpa WND2 A/B,and belonged to the B-clade associated with secondary growth. The expression levels of Cl NAC1 was the highest in male cone,and was lowest in mature leaf from one-year branch. The expression levels in semi-lignified stem was 2. 8 times as high as that of non-lignified stem,and the corresponding value in the xylem of two-years branch was about three times higher than that of bark. Through resequencing Cl NAC1 locus of 40 individuals from six geographic provenances,104 common SNPs were identified with an average frequency of 1/24 bp and a diversity level of 0. 012 53. There were 32 SNPs in the coding region,of which 25 were synonymous mutations and seven were missense mutations. There was no significant difference in the SNP diversity index( πtot,πsil,πsand πn) among the six populations,and the diversity of non-synonymous mutation( πn) in different populations was less than that of synonymous mutation diversity( πs). LD analysis showed that the length of LD decay sequence varied from 1 025 bp to 2 460 bp in six populations and the LD level within the gene has declined to be insignificant at r2> 0. 1.【Conclusion】 The Cl NAC1 might be involved in secondary wall regulation. This gene had abundant SNP mutations in the studied populations,and it was mainly subjected to purifying selection during the course of evolution. The LD declined rapidly within the Cl NAC1 with the sequence length increasing in different populations,suggesting that LD mapping based on this gene would be feasible in C. lanceolata.
【Objective】 A NAC transcription factor gene Cl NAC1 was cloned,which was related to formation of the secondary wall of Cunninghamia lanceolata. Based on tissue differential expression detection, single nucleotide polymorphism( SNP) analysis and linkage disequilibrium( LD) test of Cl NAC1 were conducted to provide an important foundation for further functional dissection and LD mapping.【Method】The c DNA sequence of Cl NAC1 was isolated based on transcriptome sequencing of C. lanceolata mixed samples, and bioinformatics characteristics were analyzed by homologous alignment and phylogenetic tree construction. The expression patterns in different organs and tissues were detected by real-time quantitative polymerase chain reaction( q PCR). The SNP variations and the patterns of LD decay among 40 clones of C. lanceolata were analyzed using MEGA 6. 0 and Dna SP 5. 0.【Result】 The isolated Cl NAC1 c DNA was 1 286 bp long with an open reading frame( ORF) of 1 092 bp,and the encoding protein possessed a NAC domain of128 amino acids residues at the N-terminus. The corresponding genomic sequence of 2 546 bp length contained three exons and three introns,and the first intron was located in the 5'untranslated region( UTR). Phylogenetic tree analysis showed that the Cl NAC1 protein was grouped with Arabidopsis thaliana NST1/2/3 and Populus trichocarpa WND2 A/B,and belonged to the B-clade associated with secondary growth. The expression levels of Cl NAC1 was the highest in male cone,and was lowest in mature leaf from one-year branch. The expression levels in semi-lignified stem was 2. 8 times as high as that of non-lignified stem,and the corresponding value in the xylem of two-years branch was about three times higher than that of bark. Through resequencing Cl NAC1 locus of 40 individuals from six geographic provenances,104 common SNPs were identified with an average frequency of 1/24 bp and a diversity level of 0. 012 53. There were 32 SNPs in the coding region,of which 25 were synonymous mutations and seven were missense mutations. There was no significant difference in the SNP diversity index( πtot,πsil,πsand πn) among the six populations,and the diversity of non-synonymous mutation( πn) in different populations was less than that of synonymous mutation diversity( πs). LD analysis showed that the length of LD decay sequence varied from 1 025 bp to 2 460 bp in six populations and the LD level within the gene has declined to be insignificant at r2> 0. 1.【Conclusion】 The Cl NAC1 might be involved in secondary wall regulation. This gene had abundant SNP mutations in the studied populations,and it was mainly subjected to purifying selection during the course of evolution. The LD declined rapidly within the Cl NAC1 with the sequence length increasing in different populations,suggesting that LD mapping based on this gene would be feasible in C. lanceolata.
