关键词: 杉木, NAC转录因子, 基因表达, SNP, 连锁不平衡

Abstract: [Objective] A NAC transcription factor gene ClNAC1 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 ClNAC1 were conducted to provide an important foundation for further functional dissection and LD mapping.[Method] The cDNA sequence of ClNAC1 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 ClNAC1 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 ClNAC1 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 ClNAC1 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 (π_tot, π_sil, π_s and π_n) among the six populations, and the diversity of non-synonymous mutation (π_n)in different populations was less than that of synonymous mutation diversity (π_s). 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 ClNAC1 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 ClNAC1 with the sequence length increasing in different populations, suggesting that LD mapping based on this gene would be feasible in C. lanceolata.

Key words: Cunninghamia lanceolata, NAC transcription factor, gene expression, single nucleotide polymorphism, linkage disequilibrium