Genome-Wide Identification of the GS3 Gene Family and the Influence of Natural Variations in BnGS3-3 on Salt and Cold Stress Tolerance in Brassica napus

Abstract

Saline-alkali stress and cold damage significantly impact the yield of Brassica napus. G proteins play a crucial role in plant resistance to abiotic stresses, and research on G proteins in Brassica napus (rapeseed) is still in its early stages. In this study, we employed bioinformatics tools to systematically investigate the basic physicochemical properties, phylogenetic relationships, distribution, gene structure, cis-regulatory elements, and expansion patterns of the GS3 gene family in Brassica napus. Additionally, reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the response of the BnGS3-3 gene to salt and low-temperature stresses. Natural variations were found in the promoter region of BnGS3-3. By conducting a promoter-driven luciferase (LUC) assay, the relationship between natural variations in the BnGS3-3 promoter and salt and cold tolerance was analyzed. Furthermore, the impact of these natural variations on flowering time, root length, and yield was explored using phenotypic data from a population. Our research results aim to provide insights into the function and molecular mechanisms of BnGS3-3 in Brassica napus, and to offer valuable genetic resources for molecular breeding to improve salt and low-temperature tolerance in Brassica napus.

Keywords: Brassica napus; GS3; gene family; low temperature stress; natural variations; salt stress.

Figure 1

Identification of GS3 gene family members in Brassica napus. (a) Amino acid sequences of GS3 gene family members in Brassica napus, different colored lines represent the intervals of different motifs; (b) Chromosomal distribution of the 5 GS3 genes in Brassica napus; (c) Analysis of the conserved domains, motifs, and gene structure of the GS3 genes in Brassica napus. The unrooted tree was inferred using the MEGA X v11.0 with the Neighbor-Joining (NJ) algorithm based on BnGS3 amino acid sequences, and branch support was assessed through 1000 bootstrap replications.

Figure 2

Prediction of cis-acting elements in the GS3 gene family of Brassica napus. The colored boxes on the left represent different cis-acting elements, with the names of each element marked below, and the numbers on the right indicate the quantities of the corresponding cis-acting elements in the Brassica napus GS3 genes.

Figure 3

Phylogenetic analysis of the GS3 gene family in Brassica napus. All syntenic blocks in the Brassica napus genome are depicted by gray lines, while the red lines highlight the gene pairs of the five genes in the soybean GS3 gene family. The heatmap of genome-wide gene density, the bar chart of genome-wide gene density, and the chromosomes are demonstrated by rings from the inside to the outside, respectively. The bar in the lower right corner indicates the magnitude of gene density in the genome-wide gene density heatmap.

Figure 4

Tissue expression analysis of GS3 genes in Brassica napus. The heatmap was constructed using TPM values (Transcripts Per Million) of gene expression across various tissues with TBtools v2.136. The darker the red color, the higher the expression level.

Figure 5

Expression heatmap of GS3 genes in Brassica napus under (a) salt and (b) cold stress. The data were normalized using log₂^{(TPM-T+1/TPM-CK)}. Red: upregulated expression; Blue: downregulated expression.

Figure 6

Phenotype and relative expression of BnGS3-3 gene in two Brassica napus varieties under salt and cold stress. (a) Phenotype of two Brassica napus varieties after 7 days of 200 mM NaCl stress; (b) Relative expression of BnGS3-3 under 200 mM NaCl stress; (c) Field performance of the two Brassica napus varieties at the 7-leaf stage during the overwintering period; (d) Relative expression of BnGS3-3 under cold stress. Each treatment’s untreated group at corresponding time points was used as the control, with BnActin2 serving as the internal reference gene. Data values represent the average of three biological replicates. Each biological replicate includes three technical replicates. Error bars indicate the standard deviation of three biological replicates. Statistical significance was analyzed using Student’s t-test, ** p < 0.01, * p < 0.05.

Figure 7

Natural variation sites in the promoter of the BnGS3-3 gene. (a) Schematic diagram of the BnGS3-3 gene promoter structure. The numbers above indicate the physical locations of the variation sites on the chromosome. (b,c) Immediate expression of activity for the two BnGS3-3 promoter haplotypes after 16 h of 0 or 300 mM NaCl treatment. The LUC reporter gene is driven by each haplotype promoter. Photos were taken using an in vivo plant imaging system; the light intensity of b is shown in c. (d,e) Immediate expression of activity for the two BnGS3-3 promoter haplotypes after 45 min at 22 °C or 4 °C. The LUC reporter gene is driven by each haplotype promoter. Photos were taken using an in vivo plant imaging system; the light intensity in d is shown in e. For presentation of LUC intensity values, the proBnGS3-1^24W232 promoter haplotype under control conditions was selected as the reference control due to its minimal fluorescence intensity value. Data values represent the average of three biological replicates. Each biological replicate includes three technical replicates. Error bars indicate the standard deviation of three biological replicates. Statistical significance was analyzed using Student’s t-test, ** p < 0.01. (f,g) Boxplots of flowering time, root length, yield, yield under low-salt conditions, and yield under high-salt conditions for natural population materials carrying the natural variation sites C02_6706660 and C02_6706551. n refers to the number of samples carrying different haplotypes, respectively. Statistical significance was analyzed using a two-sided Wilcoxon test.