-
Through bioinformatics analysis methods, a total of six CBL gene family members were identified (Fig. 1a) and named as PmCBL1~PmCBL6 (Table 1). These six CBL genes were unevenly distributed on the chromosomes. PmCBL1 was located on chromosome 1, PmCBL2 and PmCBL3 were located on chromosome 5, and PmCBL4, PmCBL5, and PmCBL6 were located on chromosome 7. In the PmCBL family, the coding sequence (CDS) of PmCBL6 was the longest, comprising 2,475 bp. PmCBL1 had the second-longest CDS with 2,418 bp, followed by PmCBL3 with 1,893 bp, PmCBL2 with 1,805 bp, PmCBL5 with 1,564 bp, and PmCBL4 with the shortest CDS of 1,252 bp. The subcellular localization prediction of these six genes was all on the cell membrane. Through analysis of conserved protein domains, it was found that PmCBLs contain six conserved motifs (Motif 1~6) (Fig. 1b). Specifically, PmCBL2, PmCBL3, PmCBL5, and PmCBL6 contained all six motifs (Motif 1~6), while PmCBL1 and PmCBL4 contained Motif 1~4 and Motif 6 but did not contain Motif 5. From the results of protein multiple sequence alignment among P. mume, A. thaliana, V. vinifera, and O. sativa, it could be observed that P. mume showed higher homology with A. thaliana and V. vinifera, while the homology with O. sativa was relatively lower (Fig. 2).
Figure 1.
Phylogenetic analysis, protein motif structure, chromosome localization, and gene structure analysis of PmCBL genes. (a) Phylogenetic analysis of PmCBL genes. (b) Protein motif structure of PmCBL genes. (c) Chromosome localization analysis of PmCBL genes. (d) Gene structure analysis of PmCBL genes.
Table 1. Members of the P. mume PmCBL gene family and their main molecular characteristics and information.
Gene name Gene ID Chromosome Position Subcelllar localization CDS (bp) Intron PmCBL1 Pm000366 Chr01 2287536~2289774 Cell membrane 2,418 5 PmCBL2 Pm018367 Chr05 17870110~17871915 Cell membrane 1,805 4 PmCBL3 Pm019754 Chr05 25555186~25557079 Cell membrane 1,893 5 PmCBL4 Pm024130 Chr07 10584318~10585570 Cell membrane 1,252 4 PmCBL5 Pm024134 Chr07 10621981~10623545 Cell membrane 1,564 5 PmCBL6 Pm024135 Chr07 10622516~10628186 Cell membrane 2,474 5 Figure 2.
Protein amino acid sequence comparison diagram. (a) Protein amino acid sequence comparison diagram between P. mume and A. thaliana. (b) Protein amino acid sequence comparison diagram between P. mume and V. vinifera. (c) Protein amino acid sequence comparison diagram between P. mume and O. sativa.
Physicochemical property analysis of the P. mume CBL gene family
-
The physicochemical properties of the six CBL homologous sequences were analyzed (Table 2). The results showed that the amino acid (aa) numbers of the PmCBL protein sequences ranged from 186 (PmCBL4) to 226 (PmCBL1), with molecular weights (MV) ranging from 21.66 kDa (PmCBL4) to 26.05 kDa (PmCBL1). The theoretical isoelectric points (pI) ranged from 4.57 to 5.14. The instability index values ranged from 35.97 to 49.06, with only PmCBL1 having an instability index below 40, indicating high stability of CBL protein. The grand average of hydropathicity (GRAVY) values for PmCBL1 ~ PmCBL6 were all negative, indicating that these six proteins were hydrophilic. The aliphatic index values ranged from 90.09~96.96, indicating that all six proteins were lipophilic. None of the six gene family members contained signal peptides or transmembrane helices.
Table 2. Physicochemical properties of P. mume CBL gene family members.
