杉木ClOFP1基因的克隆、表达及单核苷酸多态性分析

Cloning,Expression and Single Nucleotide Polymorphisms Analysis of ClOFP1 Gene in Cunninghamia lanceolata

OFP基因家族在植物的生长发育中发挥重要调控作用。为探究ClOFP1在木质部形成过程中的作用,本研究通过克隆杉木ClOFP1基因,分析其表达模式,并开展该基因在杉木群体的单核苷酸多态性(SNP)及连锁不平衡(LD)分析。结果表明,ClOFP1基因的cDNA序列长1 648 bp,开放阅读框(ORF)长1 320 bp,在氨基酸序列的376~428位有卵形蛋白家族特有的OVATE结构域。系统进化树分析结果显示,ClOFP1蛋白与水稻、拟南芥中调控细胞伸长和次生壁形成的OsOFP19、AtOFP1、AtOFP4聚为一类。ClOFP1在幼嫩叶片和茎段中优势表达,随着叶片成熟和茎段木质化程度加深其表达量递减,幼叶中的表达量是老叶中的14倍。同时,在杉木木材中,ClOFP1主要在靠近树皮的边材区域表达,而在心边过渡区表达较弱。推测ClOFP1基因作为负调控因子,参与次生木质部的形成。共检测到ClOFP1基因内存在16个常见SNP位点,平均发生频率为1/103 bp。其中编码区域有11个SNP位点,分为7个同义突变和4个错义突变。7个地理种源的SNP多样性指数差异不显著,非同义突变多样性π_(ns)小于同义突变π_(s)。连锁不平衡分析结果发现,ClOFP1基因间的LD在875 bp左右长度内迅速消失,且SNPs具有3个较强的连锁不平衡区块。综上,ClOFP1的表达与杉木茎段木质化程度负相关,ClOFP1在不同无性系中SNP变异较为丰富,位点间的LD在较短序列长度内迅速消失,表明基于该基因的LD作图是可行的。本研究结果为深入解析ClOFP1功能和开展LD作图提供了重要依据。

The OFP gene family plays an important role in regulating plant growth and development.To investigate its function in xylem formation,ClOFP1 was cloned from Cunninghamia lanceolata.Then its sequence characteristics,expression patterns,as well as single nucleotide polymorphism(SNP)and linkage disequilibrium(LD),were analyzed in this study.The cDNA sequence of the ClOFP1 gene was found to be 1648 bp in length,with an open reading frame(ORF)spanning 1320 bp.Moreover,the amino acid sequence analysis revealed the presence of an OVATE domain at positions 376-428 aa,the distinctive characteristic domain within the OVATE family protein.Phylogenetic tree analysis showed that ClOFP1 was clustered with OsOFP19,AtOFP1 and AtOFP4,which were recognized as key regulators for cell elongation and secondary wall formation in Oryza sativa and Arabidopsis thaliana.ClOFP1 was predominantly expressed in young leaves and stem segments,gradually diminishing as the leaves matured and stem segments lignified.Notably,the expression level in young leaves was 14-fold that in mature ones.Furthermore,ClOFP1 exhibited predominant expression in the sapwood region adjacent to the bark,while its expression was relatively weaker in the sap-heartwood transition zone of C.lanceolata.Based on these observations,we hypothesize that the ClOFP1 may function as a negative regulator involved in secondary xylem formation.A total of 16 common SNP sites were detected within the ClOFP1 gene within the C.lanceolata population,with an average frequency of 1/103 bp.Among these SNPs,11 were located in the coding region and could be categorized into 7 synonymous mutations and 4 missense mutations.Notably,there was no significant variation observed in the SNP diversity index across the seven geographical provenances examined,while the diversity of nonsynonymous mutations(π_(ns))was lower than that of synonymous ones(π_(s)).Linkage disequilibrium(LD)analysis revealed that the LD did not extend over the entire gene(within 875 bp),and three strong linkage blocks were identified.In conclusion,the expression of ClOFP1 exhibited a negative correlation with stem lignification degree,and the SNP variation of ClOFP1 was abundant across different clones.The LD rapidly declined within ClOFP1,indicating the feasibility of LD mapping based on this gene in C.lanceolata.This study laid a foundation for in-depth dissection of xylem development mechanisms and provided a theory basis for further molecular-assisted breeding in C.lanceolata.