The seasonal distribution of precipitation in Guizhou Province is uneven, with more precipitation in spring and summer, but the rapeseed seedlings are susceptible to drought stress in autumn and winter, which seriously affects the yield. Mustard is a special oilseed crop mainly grown in Guizhou Province. It has strong drought tolerance and can be grown in mountainous areas. It is a rich resource of drought-resistant genes. The discovery of drought-resistant genes is of critical importance for the improvement of mustard varieties. and innovation in germplasm resources. The GRF family plays a critical role in plant growth and development and the response to drought stress. At present, GRF genes have been found in Arabidopsis 2, rice (Oryza sativa) 12, rapeseed 13, cotton (Gossypium hirsutum) 14, wheat (Triticum). aestivum)15, pearl millet (Setaria italica)16 and Brassica17, but there are no reports of GRF genes detected in mustard. In this study, the GRF family genes of mustard were identified at the genome-wide level and their physical and chemical characteristics, evolutionary relationships, homology, conserved motifs, gene structure, gene duplications, cis-elements and seedling stage (four-leaf stage) were analyzed. The expression patterns under drought stress were comprehensively analyzed to provide a scientific basis for further studies on the potential function of BjGRF genes in drought response and to provide candidate genes for breeding drought-tolerant mustard.
Thirty-four BjGRF genes were identified in the Brassica juncea genome using two HMMER searches, all of which contain the QLQ and WRC domains. The CDS sequences of the identified BjGRF genes are presented in Supplementary Table S1. BjGRF01–BjGRF34 are named based on their location on the chromosome. The physicochemical properties of this family indicate that the amino acid length is highly variable, ranging from 261 aa (BjGRF19) to 905 aa (BjGRF28). The isoelectric point of BjGRF ranges from 6.19 (BjGRF02) to 9.35 (BjGRF03) with an average of 8.33, and 88.24% of BjGRF is a basic protein. The predicted molecular weight range of BjGRF is from 29.82 kDa (BjGRF19) to 102.90 kDa (BjGRF28); the instability index of BjGRF proteins ranges from 51.13 (BjGRF08) to 78.24 (BjGRF19), all are greater than 40, indicating that the fatty acid index ranges from 43.65 (BjGRF01) to 78.78 (BjGRF22), the average hydrophilicity (GRAVY) ranges from -1.07 (BjGRF31) to -0.45 (BjGRF22), all the hydrophilic BjGRF proteins have negative GRAVY values, which may be due to the lack of hydrophobicity caused by the residues. Subcellular localization prediction showed that 31 BjGRF encoded proteins could be localized in the nucleus, BjGRF04 could be localized in peroxisomes, BjGRF25 could be localized in the cytoplasm, and BjGRF28 could be localized in chloroplasts (Table 1), indicating that BjGRFs may be localized in the nucleus and play an important regulatory role as a transcription factor.
Phylogenetic analysis of GRF families in different species can help to study gene functions. Therefore, the full-length amino acid sequences of 35 rapeseed, 16 turnip, 12 rice, 10 millet and 9 Arabidopsis GRFs were downloaded and a phylogenetic tree was constructed based on 34 identified BjGRF genes (Fig. 1). The three subfamilies contain different numbers of members; 116 GRF TFs are divided into three different subfamilies (groups A~C), containing 59 (50.86%), 34 (29.31%) and 23 (19.83)% of the GRFs, respectively. Among them, 34 BjGRF family members are scattered across 3 subfamilies: 13 members in group A (38.24%), 12 members in group B (35.29%) and 9 members in group C (26.47%). In the process of mustard polyploidization, the number of BjGRFs genes in different subfamilies is different, and gene amplification and loss may have occurred. It is worth noting that there is no distribution of rice and millet GRFs in group C, while there are 2 rice GRFs and 1 millet GRF in group B, and most of the rice and millet GRFs are grouped in one branch, indicating that BjGRFs are closely related to dicots. Among them, the most in-depth studies on the GRF function in Arabidopsis thaliana provide a basis for functional studies of BjGRFs.
Phylogenetic tree of mustard including Brassica napus, Brassica napus, rice, millet and members of the Arabidopsis thaliana GRF family.
Analysis of repetitive genes in the mustard GRF family. The grey line in the background represents a synchronized block in the mustard genome, the red line represents a pair of segmented repeats of the BjGRF gene;
BjGRF gene expression under drought stress at the fourth leaf stage. qRT-PCR data are shown in Supplementary Table S5. Significant differences in data are indicated by lowercase letters.
