Genome-wide identification and expression analysis of the GASA gene family in Chinese cabbage (Brassica rapa L. ssp. pekinensis)
BMC Genomics volume 24, Article number: 668 (2023)
The Gibberellic Acid-Stimulated Arabidopsis (GASA) gene family is widely involved in the regulation of plant growth, development, and stress response. However, information on the GASA gene family has not been reported in Chinese cabbage (Brassica rapa L. ssp. pekinensis).
Here, we conducted genome-wide identification and analysis of the GASA genes in Chinese cabbage. In total, 15 GASA genes were identified in the Chinese cabbage genome, and the physicochemical property, subcellular location, and tertiary structure of the corresponding GASA proteins were elucidated. Phylogenetic analysis, conserved motif, and gene structure showed that the GASA proteins were divided into three well-conserved subfamilies. Synteny analysis proposed that the expansion of the GASA genes was influenced mainly by whole-genome duplication (WGD) and transposed duplication (TRD) and that duplication gene pairs were under negative selection. Cis-acting elements of the GASA promoters were involved in plant development, hormonal and stress responses. Expression profile analysis showed that the GASA genes were widely expressed in different tissues of Chinese cabbage, but their expression patterns appeared to diverse. The qRT-PCR analysis of nine GASA genes confirmed that they responded to salt stress, heat stress, and hormonal triggers.
Overall, this study provides a theoretical basis for further exploring the important role of the GASA gene family in the functional genome of Chinese cabbage.
Gibberellic acid (GA) is a ubiquitous plant hormone that regulates plant growth and development . In particular, DELLA protein is a key factor in the gibberellin signaling pathway that contributes to the regulation of plant growth and development processes, including epidermal hair differentiation , flower development , anther development and flowering , stress response , and root growth . Gibberellic acid-stimulated Arabidopsis (GASA), which is also known as Snakin, is downstream of DELLA and is a type of cysteine-rich peptide (CRP) . Notably, most GASA genes are regulated by GA .
In Arabidopsis, GASA proteins typically consist of 80–270 amino acids, except AtGASA14, which has a proline-rich protein (PRP) motif in the N-terminal region . The GASA proteins have three different domains: (1) an N-terminal signal peptide with 18–29 amino acids; (2) a highly variable hydrophilic region with 7–31 polar amino acid residues displaying a difference between family members both in amino acid composition and sequence length; and (3) a C-terminal GASA domain consisting of 60 amino acids, typically including 12 cysteine residues. The C-terminal GASA domain is considered a key region for maintaining the spatial structures and functions of the GASA proteins [10, 11]. The tomato gene, gibberellin-stimulated transcript 1 (GAST-1), is the first member of the GASA family to be identified , and many other genes have been identified in different species thus far [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]. With the identification of members of the GASA gene family across different species, the functions of the gene family have also been comprehensively mapped.
The GASA gene family is involved in the regulation of plant growth and development. In Arabidopsis, AtGASA4 regulates branching, floral meristems, floral organ identity, and seed growth [30, 31]. AtGASA5 is a negative regulator of flowering and stem growth . AtGASA6 can affect flowering, cell elongation and seed germination [33, 34]. AtGASA10 also affects anther and seeds . AtGASA14 controls blade expansion . The GIP (encoding the Petunia hybrida GA-induced protein), a homolog of GAST-1, inhibits flowering and stem elongation . The Gerbera GEG gene can regulate cell elongation and petal development . The rice gene, OsGASR, and the wheat gene, TaGASA7, can regulate grain size and length [38, 39]. OsGASR1 and OsGASR2 can affect panicle differentiation in rice , and TaGASR34 can affect the dormancy and germination of wheat seeds . In maize, the GASA gene family can affect lateral root development [20, 41]. Silencing the potato’s snakin-1 gene affects cell activity and changes leaf morphology . The GASA gene family in apples is involved in flower induction . VvGASA7 positively regulates seed size and yield . In strawberry, FaGAST1 and FaGAST2 synergistically regulate fruit cell development and affect fruit size . In pear, PpyGAST1 regulates bud dormancy . In the traditional Chinese medicinal plant, Salvia miltiorrhiza, SmGASA4 promotes the development of roots and flowers .