Protein name Gene ID Number of amino acids Molecular weight Thereotical pI Instability index Signal peptide PmCBL1 Pm000366 226 26052.71 4.82 49.06 NO PmCBL2 Pm018367 217 24903.52 5.14 35.97 NO PmCBL3 Pm019754 212 24429.82 4.73 46.65 NO PmCBL4 Pm024130 186 21665.75 4.6 40.14 NO PmCBL5 Pm024134 213 24720.17 4.57 40.98 NO PmCBL6 Pm024135 218 25144.71 4.86 46.47 NO Evolutionary relationship analysis of the CBL gene family in P. mume
-
To understand the evolutionary relationship between members of the P. mume CBL gene family, a phylogenetic tree was constructed using the selected six PmCBL genes, 17 OsCBLs genes from O. sativa, 16 VvCBLs genes from V. vinifera, 20 NtCBLs genes from N. tabacum and 10 AtCBLs genes from the model plant A. thaliana (Fig. 3). The results of the evolutionary analysis showed that the CBL gene family could be divided into three groups, each containing among them, P. mume Group I included PmCBL2, PmCBL3, PmCBL4, PmCBL5, and PmCBL6. Group III consisted of PmCBL1. In Group I, PmCBL4 and PmCBL5 were paralogous gene pairs. This suggested that the CBL gene in P. mume underwent expansion, expansion and replication. Some genes in AtCBLs, NtCBLs, VvCBLs and PmCBLs could be considered as orthologous gene pairs, such as AtCBL4 and PmCBL3, NtCBL14 and PmCBL2, VvCBL8 and PmCBL6. The discovery of orthologous gene pairs suggests the existence of ancient CBL genes before P. mume and A. thaliana, N. tabacum, V. vinifera were also similar.
Figure 3.
Phylogenetic tree of P. mume, A. thaliana, O. sativa, N. tabacum and V. vinifera. The green checkmark represents V. vinifera, the black square represents O. sativa, the blue circle represents A. thaliana, the red star represents N. tabacum, and the purple triangle represents P. mume.
Analysis of gene structure and chromosome localization of the P. mume CBL gene family
-
Further analysis of the gene structure of the P. mume CBL gene family revealed that the six PmCBL genes share a similar overall gene structure (Fig. 1d). Specifically, PmCBL3 had eight exons separated by introns. PmCBL4 had seven exons with approximately equal sizes separated by introns. PmCBL2 had six exons separated by introns, with two exons being relatively far apart. PmCBL5 had eight exons separated by introns, and the sizes of these eight exons were roughly similar with a similar distance between each pair of exons. PmCBL6 had eight exons separated by introns, with some exons having larger inter-exon distances. PmCBL1 also had eight exons separated by introns, and similarly, some exons had larger inter-exon distances.
Based on the results from the MG2C online tool (Fig. 1c), the six identified P. mume CBL genes were found to be located on three chromosomes. PmCBL2 and PmCBL3 were located on one chromosome, PmCBL4, PmCBL5, and PmCBL6 were located on another chromosome, and PmCBL1 was located on a separate chromosome.
Analysis of cis-acting elements in the P. mume CBL genes
-
In order to further investigate the potential functional roles of PmCBL genes, the 700 bp sequence upstream of the start codon of each PmCBL gene was extracted as its promoter region. Cis-acting element analysis was performed on this region, focusing on important elements that had been extensively studied and were associated with plant growth and development, as well as stress responses. (Table 3, Fig. 4) The results showed that a total of 17 cis-acting element types responsive to plant hormones and stress were identified, and there were differences in the types and quantities of elements among different genes. Among them, all genes contained common cis-acting elements (CAAT-box) in their promoter and enhancer regions, with slight differences in the number of elements ranging from 2~6. PmCBL3 had the highest number of six (CAAT-box elements) , while PmCBL4 had the lowest two. Two cis-acting regulatory elements involved in MeJA response (CGTCA-motif and TGACG-motif) were found, and five genes contained both of these elements, with an equal number in each gene. Five genes contained the core promoter element (TATA-box) located around the transcription start site −30, namely PmCBL1 ~ PmCBL5, but there was a large variation in the number of elements among the members, ranging from 4~11. PmCBL1 had one light-responsive element (GT1-motif), while the other genes did not contain it. There were four light-responsive elements identified: PmCBL1, PmCBL4, and PmCBL5 each contained one GATA-motif, PmCBL2 contained one I-box, PmCBL1 contained one TCCC-motif, and PmCBL3 contained one LAMP-element. Two cis-acting regulatory elements involved in zein protein metabolism regulation were found: O2-site and MBS. PmCBL3, PmCBL4, and PmCBL5 each contained one O2-site, PmCBL6 contained two O2-sites, and PmCBL4 and PmCBL6 each contained one MBS. One cis-acting regulatory element involved in light response (G-box) was identified, with PmCBL4 and PmCBL5 each containing one. One cis-acting regulatory element required for anaerobic induction ARE was found, with PmCBL2 and PmCBL5 each containing one. One cis-acting regulatory element (A-box) involved in cis-element regulation was found, with PmCBL5 and PmCBL6 each containing one. One cis-acting element involved in abscisic acid response ABRE was found, with PmCBL4 and PmCBL5 each containing one. One cis-acting element involved in auxin response (TGA-element) was found in PmCBL6. One cis-acting element involved in salicylic acid response (TCA-element) was found in PmCBL4.