As the global climate continues to change, studying how crops cope with drought stress and improving their tolerance mechanisms has become a hot research topic18. After drought, the morphological structure, gene expression and metabolic processes of plants will change, which may lead to the cessation of photosynthesis and metabolic disturbance, affecting the yield and quality of crops19,20,21. When plants sense drought signals, they produce second messengers such as Ca2+ and phosphatidylinositol, increase the intracellular calcium ion concentration and activate the regulatory network of protein phosphorylation pathway22,23. The final target protein is directly involved in cellular defense or regulates the expression of related stress genes through TFs, enhancing plant tolerance to stress24,25. Thus, TFs play a crucial role in responding to drought stress. According to the sequence and DNA binding properties of drought stress responsive TFs, TFs can be divided into different families, such as GRF, ERF, MYB, WRKY and other families26.
The GRF gene family is a type of plant-specific TF that plays important roles in various aspects such as growth, development, signal transduction and plant defense responses27. Since the first GRF gene was identified in O. sativa28, more and more GRF genes have been identified in many species and shown to affect plant growth, development and stress response8, 29, 30,31,32. With the publication of the Brassica juncea genome sequence, the identification of the BjGRF gene family became possible33. In this study, 34 BjGRF genes were identified in the whole mustard genome and named BjGRF01–BjGRF34 based on their chromosomal position. All of them contain highly conserved QLQ and WRC domains. Analysis of the physicochemical properties showed that the differences in the amino acid numbers and molecular weights of the BjGRF proteins (except BjGRF28) were not significant, indicating that the BjGRF family members may have similar functions. Gene structure analysis showed that 64.7% of the BjGRF genes contained 4 exons, indicating that the BjGRF gene structure is relatively conserved in evolution, but the number of exons in the BjGRF10, BjGRF16, BjGRP28 and BjGRF29 genes is larger. Studies have shown that the addition or deletion of exons or introns can lead to differences in gene structure and function, thereby creating new genes34,35,36. Therefore, we speculate that the intron of BjGRF was lost during evolution, which may cause changes in gene function. Consistent with existing studies, we also found that the number of introns was associated with gene expression. When the number of introns in a gene is large, the gene can quickly respond to various unfavorable factors.
Gene duplication is a major factor in genomic and genetic evolution37. Related studies have shown that gene duplication not only increases the number of GRF genes, but also serves as a means of generating new genes to help plants adapt to various adverse environmental conditions38. A total of 48 duplicate gene pairs were found in this study, all of which were segmental duplications, indicating that segmental duplications are the major mechanism for increasing the number of genes in this family. It has been reported in the literature that segmental duplication can effectively promote the amplification of GRF gene family members in Arabidopsis and strawberry, and no tandem duplication of this gene family was found in any of the species27,39. The results of this study are consistent with existing studies on Arabidopsis thaliana and strawberry families, suggesting that the GRF family can increase the number of genes and generate new genes through segmental duplication in different plants.
In this study, a total of 34 BjGRF genes were identified in mustard, which were divided into 3 subfamilies. These genes showed similar conserved motifs and gene structures. Collinearity analysis revealed 48 pairs of segment duplications in mustard. The BjGRF promoter region contains cis-acting elements associated with light response, hormonal response, environmental stress response, and growth and development. The expression of 34 BjGRF genes was detected at the mustard seedling stage (roots, stems, leaves), and the expression pattern of 10 BjGRF genes under drought conditions. It was found that the expression patterns of BjGRF genes under drought stress were similar and may be similar. involvement in drought Forcing regulation. BjGRF03 and BjGRF32 genes may play positive regulatory roles in drought stress, while BjGRF06 and BjGRF23 play roles in drought stress as miR396 target genes. Overall, our study provides a biological basis for future discovery of BjGRF gene function in Brassicaceae plants.
The mustard seeds used in this experiment were provided by Guizhou Oil Seed Research Institute, Guizhou Academy of Agricultural Sciences. Select the whole seeds and plant them in soil (substrate: soil = 3:1), and collect the roots, stems and leaves after the four-leaf stage. The plants were treated with 20% PEG 6000 to simulate drought, and the leaves were collected after 0, 3, 6, 12 and 24 hours. All plant samples were immediately frozen in liquid nitrogen and then stored in a -80°C freezer for the next test.
All data obtained or analyzed during this study are included in the published article and supplementary information files.
Post time: Jan-22-2025