The GASA gene family also responds to biotic and abiotic stress in plants. The overexpression of AtGASA5 negatively regulates heat tolerance in Arabidopsis thaliana , while AtGASA14 controls plant resistance to abiotic stress . Leaf expression of the gerbera gene, PRGL (a GAST1-like gene), is induced by injury . Rice contains multiple GASA genes that respond to abiotic stress . TaGASR1 improves wheat tolerance to heat and oxidative stress . The overexpression of snakin-1 increases potato resistance to fungal and bacterial diseases, and these findings have been verified in lettuce, tomato, and Peltophorum dubium [47,48,49,50]. The GASA gene in rubber tree plays a role in fungal pathogen resistance .
Although many studies have been conducted on GASA genes across various species, such studies have not been conducted in the context of the Chinese cabbage. The Chinese cabbage is a nutrient-rich cruciferous crop that originates from China. Evolutionarily, the Chinese cabbage is closely related to the model plant, A. thaliana. Fifteen GASA genes have been identified in A. thaliana, and their functions have been verified . Thus, this study was based on the homology between Chinese cabbage and A. thaliana. Briefly, herein, we identified members of the GASA gene family at the level of the whole-genome of Chinese cabbage and conducted detailed bioinformatics analysis, including chromosome location and gene structure, sequence homology, evolutionary history, synchrony analysis, cis-acting element analysis, protein structure analysis, and subcellular localization. In addition, expression differences and stress responses of the members of the GASA gene family in different parts of Chinese cabbage were clarified, laying a foundation for further studies of GASA family genes in Chinese cabbage.
Results and discussion
Genome-Wide identification and protein features of GASA genes in Chinese cabbage
In this study, 15 GASA genes were identified in the genome network of the Chinese cabbage (Table 1). They were named BrGASA1 to BrGASA15, according to the top-to-bottom position of chromosomes A01-A10. BrGASA genes were unevenly distributed across seven chromosomes of the Chinese cabbage genome (Fig. 1). Specifically, three BrGASA genes were found on chromosomes A01, A02, and A09; two on chromosomes A03 and A08; and only one BrGASA gene was found on chromosomes A06 and A10. In previous reports, the GASA genes were found to be randomly distributed in the chromosomes of species such as Arabidopsis , whereas they were found to be unevenly distributed in those of potato, apple, grapevine, Zea mays, Glycine max, Populus [20, 23, 26,27,28, 42]. Previous reports have shown that the Brassica ancestors experienced extensive gene loss after the Whole-Genome Triplication (WGT) event . Therefore, we believe that the uneven distribution of GASA gene on the chromosomes of the Chinese cabbage genome is closely related to gene loss. Protein characteristics, including molecular weight, isoelectric point, instability index, grand average of hydropathicity (GRAVY), major amino acid content, and aliphatic index, were analyzed using the ExPASy program (Table 2). The number of amino acids in the BrGASA protein was between 64 and 283, with BrGASA7 encoding the longest protein with highest molecular weight (30.17 kDa), and BrGASA2 encoding the shortest protein with lowest molecular weight (7.14 kDa). The maximum difference in molecular weight between BrGASA2 and BrGASA7 suggests that there could be structural and functional differences between these two genes . The average length of the BrGASA proteins was 124 