关键词: 杉木, 心材形成, 过渡区, 双功能核酸酶BFN

Abstract:

Objective: In this study, the bifunctional nuclease gene family was identified, their protein-sequence characteristics and tissue-specific expression patterns were analyzed, and candidate molecular marker genes for the transition zone were screened, aiming to lay a foundation for clarifying the mechanism of heartwood formation in Chinese fir and provide a reference for transition-zone identification in other heartwood-forming species. Method: Bioinformatic methods were employed to identify the ClBFNs and to analyze the protein physicochemical properties, subcellular localization, sequence features and phylogeny. Expression pattern of ClBFNs in different tissues was analyzed and the candidate marker genes of transition zone were preliminarily screened. Annual rings from increment cores were taken, the RNA of each ring was extracted, and their RNA was analyzed by semi-quantitative RT-PCR (SqRT-PCR) and validated by qRT-PCR to refine the candidates. DAPI staining was used to observe nuclei of ray parenchyma cells in the wood-core transition zone and sapwood. Result: Four ClBFNs genes were identified, encoding proteins of 31.95 to 34.88 kDa. All proteins contained the S1-P1 nuclease domain and were acidic. Phylogenetically, ClBFN1 was clustered with PsBFN, PaBFN4, PtabBFN3, and PtaeBFN3 on the same evolutionary branch, whereas ClBFN2, ClBFN3, ClBFN4 were grouped with PtabBFN1 and PtaeBFN2. The semi- and quantitative PCR results showed that ClBFN2 was only expressed in the roots, while the other ClBFNs were expressed in the bark, roots, xylem, young needles, and mature needles. ClBFN1 exhibited high expression in roots and xylem but low expression in needles and bark. ClBFN3 was predominant in roots and needles, with lower expression in xylem and the lowest in the bark. ClBFN4 had lower expression in bark and xylem than in needles, with the highest level in mature needles. Only ClBFN1 showed a consistent expression pattern in the transition zone of the increment core. Radial section anatomy revealed that the length-to-width ratio of ray parenchyma nuclei decreased from the sapwood to the transition zone, and the nuclei of the ray parenchyma cells in the transition zone were shrunken and rounded, which are typical PCD hallmarks. Accordingly, ClBFN1 was designated as a molecular marker for the sapwood–heartwood transition zone in Chinese fir. Conclusion: Chinese fir possesses four BFN genes with conserved protein domains. During the heartwood formation, the family member ClBFN1 participates in PCD and responds specifically at the sapwood–heartwood transition zone, making it a molecular marker gene for the transition zone.

Key words: Cunninghamia lanceolata, heartwood formation, transition zone, bifunctional nuclease BFN