杉木次生壁发育调控转录因子基因ClNAC40的生物学功能  

Biological Functional Analysis of Transcription Factor Gene ClNAC40 Regulating Secondary Cell Wall Development in Cunninghamia lanceolata

作　　者：刘明彤 庄和必 王紫彤 石帅宾 刘晓娟 林二培[1] 胡现铬 黄华宏[1] Liu Mingtong;Zhuang Hebi;Wang Zitong;Shi Shuaibin;Liu Xiaojuan;Lin Erpei;Hu Xiange;Huang Huahong(The State Key Laboratory of Subtropical SilvicultureZhejiang Agricultural and Forestry University,Hangzhou 311300)

机构地区：[1]浙江农林大学亚热带森林培育国家重点实验室,杭州311300

出　　处：《林业科学》2025年第2期131-141,共11页Scientia Silvae Sinicae

基　　金：国家自然科学基金项目(32271919);浙江省农业新品种选育项目(2021C02070-8)。

摘　　要：【目的】在挖掘杉木次生壁发育相关NAC调控因子基础上,分析其生物信息学特征、表达模式,以及在拟南芥中过表达的作用,以期为杉木次生壁发育分子机制解析和分子辅助育种提供重要参考价值。【方法】基于杉木不同器官和组织的转录组测序数据,通过共表达网络分析(WGCNA)筛选与木质化相关的NAC基因,克隆其全长序列,进行序列比对和系统进化树分析;利用实时荧光定量PCR技术(RT-qPCR)分析其在不同组织和应压木形成中的表达模式;构建NAC基因过表达重组载体,利用花序侵染法获得转基因拟南芥,并进行转基因植株花序茎横切面染色观察和木质素合成相关基因的表达分析。【结果】分离得到1个杉木NAC基因(ClNAC40),其cDNA序列长度为1556 bp,开放阅读框(ORF)为1344 bp,编码447个氨基酸。获得ClNAC40基因组序列共3430 bp,由6个外显子和5个内含子组成。进化树分析发现ClNAC40与已报道的次生发育相关NAC转录因子聚为一类。ClNAC40基因在木质部(XY)优势表达,而在根(RT)、雌球花(FC)、雄球花(MC)和皮层(BK)中的表达量相对较低;同时,在茎中表达呈现随木质化程度升高而递增趋势,在木质化茎(S3)中的表达量是未木质化茎(S1)的3.2倍。在应压木诱导形成过程中,ClNAC40在应压木中的表达量呈上升趋势,斜放处理30天和60天时的表达水平均显著高于直立木(对照)。在拟南芥中过表达Cl NAC40导致花序茎的高度和直径均明显增加;花序茎横切面经间苯三酚染色,显色较野生型深且染色区域占比更大,表明木质素沉积更多。RT-qPCR分析结果表明,ClNAC40过表达显著提高了拟南芥木质素合成途径关键酶基因的表达。【结论】杉木ClNAC40基因通过激活木质素生物合成相关酶基因表达,参与调控次生壁的发育。【Objective】The NAC transcription factor plays a pivotal role in plant,specifically in regulating secondary cell wall(SCW)development.In this study,we cloned a NAC gene associated with SCW development in Cunninghamia lanceolata and conducted an extensive investigation into its bioinformatics characteristics,expression patterns,and the effects of overexpression in Arabidopsis thaliana.This study aims to provide important reference value for elucidating the molecular mechanisms underlying SCW development and facilitating molecular-assisted breeding in C.lanceolata.【Method】Based on the transcriptome sequencing data from different organs and tissues of C.lanceolata,a co-expression network analysis was conducted to identify the NAC gene associated with lignification.Subsequently,the full-length sequence of this gene was cloned and subjected to sequence alignment and phylogenetic tree analysis.Real-time quantitative PCR(RT-qPCR)was used to investigate its expression patterns in different tissues and during the process of compression wood formation.Furthermore,an overexpression recombinant vector for the NAC gene was constructed and transformed into A.thaliana via inflorescence infestation.The cross sections of the inflorescence stem from transgenic plants were stained,and the expression of key enzyme genes involved in lignin biosynthesis was analyzed.【Result】A ClNAC40 gene was isolated from C.lanceolata,with the cDNA sequence length of 1556 bp and an open reading frame(ORF)of 1344 bp that can encode 447 amino acids.A total of 3430 bp of the ClNAC40 genome sequence was obtained,consisting of six exons and five introns.Evolutionary tree analysis revealed that ClNAC40 was clustered with the reported NAC transcription factors involved in SCW formation.The ClNAC40 was predominantly expressed in xylem(XY),while it exhibited relatively lower levels in root(RT),female cone(FC),male cone(MC),and bark(BK).Additionally,the expression in the stems was positively correlated with the degree of lignification,with a 3.2-fol

关 键 词：杉木 NAC转录因子 基因功能 次生壁发育 

分 类 号：S718.46[农业科学—林学]