amino acids, whereas the average molecular weight was 13.35 kDa. Overall, BrGASA was a low-molecular-weight protein, consistent with the results of Arabidopsis . Furthermore, the isoelectric point ranged from 6.67 (BrGASA1) to 10.14 (BrGASA7), and the instability index ranged from 26.86 (BrGASA2) to 66.48 (BrGASA4). According to the GRAVY values, Chinese cabbage GASA proteins were hydrophilic. Meanwhile, the aliphatic index values of Chinese cabbage GASA proteins ranged from 27.08 (BrGASA2) to 35.57 (BrGASA1). The main amino acid residues of Chinese cabbage GASA proteins were Cys, Lys, and Gly. In apple, the main amino acid residues of GASA proteins were mainly Cys, Lys and Leu . Predicting the subcellular locations of proteins can provide important clues regarding gene function . In our prediction, the Chinese cabbage GASA proteins were mainly located in the extracellular environment, besides Golgi apparatus, chloroplast, cytoplasm, etc. (Table 2). The localization results of GASA proteins in cotton indicated that they were mostly located extracellular, while a few were in the nucleus and plasma membrane . Not all GASA proteins in potato were located extracellular, and the localization results showed differences. The signal of GASA-GFP fusion protein in rubber tree showed that all proteins could signal in the cytoplasm , while GASA protein in Populus was found in four positions: Golgi apparatus, cell wall, cell membrane and nucleus . The tertiary structure of proteins facilitates the accurate characterization of protein functions . Based on predictions of the tertiary spatial structures of the Chinese cabbage GASA gene family, we found that the protein structures mainly consisted of random coils and α-helix composition, but the β-fold structures were also present (Fig. 2). The similar structural characteristics have also been found in the GASA proteins of apple, grape, poplar, and cotton [23, 27,28,29].
Analysis of phylogenetic relationship, Gene structure and conserved motifs of GASA Proteins in Chinese Cabbage
Phylogenetic analysis can help us understand the evolutionary relationships among genes . To classify the GASA gene family, we constructed a phylogenetic tree based on the GASA protein sequences of Arabidopsis and Chinese cabbage (Fig. 3). The analysis included 30 GASA proteins: 15 from Chinese cabbage and 15 from Arabidopsis. There was a similarity between the GASA genes of Chinese cabbage and Arabidopsis, indicating that these genes might also have functional similarities. As shown in Fig. 3, the proteins were divided into three groups, named subfamily A, B, and C, consisting of 10, 9, and 11 GASA proteins, respectively. Five Chinese cabbage proteins (i.e., BrGASA1, BrGASA3, BrGASA4, BrGASA5, and BrGASA10) and five Arabidopsis proteins (i.e., AtGASA4, AtGASA5, AtGASA6, AtGASA12, and AtGASA15) clustered in subfamily A, while five Chinese cabbage proteins (i.e., BrGASA2, BrGASA8, BrGASA9, BrGASA11, and BrGASA12) and four Arabidopsis proteins (i.e., AtGASA7, AtGASA8, AtGASA10, and AtGASA14) clustered in subfamily B. Five Chinese cabbage proteins (i.e., BrGASA6, BrGASA7, BrGASA13, BrGASA14, and BrGASA15) and six Arabidopsis proteins (i.e., AtGASA1, AtGASA2, AtGASA3, AtGASA9, AtGASA11, and AtGASA13) clustered in subfamily C. In previous studies, Arabidopsis GASA proteins were divided into three subfamilies based on its own homology . Subsequently, phylogenetic trees constructed by other species based on the Arabidopsis GASA proteins were also divided into three subfamilies, such as maize, rice, apples, wheat, soybeans, grapes, and poplars [20, 23, 26,27,28, 40].
