| Masson pine(Pinus massoniana Lamb.)is a unique coniferous species in China,characterized by rapid growth,strong adaptability,wide distribution and versatile uses.It is an important tree species for both timber and rosin production,as well as a pioneer in afforestation efforts in southern China.It is crucial to enhance the yield and quality of Masson pine wood while cultivating superior wood varieties.However,traditional breeding methods are long time cycle and may not meet the demand for high-quality materials in a timely manner.Therefore,modern molecular technology can aid in achieving the goal of high-yield and high-quality plantations.It has been demonstrated that the NAC transcription factor(TF)plays a crucial regulatory role in the biosynthesis and deposition of secondary cell walls.Therefore,investigating the NAC TF in Masson pine and exploring its regulatory network for secondary wall formation can provide valuable reference information for enhancing wood properties of this species.In this study,the NAC gene family was analyzed in the transcriptome of Masson pine to identify potential regulators of wood formation,leading to the discovery of PmSND4 as a putative TF involved in this process.The main research findings are presented below:1.The phylogenetic tree of the PmSND4 gene and Arabidopsis thaliana was constructed,revealing that PmSND4 belongs to the ONAC3 subgroup of NAC TFs in Masson pine.Tissue-specific quantitative analysis demonstrated that PmSND4 expression is not limited to specific tissues but rather highly expressed in mature needle and stems.The GUS overexpression vector was constructed to drive the expression of this gene promoter,and its localization was observed.The results further indicated that this gene exhibited no tissue specificity.Subcellular localization analysis revealed that the TF was localized in the nucleus.Yeast autoactivation assay demonstrated that PmSND4 lacked transcriptional autoactivation.2.By analyzing the codon preference of PmSND4,the poplar transformation receptor material was selected,and the PmSND4 gene was optimized according to the usage pattern of the poplar codon.Leaf disk method was used to transform the optimized PmSND4 into poplar,and transgenic materials were obtained by RT-PCR and qRT-PCR.Phenotypic analysis of overexpressed plants showed that compared with the wild type,the plant height and stem diameter of transgenic plants increased,and the leaf size also increased to a certain extent.By sectioning,staining and electron microscopy scanning of the stem segments between the 9th segments,it was found that the cell wall thickness of the overexpressed strain increased,and the lignin deposition increased significantly.The content of lignin and hemicellulose increased significantly,but the content of cellulose did not change significantly.It is speculated that PmSND4 can participate in the biosynthesis of secondary walls.3.The promoter of PmMYB4 was cloned,and its promoter was divided into 7 sequences according to the SNBE site.Yeast single hybridization and double luciferase assay were conducted with PmSND4,respectively,to explore the function of PmSND4 gene in the secondary wall regulatory network.It was found that PmSND4 could bind to the PmMYB4 promoter,and that the PmMYB4 promoter containing two SNBE element sites had stronger binding ability to PmSND4.In the promoter sequence containing one SNBE element,the SNBE site near the open reading frame had stronger binding ability.Therefore,it is speculated that PmSND4 can bind to the promoter of PmMYB4 to participate in the regulation of secondary wall biosynthesis.In conclusion,PmSND4 is involved in the regulation of plant secondary wall by binding to the promoter of PmMYB4,and by this way,it activates PmMYB4 to promote the biosynthesis of secondary wall and ultimately achieve wood formation. |
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