引文
蔡秀玲,王宗阳,郑霏琴,等.1997.水稻蜡质基因5’非翻译区一个与调控有关的内含子.植物生理学报,23(3):257-261.(Cai X L,Wang Z Y,Zheng F Q,et al.1997.A regulation-related introl in 5'untranslated region of rice Waxy gene.Acta Phytophysiologica Sinica,23(3):257-261.[in Chinese])
高燕.2011.杉木木材形成过程扩展蛋白基因的克隆与表达分析.林业科学,47(11):44-51.(Gao Y.2011.Cloning and expression analysis of expansin genes during wood formation in Chinese fir.Scientia Silvae Sinicae,47(11):44-51.[in Chinese])
巩琛锐,王璐,杜庆章,等.2013.毛白杨S-腺苷高半胱氨酸水解酶基因PtS AHHA的克隆、表达及SNP关联分析.林业科学,49(11):67-76.(Gong C R,Wang L,Du Q Z,et al.2013.Isolation,expression,and association genetics of the S-adenosyl-L-homocysteine hydrolase gene A(PtS AHHA)in Populus tomentosa.Scientia Silvae Sinicae,49(11):67-76.[in Chinese])
李玉岭,周厚君,林二培,等.2013.光皮桦BlS PL1转录因子基因的克隆、表达及单核苷酸多态性分析.林业科学,49(9):52-61.(Li Y L,Zhou H J,Lin E P,et al.2013.Isolation,expression and single nucleotide polymorphisms analysis of transcription factor(BlS PL1)from Betula luminifera.Scientia Silvae Sinicae,49(9):52-61.[in Chinese])
李伟,韩蕾,钱永强,等.2011.植物NAC转录因子的种类、特征及功能.应用与环境生物学报,17(4):596-606.(Li W,Han L,Qian Y Q,et al.2011.Characteristics and functions of NAC transcription factors in plants.Chinese Journal of Applied and Environmental Biology,17(4):596-606.[in Chinese])
潘炜,田佳星,杜庆章,等.2011.毛白杨蔗糖合酶基因PtS US1的克隆、表达及单核苷酸多态性分析.林业科学,47(3):52-58.(Pan W,Tian J X,Du Q Z,et al.2011.Isolation,expression and single nucleotide polymorphisms analysis of sucrose synthase gene(PtS US1)from Populus tomentosa.Scientia Silvae Sinicae,47(3):52-58.[in Chinese])
庞涛,郭丽丽,夏新莉,等.2011.CBL1基因5’非翻译区内含子在旱生植物沙冬青中的作用.北京林业大学学报,33(6):157-165.(Pang T,Guo L L,Xia X L,et al.2011.Effects of 5'UTR intron of AmC BL1 gene in a xerophyte Ammopiptanthus mongolicus.Journal of Beijing Forestry University,33(6):157-165.[in Chinese])
王钰,童再康,黄华宏,等.2010.光皮桦总RNA的提取及Actin基因的克隆.浙江林业科技,30(4):32-36.(Wang Y,Tong Z K,Huang H H,et al.2010.Extracting of total RNAfrom Betula luminifera and the cloning of Actin gene.Journal of Zhejiang Forestry Science and Technology,30(4):32-36.[in Chinese])
谢媛,田云,卢向阳.2012.单核苷酸多态性及其在水稻中的应用(综述).亚热带植物科学,41(1):68-72.(Xie Y,Tian Y,Lu X Y.2012.Single nucleotide polymorphism and its application in rice(Review).Subtropical Plant Science,41(1):68-72.[in Chinese])
谢一青,李志真,黄儒珠,等.2006.光皮桦基因组DNA提取方法比较.浙江林学院学报,23(6):664-668.(Xie Y Q,Li Z Z,Huang R Z,et al.2006.Comparison of methods of extracting genomic DNA from Betula luminifera.Journal of Zhejiang Forestry College,23(6):664-668.[in Chinese])
徐煲铧,杨晓慧,李百炼,等.2009.毛白杨纤维素合酶基因PtC esA 4的克隆、表达及单核苷酸多态性分析.林业科学,45(5):1-10.(Xu B H,Yang X H,Li B L,et al.2009.Isolation,expression and single nucleotide polymorphisms analysis of cellulose synthase gene(PtC esA 4)from Populus tomentosa.Scientia Silvae Sinicae,45(5):1-10.[in Chinese])
徐莉莉,童再康,林二培,等.2013.杉木苯丙氨酸解氨酶基因ClP AL的克隆与表达分析.林业科学,49(12):64-72.(Xu L L,Tong Z K,Lin E P,et al.2013.Isolation and expression of ClP AL genes in Chinese fir(Cunninghamia lanceolata).Scientia Silvae Sinicae,49(12):64-72.[in Chinese])
杨晓慧,张有慧,张志毅,等.2009.毛白杨干细胞决定基因Wuschel的克隆及其单核苷酸多态性分析.林业科学,45(1):43-49.(Yang X H,Zhang Y H,Zhang Z Y,et al.2009.Isolation and single nucleotide polymorphisms analysis of stem cell organizer gene Wuschel in Populus tomentosa.Scientia Silvae Sinicae,45(1):43-49.[in Chinese])
Aminoroaya K,Sadeghi A A,Ansaripirsaraei Z,et al.2016.The effects of cyclical higher incubation temperatures on body and organs weights,thyroid hormones and HSP70 gene expression of newly hatched broiler chicks.Kafkas Universitesi Veteriner Fakultesi Dergisi,22(4):613-618.