An unrooted tree was constructed to explore further the phylogenetic relationships of the GASA proteins in the Chinese cabbage. Similar to the above-described phylogenetic tree data, the Chinese cabbage GASA proteins were divided into three groups in this new analysis (Fig. 4A). The number, location, and length of the exons are closely related to gene homology, and research on exons and introns indicate differences in structure and function between genes [28, 53]. To illustrate the diversity of GASA genes in the Chinese cabbage, we compared the arrangement of introns and exons according to their phylogenetic relationships (Fig. 4B). The BrGASA genes in subfamily A (green) and C (pink) had 3–4 exons, whereas the BrGASA genes in subfamily B (blue) had 1–2 exons. The number of exons of the BrGASA gene in the same subfamily was similar, which indicated that the structure of these genes was conserved and that there was a close evolutionary relationship between genes [28, 40]. Subfamily A (green) was more conserved in gene structure than the other subfamilies, which indicated that the production rate of introns in these genes was higher at the early stage of evolution stage . Other subfamilies might have acquired exon during evolution, resulting in the difference in the number of exon-intron. In studies of other species, the number and structure of exons in the same gene subfamily also exhibited similarities, such as in apple, grape, poplar, and potato [23, 27, 28, 55]. On the other hand, in monocotyledonous wheat, the GASA gene had the same number (2–4) and structure of exons . These results further verified the homology in the phylogenetic analysis. The predicted motifs of BrGASA proteins were highly conserved with three structural motifs at the C-terminus, consisting of motifs 1, 2, and 3 (Fig. 4C). Homologous BrGASA genes also had similar protein structures, such as BrGASA8/BrGASA12 in subfamily B; and BrGASA7/BrGASA15, BrGASA6/BrGASA13 in subfamily C. Four and 10 motifs were found in apple and poplar GASA proteins, respectively, and proteins from the same subfamily had similar length and number [23, 28]. In rubber tree, GASA proteins had 10 motifs, of which three motifs existed in all proteins, whereas some proteins had specific motifs, indicating differences in protein function .
Evolutionary relationships and collinearity analysis among GASA genes
According to our results, the 15 BrGASA genes were randomly distributed on 7 of the 10 chromosomes of Chinese cabbage (Fig. 1). Three whole-genome duplication (WGD) pairs of BrGASA genes (BrGASA7/15, BrGASA8/12, and BrGASA9/11) were distributed across five chromosomes (Fig. 5, Table S1). Synteny analysis of the GASA genes in the Chinese cabbage and Arabidopsis genomes showed that most of the GASA genes in the Chinese cabbage were homologous to those in the Arabidopsis genome (Fig. 6, Table S2). Most homologous genes on the seven chromosomes hosting the GASA genes were located on chromosome 1 of Arabidopsis (five genes), followed by chromosome 5 (three genes). Finally, two genes were located on chromosome 2, with one homologous gene on chromosomes 3 and 4.
Gene replication promotes the evolution of the genome and genetic system as well as the diversity of gene structure and function in the gene family . Four gene replication modes can help in the evolution of gene families, namely: WGD, tandem duplication, segmental duplication, and transposition duplication (TRD) . To illustrate the expansion pattern of BrGASA genes, we analyzed duplication events in the genomes of Chinese cabbage (Table 3). Six genes made up three WGD pairs (BrGASA7/15, BrGASA8/12, and BrGASA9/11), which indicated that they all had a common ancestor. In addition to WGD duplication, four pairs of TRD, BrGASA10/5, BrGASA13/6, BrGASA1/5, and BrGASA2/8 were observed among seven genes. This indicates that both WGD and TRD contributed to expanding of the GASA gene family in the Chinese cabbage. In general, WGD is believed to contribute significantly toward the evolution of morphological and physiological diversity, and TRD is important within the context of single-gene replication . New genes evolve through selection and mutation . Studies on other species have shown that the GASA gene in wheat underwent large scale duplication or tandem duplication events . The subfamily of cotton GASA genes that has underwent WGD replication was highly conserved during evolution . Segmental duplication was a common replication method in the GASA gene family, which has been found in the results of apple, soybean, and grape [23, 26, 27]. The ratio of non-synonymous (Ka) to synonymous (Ks) can be used to describe the evolution history . In our study, the Ka/Ks value of each repeat pair was much smaller than 1, indicating that these genes were selected for purification . Similarly, only one pair of homologous genes in wheat had a Ka/Ks value greater than 1, and most genes were in the purifying selection . In conclusion, environmental changes had little impact on the GASA gene family evolution in Chinese cabbage. In addition, the average Ka/Ks values were 0.162 and 0.318 for the TRD- and WGD-duplicated gene pairs, respectively, indicating that TRD-duplicated genes could be more conserved.