Table 3. Analysis of cis-acting elements in the P. mume CBL gene family members.
Gene PmCBL1 PmCBL2 PmCBL3 PmCBL4 PmCBL5 PmCBL6 Gene ID Pm000366 Pm018367 Pm019754 Pm024130 Pm024134 Pm024135 CAAT-box 3 4 6 2 4 4 CGTCA-motif 2 1 1 2 1 TGACG-motif 2 1 1 2 1 TATA-box 4 4 4 11 8 GT1-motif 1 TCCC-motif 1 GATA-motif 1 1 1 ARE 1 1 G-box 1 1 MBS 1 1 TGA-element 1 A-box 1 1 I-box 1 O2-site 1 1 1 2 LAMP-element 1 ABRE 1 1 TCA-element 1 CAAT-box was a common cis-acting element in the promoter and enhancer regions. CGTCA-motif/TGACG-motif was cis-acting regulatory elements involved in MeJA response. TATA-box was a core promoter element located around the transcription start site (-30). GT1-motif was a light-responsive element. TCA-element was a cis-acting element involved in salicylic acid response. TGA-element was an element involved in auxin response. ABRE was a cis-acting element involved in abscisic acid response. A-box was a cis-acting regulatory element. ARE was a cis-acting element required for anaerobic induction. G-box was a cis-acting element involved in light response. O2-site/MBS was cis-acting regulatory elements involved in zein protein metabolism regulation. TCCC-motif/GATA-motif/I-box/LAMP-element was part of light-responsive elements. Gene expression of the P. mume CBL gene family
-
By analyzing transcriptome data from different parts of the P. mume, tissue-specific expression patterns were observed in different members of the P. mume CBL gene family (Fig. 5). PmCBL1 showed higher expression levels in flower buds, PmCBL2 exhibited higher expression levels in roots, PmCBL3 and PmCBL6 had higher expression levels in leaves, and PmCBL4 and PmCBL5 showed higher expression levels in stems. From this, it could be observed that PmCBL3 and PmCBL6 had similar expression patterns, while PmCBL4 and PmCBL5 had similar expression patterns. This suggested that they belonged to the same Group I subfamily.
By analyzing the transcriptome data of P. mume during wintering, the wintering process of P. mume could be divided into three stages: the early stages of overwintering, which was November (DD); midwinter, which was December (NDD); late overwintering which was January (LD); and naturalness, which was February (NF). The expression patterns of the P. mume CBL gene family could be obtained (see Fig. 6), with different genes showing different expression patterns. PmCBL1 showed an upregulation trend in all three stages compared to the NF stage, reaching its peak in the LD stage, with expression levels 1.23 times higher than the NF stage. PmCBL2 exhibited both upregulation and downregulation trends in the three stages compared to the NF stage. It reached its maximum expression level in the NDD stage, being 1.22 times higher than the NF stage. However, it reached its minimum expression level in the LD stage, showing a downregulation of 1.3 times compared to the NF stage. Overall, PmCBL2 displayed a downregulation trend. PmCBL3 showed an upregulation trend in all three stages compared to the NF stage, with the highest expression level observed in the DD stage, being 1.06 times higher than the NF stage. PmCBL4 showed a downregulation trend in all three stages compared to the NF stage, with the minimum expression level observed in the DD stage, showing a downregulation of 1.20 times compared to the NF stage. PmCBL5 exhibited a downregulation trend relative to the NF stage, reaching its minimum expression level in the NDD stage, showing a downregulation of 8.28 times compared to the NF stage. PmCBL6 showed an upregulation trend relative to the NF stage, with an upregulation of 1.42 times compared to the NF stage. PmCBL4 and PmCBL5 were known to had significant roles in the wintering process of P. mume.
