Isolation and functional analysis of the Larix olgensis LoNAC3 transcription factor gene

Abstract

Background: Larch is an important timber tree species. The traditional methods of tree genetic breeding have been progressing slowly. It is necessary to carry out gene function analysis and genetically modified breeding research. The NAC transcription factor family is a plant-specific transcription factor family with various biological functions, as shown in recent research. However, there are few studies on the NAC gene among gymnosperm coniferous species.

Results: LoNAC3 with complete cds was identified and isolated from the cDNA of Larix olgensis based on transcriptome data. The cDNA length of LoNAC3 is 1185 bp, encoding 394 amino acids, with a conserved NAM domain located at the N-terminus, and subcellular localization in the nucleus. The results of real-time quantitative PCR analysis showed that at different growth stages and in different tissues of L. olgensis, the relative expression level of LoNAC3 was highest in the needles. After drought, salt, alkali stress and hormone treatment, expression was induced to different degrees. The expression level of LoNAC3 was significantly increased under drought and salt conditions. The relative expression level changed under methyl jasmonate (MeJA) and abscisic acid (ABA) treatment. By observing the phenotype of overexpressed LoNAC3 tobacco, it was found that overexpressed tobacco is shorter and blooms earlier than wild-type tobacco. Under abiotic stress, LoNAC3 overexpressed tobacco has lower germination rates and poorer growth status. Transgenic tobacco under stress treatment has a higher malondialdehyde (MDA) content than wild-type tobacco, while peroxidase (POD) activity is lower than wild-type tobacco.

Conclusions: Through the analysis of LoNAC3 sequence and promoter expression, it can be concluded that LoNAC3 is involved in the drought and salt stress response processes of L. olgensis, and is induced by ABA and MeJA expression. Overexpression of LoNAC3 leads to stunted tobacco growth and negatively regulates its tolerance to drought and salt stress through the reactive oxygen species pathway. The preliminary analysis of the expression pattern and function of the LoNAC3 can provide a theoretical basis and high-quality materials for genetic improvement of larch in later stages.

Keywords: Larix olgensis; LoNAC3; Bioinformatics analysis; Expression pattern; Functional validation.

Fig. 1

Bioinformatics analysis of LoNAC3. A Subgroup classification analysis of LoNAC3. B The phylogenetic tree and motif analysis of LoNAC3

Fig. 2

Analysis of LoNAC3 promoter and gene expression patterns. A The cis-acting elements in LoNAC3 promoter sequence. B The results of tobacco GUS staining. C Expression analysis of LoNAC3 in different growth stages of L. olgensis. D Expression analysis of LoNAC3 under different stress treatment. E Expression analysis of LoNAC3 under different hormone treatment

Fig. 3

Obtaining and phenotypic observation of overexpressed tobacco. A Co-cultivation. B Induced resistant differentiated buds. C Rooted tissue culture seedlings. D Transplanted into soil. E Selection of transgenic tobacco seed resistance (25mg.L⁻¹ Hyg). F PCR detection of VB191104-LoNAC3 transgenic tobacco, M: DL2000 Marker, 1: negative control (DNA of wild-type tobacco), 2: positive control (Agrobacterium tumefaciens of VB191104-LoNAC3), 3 ~ 11: DNA of OE1 ~ OE9(The original, unprocessed gel image can be found in the additional file.). G Phenotype of tobacco. H Relative expression levels of VB191104-LoNAC3 transgenic tobacco. I Plant height of tobacco with different asexual strains. J Ground diameter of tobacco with different asexual strains. Significance was determined by the least significant difference, asterisks indicate statistically significant differences from WT (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 4

Analysis of stress resistance in genetically modified tobacco seeds. A Sowing tobacco seeds in 1/2MS medium containing 0.2 M mannitol for 10 days. B Sowing tobacco seeds in 1/2MS medium containing 0.1 M NaCl for 10 days. C The germination rate of Tobacco in 1/2MS medium with 0.2 M mannitol. D The germination rate of Tobacco in 1/2MS medium with 0.1 M NaCl. E-F Root length of tobacco under stress treatment. Significance was determined by the least significant difference, asterisks indicate statistically significant differences from WT (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

Analysis of stress resistance in tobacco. A Determination of POD activity and MDA content in various tobacco strains before and after treatment. B DAB and NBT staining of tobacco leaves before and after treatment. C Detection of ROS-related gene expression levels before and after treatment. Significance was determined by the least significant difference, asterisks indicate statistically significant differences from WT (*P < 0.05, **P < 0.01, ***P < 0.001)