Cis‑acting element analysis of BrGASA genes in the Chinese cabbage
Cis-acting elements in gene promoter regions can help us explore gene function . To predict the potential biological function of the GASA genes in Chinese cabbage, we analyzed the cis-acting elements of their promoter (2 kb upstream). As shown in Fig. 7, the identified cis-acting elements were divided into three categories: plant growth and development, phytohormone responses, and stress responses. This indicated that BrGASA genes might participate in and affect these three biological activities. These three types of elements were also common in the GASA gene promoter sequences of other species [23, 26,27,28,29, 40, 52]. The cis-acting elements detected in most BrGASA gene promoters associated with plant growth and development were light-responsive elements, such as Box4 and G-box, indicating that GASA proteins might participate in light response in Chinese cabbage. Light responsive elements were the main components on the GASA gene promoter in Glycine max . In addition to light responsive elements, there were other elements related to growth and development in some genes, including the CAT-box related to meristem expression, GCN4-motif related to endosperm expression, and HD-Zip1 related to the differentiation of palisade mesophyll cells. In a previous study, endosperm (AAGAA motif) expression and meristem activation (CCGTCC box) elements were found in grape GASA genes . Meanwhile, we found that Chinese cabbage GASA genes all contain hormone-responsive elements, mainly abscisic acid (ABA) and methyl jasmonate (MeJA) elements. On the other hand, some genes contained elements related to the GA and salicylic acid (SA) responses, which may be because BrGASA genes are involved in the signaling pathways of these hormones . In Arabidopsis, the most common hormone responsive elements on GASA genes were GA (GARE) and ABA (ABRE) . Additionally, the BrGASA genes had rich stress response element, which was similar to the fact that the GASA gene family was involved in stress response research in other species. For example, in the rice GASA gene, elements that respond to low temperature (LTR) and drought (MBS) were the most common . Among the stress response elements, all genes except BrGASA15 had ARE elements related to anaerobic induction. Six genes contained LTR elements related to low temperature stress response and TC-rich repeat elements related to defense and stress response. AT-rich sequences were detected in four genes. BrGASA5 and BrGASA13 also contained drought-inducible MBS. DRE related to dehydration, low temperature, and salt stress were found only in BrGASA8. The above mentioned stress response elements were also present in the GASA gene promoter regions of apple, Glycine max, and grape [23, 26, 27].
Expression Patterns of GASA genes in different tissues of Chinese cabbage
The expression profiles of genes in different plant organs and parts can provide clues to their respective functions [33, 42]. We then investigated the expression of each BrGASA gene using published RNA-seq data from six different Chinese cabbage tissues during vegetative and reproductive development. The expression levels of each gene were normalized using the FPKM method. Among the 15 GASA genes in Chinese cabbage, the transcription levels (FPKM value) of 10 genes were determined in each tissue sample (Fig. 8). The expression of the other five BrGASA genes (BrGASA1, BrGASA2, BrGASA10, BrGASA11, and BrGASA15) was not detected in the RNA-seq libraries, which might be because they have no expression or spatiotemporal modes. In our study, BrGASA13, BrGASA14, and BrGASA8 were highly expressed in the flowers. BrGASA6, BrGASA14, and BrGASA9 were highly expressed in the siliques. BrGASA5 and BrGASA6 were highly expressed in the roots. BrGASA4 and BrGASA12 were highly expressed in the callus. BrGASA12 was highly expressed in the stems. BrGASA6, BrGASA13, BrGASA14, BrGASA12, BrGASA4, BrGASA5, and BrGASA7 were highly expressed in two or more different tissues. Noteworthily, AtGASA15 was homologous to BrGASA3, whereas AtGASA15 was expressed not only in leaves, but also in stems and flowers , which might suggest differences in gene function. In studies of other species, the GASA genes in Arabidopsis were expressed in root, stem, leave, flower, and developing silique , whereas most of the GASA genes in poplar were expressed in stem and root . All GASA genes in apple were expressed more in flower, leave, and fruit than in stem and seedlings . The GASA genes in soybean were mainly expressed in flower , whereas those of grape were specifically expressed in seeds . Some cotton GASA genes had higher expression levels in fiber . Finally, most of the GASA genes in wheat were highly expressed in embryo and anther .