Demura T,Fukuda H.2007.Transcriptional regulation in wood formation.Trends in Plant Science,12(2):64-70.
Fu Y X,Li W H.1993.Statistical tests of neutrality of mutations.Genetics,133(3):693-709.
Grant E H,Fujino T,Beers E P,et al.2010.Characterization of NACdomain transcription factors implicated in control of vascular cell differentiation in Arabidopsis and Populus.Planta,232(2):337-352.
Groover A T.2005.What genes make a tree a tree?.Trends in Plant Science,10(5):210-214.
Groover A,Robischon M.2006.Developmental mechanisms regulating secondary growth in woody plants.Current Opinion in Plant Biology,9(1):55-58.
Huang H H,Xu L L,Tong Z K,et al.2012.De novo characterization of the Chinese fir(Cunninghamia lanceolata)transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis.BMC Genomics,13(1):648-671.
Kalluri U C,Joshi C P.2004.Differential expression patterns of two cellulose synthase genes are associated with primary and secondary cell wall development in Aspen trees.Planta,220(1):47-55.
Kraakman A W,Niks R E,Petra M B,et al.2004.Linkage disequilibrium mapping of yield and yield stability in modern spring barley cultivars.Genetics,168(1):435-446.
Lalague H,Csillery K,Oddou-muratorio S,et al.2014.Nucleotide diversity and linkage disequilibrium at 58 stress response and phenology candidate genes in a European beech(Fagus sylvatica)population from southeastern France.Tree Genetics&Genomes,10(1):15-26
Lu M M,Krutovsky K V,Nelson C D,et al.2016.Exome genotyping,linkage disequilibrium and population structure in loblolly pine(Pinus taeda L.).BMC Genomics,17(1):730-740.
Mitsuda N,Seki M,Shinozaki K,et al.2005.The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence.Plant Cell,17(11):2993-3006.
Nordborg M,Borevitz J O,Bergelson J,et al.2002.The extent of linkage disequilibrium in Arabidopsis thaliana.Nature Genetics,30(2):190-193.
Remington D L,Thornsberry J M,Matsuoka Y,et al.2001.Structure of linkage disequilibrium and phenotypic associations in the maize genome.Proceedings of the National Academy of Sciences of the United States of America,98(20):11479-11484.
Salinas M,Xing S,H9hmann S,et al.2012.Genomic organization,phylogenetic comparison and differential expression of the SBP-box family of transcription factors in tomato.Planta,235(6):1171-1184.
Tajima F.1989.Statistical method for testing the neutral mutation hypothesis by DNA polymorphism.Genetics,123(3):585-595.
Wu L,Joshi C P,Chiang V L.2000.A xylem-specific cellulose synthase gene from Aspen(Populus tremuloides)is responsive to mechanical stress.Plant Journal,22(6):495-502.
Yang L,Hou Y,Zhao X,et al.2015.Identification and characterization of a wood-associated NAC domain transcription factor PtoV NS11from Populus tomentosa Carr.Trees,29(4):1091-1101.
Zhong R,Ye Z H.2010.Functional characterization of poplar woodassociated NAC domain transcription factors.Plant Physiology,152(2):1044-1055.
Zhong R,Ye Z H.2015.Secondary cell walls:biosynthesis,patterned deposition and transcriptional regulation.Plant&Cell Physiology,56(2):195-214.
