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

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.

Function of MYB8 in larch under PEG simulated drought stress.

Larch, a prominent afforestation, and timber species in northeastern China, faces growth limitations due to drought. To further investigate the mechanism of larch's drought resistance, we conducted full-length sequencing on embryonic callus subjected to PEG-simulated drought stress. The sequencing results revealed that the differentially expressed genes (DEGs) primarily played roles in cellular activities and cell components, with molecular functions such as binding, catalytic activity, and transport activity. Furthermore, the DEGs showed significant enrichment in pathways related to protein processing, starch and sucrose metabolism, benzose-glucuronic acid interconversion, phenylpropyl biology, flavonoid biosynthesis, as well as nitrogen metabolism and alanine, aspartic acid, and glutamic acid metabolism. Consequently, the transcription factor T_transcript_77027, which is involved in multiple pathways, was selected as a candidate gene for subsequent drought stress resistance tests. Under PEG-simulated drought stress, the LoMYB8 gene was induced and showed significantly upregulated expression compared to the control. Physiological indices demonstrated an improved drought resistance in the transgenic plants. After 48 h of PEG stress, the transcriptome sequencing results of the transiently transformed LoMYB8 plants and control plants exhibited that genes were significantly enriched in biological process, cellular component and molecular function. Function analyses indicated for the enrichment of multiple KEGG pathways, including energy synthesis, metabolic pathways, antioxidant pathways, and other relevant processes. The pathways annotated by the differential metabolites mainly encompassed signal transduction, carbohydrate metabolism, amino acid metabolism, and flavonoid metabolism.

Phylogeny and expression patterns of ERF genes that are potential reproductive inducers in hybrid larch.

BACKGROUND: Larch is an important component of northern forests and a major cultivated tree species in restoration of forest cover using improved seed material. In recent years, the continuous low seed production has severely affected the production of improved variety seedlings and natural regeneration. However, research on the reproductive growth of gymnosperms is extremely scarce. RESULTS: In this study, based on differential transcriptome analysis of two asexual reproductive phases, namely high-yield and low-yield, we further screened 5 ERF family genes that may affect the reproductive development of larch. We analyzed their genetic relationships and predicted their physicochemical properties. The expression patterns of these genes were analyzed in different tissues, developmental stages, hormone treatments, and environmental conditions in hybrid larch. CONCLUSION: The results showed that all 5 genes were induced by low temperature and ABA, and their expression patterns in different tissues suggested a suppressive role in the development of female cones in larch. Among them, LkoERF3-like1 and LkoERF071 may be involved in the flowering age pathway. This study enriches the scarce research on reproductive development in gymnosperms and provides a theoretical basis and research direction for regulating the reproductive development of larch in seed orchards.

Anatomical observation and transcriptome analysis of buds reveal the association between the AP2 gene family and reproductive induction in hybrid larch (Larix kaempferi × Larix olgensis).

Hybrid larch is an excellent afforestation species in northern China. The instability of seed yield is an urgent problem to be solved. The biological characteristics related to seed setting in larch are different from those in angiosperms and other gymnosperms. Studying the developmental mechanism of the larch sporophyll can deepen our understanding of conifer reproductive development and help to ensure an adequate supply of seeds in the seed orchard. The results showed that the formation of microstrobilus primordia in hybrid larch could be observed in anatomical sections collected in the middle of July. The contents of endogenous gibberellin 3 (GA3) and abscisic acid (ABA) were higher and the contents of GA4, GA7, jasmonic acid and salicylic acid were lower in multiseeded larch. Transcriptome analysis showed that transcription factors were significantly enriched in the AP2 family. There were 23 differentially expressed genes in the buds of the multiseeded and less-seeded types, and the expression of most of these genes was higher in the buds than in the needles. We conclude that mid-July is the early stage of reproductive organ development in hybrid larch and is suitable for the study of reproductive development. GA3 and ABA may be helpful for improving seed setting in larch, and 23 AP2/EREBP family genes are involved in the regulation of reproductive development in larch.

The diagnostic significance of 64-slice spiral CT combined with serological CA19-9, Bcl-2, CYFRA21-1 detection in thoracic esophageal carcinoma.

BACKGROUND: To analyze the clinical value of multi-slice spiral computed tomography (MSCT) combined with carbohydrate antigen 19-9 (CA19-9), B-cell leukemia/lymphoma-2 protein (Bcl-2), and cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) detection in the diagnosis of thoracic esophageal cancer. METHODS: The clinical data of 74 patients with thoracic esophageal cancer admitted to the Dazu District People's Hospital in Chonqing, China, from December 2019 to December 2020 were collected (esophageal cancer group), and their computed tomography (CT) signs were analyzed. Another 55 healthy people who underwent physical examination during the same period in the hospital were selected for the healthy group. The serum levels of CA19-9, Bcl-2, and CYFRA21-1 in the different populations were compared, using the receiver operating characteristic (ROC) curve to analyze the value of MSCT combined with CA19-9, Bcl-2, and CYFRA21-1 detection in the diagnosis of thoracic esophageal cancer. RESULTS: The serum levels of CA19-9, Bcl-2, and CYFRA21-1 in patients of the esophageal cancer group were significantly higher than those in the healthy group (P<0.05). The serum levels of CA19-9, Bcl-2, and CYFRA21-1 in patients with poorly differentiated, stage III-IV carcinoma and lymph node metastasis were significantly higher than in those patients with moderately well-differentiated, stage I-II carcinoma and no lymph node metastasis (P<0.05). The CT scans of patients in the esophageal cancer group showed esophageal walls with irregular, needle-shaped, circular, or localized eccentric thickening and narrowed lumens, which were dilated above the cancerous lesions. Some tumors compressed adjacent organs to deform and shift the organs, resulting in the disappearance of surrounding fat layers. Enhanced scans showed mild or moderate enhancement, with large-diameter lesions unable to enhance central, low-density, necrotic areas. The ROC curve showed that the area under the curve (AUC) and the sensitivity and specificity of MSCT combined with CA19-9, Bcl-2, and CYFRA21-1 detection were all higher than for esophageal lesions detected by individual indicators. CONCLUSIONS: CA19-9, Bcl-2, and CYFRA21-1, which are abnormally expressed in patients with esophageal cancer, may be related to the occurrence and development of esophageal cancer. MSCT combined with CA19-9, Bcl-2, and CYFRA21-1 detection appears to enhance the diagnosis of esophageal cancer.