Relative expression of nine BrGASA genes in Chinese cabbage
Next, the expression patterns of nine BrGASA genes were analyzed by qRT-PCR at four time points under abiotic stress and different hormonal treatments (Fig. 9). Under the salt stress of 2% NaCl, all genes except BrGASA15 were significantly up-regulated, and the expression level of most genes was up-regulated considerably, in particular at 0.5 h time point, and then down-regulated at the last two time points. The expression level of BrGASA14 was the highest at 0.5 h, approximately 6.8 times higher than that at baseline (0 h). The expression trends of BrGASA7 and BrGASA8 differed from those of the other genes. Specifically, BrGASA7 was significantly up-regulated at 6 h after treatment, while BrGASA8 was up-regulated at 0.5 and 6 h after treatment. Upon high temperature treatment, the expression of BrGASA3 was significantly down-regulated, whereas the other genes were significantly up-regulated and showed differences at different time points. Only BrGASA8 was up-regulated considerably at 0.5 h after treatment, whereas BrGASA6, BrGASA13, and BrGASA14 were significantly up-regulated at 3 h, and BrGASA4, BrGASA5, BrGASA7, and BrGASA15 were significantly up-regulated at 6 h after treatment, among which those of BrGASA15 and BrGASA7 were approximately 6- and 29-times greater than those at baseline, respectively.
It is important to note that the expression of GASA genes is controlled by GA . Hormone treatment with 500 mg/L GA3 induced significant upregulation of all BrGASA genes, except for BrGASA3, which was down-regulated. Noteworthily, the expression of these genes varied in a time-dependent manner. The expression levels of BrGASA6, BrGASA8, and BrGASA13 were significantly up-regulated at 0.5 h after treatment, whereas BrGASA4, BrGASA7, and BrGASA15 were up-regulated 3 h after treatment, with BrGASA7 reaching the highest expression. Only BrGASA14 was significantly up-regulated at 6 h after treatment. In studies of other species, the GASA gene had also responded to exogenous GA treatment, such as ZmGSL4/6 in maize , TaGASR34 in wheat , AtGASA14 in Arabidopsis , HbGASA16 in rubber tree , and most MdGASA genes in apples had been down-regulated after GA treatment . Several studies had shown that GASA was regulated by ABA [29, 34, 43]. In Arabidopsis, AtGASA2/3/5/14 responded to ABA. A few HbGASA genes (3/16) responded to ABA . In contrast, the expression of five genes (BrGASA3, BrGASA4, BrGASA5, BrGASA6, and BrGASA13) was significantly down-regulated following ABA treatment. BrGASA7, BrGASA14, and BrGASA15 levels were up-regulated 3 h after treatment, of which BrGASA7 had the highest expression. BrGASA8 also achieved a significantly higher expression upon ABA exposure for 6 h (approximately 4.7 times higher than at baseline). The different responses induced by gibberellin and ABA may be due to these hormone antagonist effects . MeJA was a natural hormone that affects the stress response of plants . After treatment with MeJA, BrGASA3, BrGASA4, BrGASA5 and BrGASA13 were significantly down-regulated: The expression levels of BrGASA6, BrGASA7, and BrGASA8 increased dramatically at 0.5 h after treatment and then showed a downward trend over time. However, an opposite trend was observed for BrGASA14, whose expression significantly increased at 3 and 6 h after treatment compared with baseline. In addition, the expression level of BrGASA15 increased, but the difference was not statistically significant. In addition to the three hormones above, GASA also responded to other hormones. For example, the MdGASA gene in apple mainly responded to 6-BA treatment and upregulates expression , wheraes HbGASA gene in rubber tree responded to ethylene and jasmonic acid treatment . These findings show that BrGASA is involved in and regulated by abiotic stress and hormone responses in the Chinese cabbage.