高燕.2011.杉木木材形成过程扩展蛋白基因的克隆与表达分析.林业科学,47(11):44-51.(Gao Y.2011.Cloning and expression analysis of expansin genes during wood formation in Chinese fir.Scientia Silvae Sinicae,47(11):44-51.[in Chinese])
巩琛锐,王璐,杜庆章,等.2013.毛白杨S-腺苷高半胱氨酸水解酶基因PtS AHHA的克隆、表达及SNP关联分析.林业科学,49(11):67-76.(Gong C R,Wang L,Du Q Z,et al.2013.Isolation,expression,and association genetics of the S-adenosyl-L-homocysteine hydrolase gene A(PtS AHHA)in Populus tomentosa.Scientia Silvae Sinicae,49(11):67-76.[in Chinese])
李玉岭,周厚君,林二培,等.2013.光皮桦BlS PL1转录因子基因的克隆、表达及单核苷酸多态性分析.林业科学,49(9):52-61.(Li Y L,Zhou H J,Lin E P,et al.2013.Isolation,expression and single nucleotide polymorphisms analysis of transcription factor(BlS PL1)from Betula luminifera.Scientia Silvae Sinicae,49(9):52-61.[in Chinese])
李伟,韩蕾,钱永强,等.2011.植物NAC转录因子的种类、特征及功能.应用与环境生物学报,17(4):596-606.(Li W,Han L,Qian Y Q,et al.2011.Characteristics and functions of NAC transcription factors in plants.Chinese Journal of Applied and Environmental Biology,17(4):596-606.[in Chinese])
潘炜,田佳星,杜庆章,等.2011.毛白杨蔗糖合酶基因PtS US1的克隆、表达及单核苷酸多态性分析.林业科学,47(3):52-58.(Pan W,Tian J X,Du Q Z,et al.2011.Isolation,expression and single nucleotide polymorphisms analysis of sucrose synthase gene(PtS US1)from Populus tomentosa.Scientia Silvae Sinicae,47(3):52-58.[in Chinese])
庞涛,郭丽丽,夏新莉,等.2011.CBL1基因5’非翻译区内含子在旱生植物沙冬青中的作用.北京林业大学学报,33(6):157-165.(Pang T,Guo L L,Xia X L,et al.2011.Effects of 5'UTR intron of AmC BL1 gene in a xerophyte Ammopiptanthus mongolicus.Journal of Beijing Forestry University,33(6):157-165.[in Chinese])
王钰,童再康,黄华宏,等.2010.光皮桦总RNA的提取及Actin基因的克隆.浙江林业科技,30(4):32-36.(Wang Y,Tong Z K,Huang H H,et al.2010.Extracting of total RNAfrom Betula luminifera and the cloning of Actin gene.Journal of Zhejiang Forestry Science and Technology,30(4):32-36.[in Chinese])
谢媛,田云,卢向阳.2012.单核苷酸多态性及其在水稻中的应用(综述).亚热带植物科学,41(1):68-72.(Xie Y,Tian Y,Lu X Y.2012.Single nucleotide polymorphism and its application in rice(Review).Subtropical Plant Science,41(1):68-72.[in Chinese])
谢一青,李志真,黄儒珠,等.2006.光皮桦基因组DNA提取方法比较.浙江林学院学报,23(6):664-668.(Xie Y Q,Li Z Z,Huang R Z,et al.2006.Comparison of methods of extracting genomic DNA from Betula luminifera.Journal of Zhejiang Forestry College,23(6):664-668.[in Chinese])
徐煲铧,杨晓慧,李百炼,等.2009.毛白杨纤维素合酶基因PtC esA 4的克隆、表达及单核苷酸多态性分析.林业科学,45(5):1-10.(Xu B H,Yang X H,Li B L,et al.2009.Isolation,expression and single nucleotide polymorphisms analysis of cellulose synthase gene(PtC esA 4)from Populus tomentosa.Scientia Silvae Sinicae,45(5):1-10.[in Chinese])
徐莉莉,童再康,林二培,等.2013.杉木苯丙氨酸解氨酶基因ClP AL的克隆与表达分析.林业科学,49(12):64-72.(Xu L L,Tong Z K,Lin E P,et al.2013.Isolation and expression of ClP AL genes in Chinese fir(Cunninghamia lanceolata).Scientia Silvae Sinicae,49(12):64-72.[in Chinese])
杨晓慧,张有慧,张志毅,等.2009.毛白杨干细胞决定基因Wuschel的克隆及其单核苷酸多态性分析.林业科学,45(1):43-49.(Yang X H,Zhang Y H,Zhang Z Y,et al.2009.Isolation and single nucleotide polymorphisms analysis of stem cell organizer gene Wuschel in Populus tomentosa.Scientia Silvae Sinicae,45(1):43-49.[in Chinese])
Aminoroaya K,Sadeghi A A,Ansaripirsaraei Z,et al.2016.The effects of cyclical higher incubation temperatures on body and organs weights,thyroid hormones and HSP70 gene expression of newly hatched broiler chicks.Kafkas Universitesi Veteriner Fakultesi Dergisi,22(4):613-618.