Figure 6.
The expression pattern of PmCBLs genes during overwintering. DD, November; NDD, December; LD, January; NF, February.
Quantitative analysis of gene expression
-
To further investigate the expression patterns of PmCBL genes in response to low temperature stress, qRT-PCR experiments were conducted to examine the expression levels of PmCBLs under cold treatment. During the treatment at 4 °C, the expression levels of PmCBL genes showed an upregulation or downregulation trend over a 24-h time course (Fig. 7). PmCBL1, PmCBL2, PmCBL3, PmCBL5, and PmCBL6 showed an upregulation trend in their expression levels. Among them, PmCBL1 and PmCBL5 exhibited the highest expression levels at 24 h, being 12.18-fold and 50.11-fold higher than the pre-treatment levels, respectively. PmCBL2 and PmCBL6 reached their maximum expression levels at 6 h, with a 5.50-fold and 8.90-fold increase, respectively, compared to the pre-treatment levels. PmCBL3 showed the highest expression level at 12 h, which was 11.22-fold higher than the pre-treatment level. PmCBL4 showed a downregulation trend compared to the pre-treatment levels.
-
All data generated or analyzed during this study are included in this published article.
-
About this article
Cite this article
Liu H, Hao L, Zhang X, Zhang Y, Wang H, et al. 2024. Identification of the Calcineurin B-like gene family and gene expression patterns in response to low temperature stress in Prunus mume. Tropical Plants 3: e010 doi: 10.48130/tp-0024-0010
Identification of the Calcineurin B-like gene family and gene expression patterns in response to low temperature stress in Prunus mume
- Received: 22 October 2023
- Accepted: 27 February 2024
- Published online: 03 April 2024
Abstract: The CBL gene family is an important family in the Ca2+ mediated signal transduction pathway in plants and plays a crucial role in plant stress responses and growth development. However, research on the response of members of the Prunus mume CBL gene family to low temperature stress remains scarce. In this study, we systematically analyzed the protein physicochemical properties, chromosome localization, phylogenetic evolution, gene structure, conserved domains, cis-acting elements, and gene expression patterns in response to low temperature stress of members of the P. mume CBL gene family using bioinformatics tools. Six PmCBL gene family members were identified in the P. mume genome. Phylogenetic trees were constructed, revealing three subfamilies named Group I, Group II, and Group III. In the P. mume gene family, PmCBL4 and PmCBL5 were paralogous genes. The members of the P. mume CBL gene family were unevenly distributed on three chromosomes. The CBL encoding protein, the number of isoelectric points (pI), the number of introns and exons of the six gene families were different. Analysis of the upstream 700 bp promoter sequences of the P. mume CBL gene family revealed the presence of various types of cis-acting elements involved in non-biological stress responses. Among the six identified genes, each gene exhibited different expression patterns in response to low temperature. Among them, the up-regulated expression of PmCBL5 was the largest, and the expression of PmCBL1, PmCBL3 and PmCBL5 showed the up-regulated trend. These results indicated that PmCBL1, PmCBL3, PmCBL5, and PmCBL6 were key genes involved in the response of P. mume to low temperature stress. This study provided comprehensive and systematic analysis of the P. mume CBL gene family members and identified key genes involved in the response to low temperature stress, thereby providing genetic resources for molecular breeding programs aimed at enhancing cold resistance in P. mume.
-
Key words:
- Prunus mume /
- CBL gene family /
- Low temperature stress /
- Gene identification