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 ClNAC 1 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 ClNAC 1 were conducted to provide an important foundation for further functional dissection and LD mapping.【Method】The cDNA sequence of ClNAC 1 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(qPCR).The SNP variations and the patterns of LD decay among 40 clones of C.lanceolata were analyzed using MEGA 6.0 and DnaSP 5.0.【Result】The isolated ClNAC 1 cDNA was 1 286 bp long with an open reading frame(ORF)of 1 092 bp,and the encoding protein possessed a NAC domain of 128 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 ClNAC1 protein was grouped with Arabidopsis thaliana NST1/2/3 and Populus trichocarpa WND2A/B,and belonged to the B-clade associated with secondary growth.The expression levels of ClNAC 1 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 ClNAC 1 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,πs andπn)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 r 2>0.1.【Conclusion】The ClNAC 1 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 ClNAC 1 with the sequence length increasing in different populations,suggesting that LD mapping based on this gene would be feasible in C.lanceolata.
作者
魏明科
俞金健
黄晓龙
刘琼瑶
黄华宏
林二培
童再康
Wei Mingke;Yu Jinjian;Huang Xiaolong;Liu Qiongyao;Huang Huahong;Lin Erpei;Tong Zaikang(The State Key Laboratory of Subtropical Silviculture Zhejiang Agricultural and Forestry University Hangzhou 311300)
出处
《林业科学》
EI
CAS
CSCD
北大核心
2018年第9期49-59,共11页
Scientia Silvae Sinicae
基金
国家重点研发计划项目(2017YFD0600201)
浙江省农业新品种选育重大科技专项(2016C02056-5)
关键词
杉木
NAC转录因子
基因表达
SNP
连锁不平衡
Cunninghamia lanceolata
NAC transcription factor
gene expression
single nucleotide polymorphism
linkage disequilibrium