Demura T,Fukuda H.2007.Transcriptional regulation in wood formation.Trends in Plant Science,12(2):64-70.
Fu Y X,Li W H.1993.Statistical tests of neutrality of mutations.Genetics,133(3):693-709.
Grant E H,Fujino T,Beers E P,et al.2010.Characterization of NACdomain transcription factors implicated in control of vascular cell differentiation in Arabidopsis and Populus.Planta,232(2):337-352.
Groover A T.2005.What genes make a tree a tree?.Trends in Plant Science,10(5):210-214.
Groover A,Robischon M.2006.Developmental mechanisms regulating secondary growth in woody plants.Current Opinion in Plant Biology,9(1):55-58.
Huang H H,Xu L L,Tong Z K,et al.2012.De novo characterization of the Chinese fir(Cunninghamia lanceolata)transcriptome and analysis of candidate genes involved in cellulose and lignin biosynthesis.BMC Genomics,13(1):648-671.
Kalluri U C,Joshi C P.2004.Differential expression patterns of two cellulose synthase genes are associated with primary and secondary cell wall development in Aspen trees.Planta,220(1):47-55.
Kraakman A W,Niks R E,Petra M B,et al.2004.Linkage disequilibrium mapping of yield and yield stability in modern spring barley cultivars.Genetics,168(1):435-446.
Lalague H,Csillery K,Oddou-muratorio S,et al.2014.Nucleotide diversity and linkage disequilibrium at 58 stress response and phenology candidate genes in a European beech(Fagus sylvatica)population from southeastern France.Tree Genetics&Genomes,10(1):15-26
Lu M M,Krutovsky K V,Nelson C D,et al.2016.Exome genotyping,linkage disequilibrium and population structure in loblolly pine(Pinus taeda L.).BMC Genomics,17(1):730-740.
Mitsuda N,Seki M,Shinozaki K,et al.2005.The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence.Plant Cell,17(11):2993-3006.
Nordborg M,Borevitz J O,Bergelson J,et al.2002.The extent of linkage disequilibrium in Arabidopsis thaliana.Nature Genetics,30(2):190-193.
Remington D L,Thornsberry J M,Matsuoka Y,et al.2001.Structure of linkage disequilibrium and phenotypic associations in the maize genome.Proceedings of the National Academy of Sciences of the United States of America,98(20):11479-11484.
Salinas M,Xing S,H9hmann S,et al.2012.Genomic organization,phylogenetic comparison and differential expression of the SBP-box family of transcription factors in tomato.Planta,235(6):1171-1184.
Tajima F.1989.Statistical method for testing the neutral mutation hypothesis by DNA polymorphism.Genetics,123(3):585-595.
Wu L,Joshi C P,Chiang V L.2000.A xylem-specific cellulose synthase gene from Aspen(Populus tremuloides)is responsive to mechanical stress.Plant Journal,22(6):495-502.
Yang L,Hou Y,Zhao X,et al.2015.Identification and characterization of a wood-associated NAC domain transcription factor PtoV NS11from Populus tomentosa Carr.Trees,29(4):1091-1101.
Zhong R,Ye Z H.2010.Functional characterization of poplar woodassociated NAC domain transcription factors.Plant Physiology,152(2):1044-1055.
Zhong R,Ye Z H.2015.Secondary cell walls:biosynthesis,patterned deposition and transcriptional regulation.Plant&Cell Physiology,56(2):195-214.