Abstract:

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 1 556 bp and an open reading frame (ORF) of 1 344 bp that can encode 447 amino acids. A total of 3 430 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-fold increase in expression level observed in lignified stems (S3) compared to non-lignified stems (S1). During the formation of compression wood, the expression of ClNAC40 was upregulated, with significantly higher levels observed in compression wood compared to upright wood after 30 and 60 days of oblique treatment. The overexpression of ClNAC40 in A. thaliana resulted in a significant enhancement in plant height and inflorescence stem diameter. Subsequently, the phloroglucinol staining of the cross section of the inflorescence stem exhibited a deeper color compared to the wild type, accompanied by a larger staining area, indicating an increase in lignin deposition. The RT-qPCR analysis showed that the overexpression of ClNAC40 significantly upregulated the expression of key enzyme genes involved in the lignin biosynthesis in A. thaliana. Conclusion: The ClNAC40 in C. lanceolata participates in regulating SCW development by activating the transcription of lignin biosynthesis-related enzyme genes.

Key words: Cunninghamia lanceolata, NAC transcription factors, gene function, secondary cell wall development