In this study, 15 GASA genes were identified in the Chinese cabbage genome and were divided into three subfamilies. The GASA genes were distributed unevenly on 10 Chinese cabbage chromosomes. A total of four pairs of GASA genes were found to originate from tandem duplication (TRD), and three pairs of GASA genes originated from whole-genome duplication (WGD). Cis-acting elements of the GASA promoters were involved in plant development, hormonal and stress responses. The members of GASA genes showed differential expression patterns in diverse tissues, and differential responses were also found under different abiotic and hormonal stress. The results of our study provide valuable clues for studying of GASA genes in Chinese cabbage. We will further analyze the molecular mechanism of the GASA genes response to hormones and stress in Chinese cabbage, and lay the foundation for improving the cultivation, yield, and quality of Chinese cabbage through biological breeding.
Identification of GASA family genes in Chinese cabbage
Brassica rapa (v3.5) data were downloaded from the Chinese cabbage database (http://brassicadb.cn), and the Hidden Markov Model (HMM) profile of the GASA domain (PF02704) was obtained from the Pfam database (http://pfam.xfam.org/). The multi-transcript gene was filtered according to the gff gene annotation file, and the longest mRNA sequence was selected as the representative of the gene. The hmmsearch program in the HMMER (v 3.1b2) software package was used to detect protein sequences containing the GASA domain (PF02704). The Arabidopsis genome information was downloaded from the Arabidopsis database (http://arabidopsis.org) using Diamond (v0.9.24.125) to build a database of 15 known GASA family genes in Arabidopsis and conduct a blastp comparison to identify homologous genes. Parameter settings: e-value 1e-20. Fifteen GASA family genes were obtained using the abovementioned methods.
Physicochemical Properties, phylogeny, and Synteny Analysis
All identified BrGASA protein, coding, and genomic sequences, as well as related information regarding the start-end position of the gene, number of amino acids, and chromosome location, were downloaded from the Chinese cabbage database. Information on the physicochemical properties of GASA proteins was obtained from the online ExPASy program (https://web.expasy.org/protparam/) using protein sequences . In silico analysis of subcellular location and tertiary structure of proteins was performed using online programs: The WOLF PSORTII program (https://wolfpsort.hgc.jp/)  and PHYRE server v2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index), respectively. A phylogenetic tree was constructed using Fast Tree software based on the neighbor-joining method with a bootstrap test of 1,000 replicates. The occurrence of replication events and synteny of GASA genes in Chinese cabbage were analyzed and visualized using MCScanX , DupGen_finder , and TBtools .
Exon–Intron, Gene structure, conserved Motif, and promoter analysis
Structural information, such as the number of introns and exons of the Chinese cabbage GASA gene family members, was obtained from the protein annotation files retrieved from the Chinese cabbage database. The gene structure was determined according to the corresponding sequence, and the gene structure map was generated using TBtools software (v 0.665). The MEME platform (https://meme-suite.org/meme/doc/meme.html) was used to identify conserved motifs in the BrGASA proteins  (default parameters with the maximum number of motifs set to 10). Furthermore, the 2 kb region upstream of the start codon of candidate BrGASA genes was examined for the presence of cis-elements. The PlantCARE program (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to search for regulatory elements.
Tissue-specific gene expression analysis of BrGASA genes
For the expression profiling of the GASA genes in Chinese cabbage, we utilized the Illumina RNA-Seq data previously generated and analyzed by Tong et al. . Six tissues including callus, root, stem, leaf, flower, and silique of the Chinese cabbage cultivar Chiifu were studied. The transcript abundance is expressed as fragments per kilobase of exon model per million mapped reads (FPKM). The expression profiles of the Chinese cabbage GASA genes from each sample were clustered and a heatmap was drawn using the HemI program (http://hemi.biocuckoo.cn/). After normalization using the default linear method, the expression data were clustered using the hierarchical average linkage algorithm and the Euclidean distance similarity metric algorithm on both the horizontal and vertical axes.
Plant Growth conditions, treatments, and Sampling
The wild-type ‘FT’ was a double haploid line obtained by microspore culture from the Chinese cabbage variety ‘Fukuda 50’, screened by Shenyang Greenstar Chinese cabbage research institute (Shenyang, China). In September 2022, the seeds stored in our laboratory were placed in a moist petri dish for germination at room temperature. Under routine management, the sprouting seeds were sown into a 32-well seedling tray in a solar greenhouse at Shenyang Agricultural University (Shenyang, China). The solar greenhouse was monitored at 41 degrees north latitude and 123.33 degrees east longitude. After sowing for four weeks, the uniform seedlings were selected and treated with a high temperature of 35 ℃, 2% NaCl, 500 mg/L GA3, 100 µmol/L ABA, 100 µmol/L MeJA and under natural drought conditions for 0 h, 0.5 h, 3 h, and 6 h [19, 28, 68]. The leaves treated with different treatments for 0 h, 0.5 h, 3 h, and 6 h were sampled, which were frozen with liquid nitrogen and stored at − 80 ℃ for the following experiment. Three biological replicates were performed for each treatment.
qRT- PCR expression analysis
Total RNA was extracted from Chinese cabbage leaves using the RNAsimple Total RNA Kit (TIANGEN BIOTECH, Beijing, China). First-strand cDNA fragments were synthesized from total RNA using the FastKing RT Kit (TIANGEN BIOTECH, Beijing, China). Before the subsequent PCR reaction, the cDNA samples were stored at -20 ℃. The BrGASA CDS sequence and Primer Premier software (version 5.0) were used for primer design and synthesis by the biology company (Sangon Biotech (Shanghai, China). BrActin (BraA10g027990.3 C), was used as an internal reference gene . The primer sequences are shown in Table S3. The amplification reaction contained 10 × diluted cDNA 1 µL, upstream primers 0.2 µL, downstream primers 0.2 µL, SybrGreen qPCR Master Mix 5 µL, and ddH2O 3.6 µL. The PCR cycling conditions included an initial polymerase activation step of 95 ℃ for 3 min, followed by 45 cycles of 95 ℃ for 15 s and 60 ℃ for 30 s. Three biological replicates were performed for each sample. The relative expression levels of the BrGASA gene are represented in the form of relative changes by the 2−∆∆Ct method . The statistical analysis was performed using Microsoft Excel 2019 and SPSS 26.0.
The datasets supporting the conclusions of this article are included within the article and its additional files. Genomic sequences and gene annotation information of Brassica rapa were downloaded from http://brassicadb.cn/#/.
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We would like to thank Editage (www.editage.cn) for English language editing.
This research was funded by the National Natural Science Foundation of China (32002033), Liaoning Province Scientific Research Funding Project (LSNQN202019) and China Postdoctoral Science Foundation (2022MD723806).
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Sun, B., Zhao, X., Gao, J. et al. Genome-wide identification and expression analysis of the GASA gene family in Chinese cabbage (Brassica rapa L. ssp. pekinensis). BMC Genomics 24, 668 (2023). https://doi.org/10.1186/s12864-023-09773-9