There is no trouble in the eighth day of the first month
Edit丨No worries about the eighth day of the first month
BAG, in eukaryotes, is a conserved helper protein, an anti-apoptotic protein that interacts with the B-cell lymphoma-2 protein.
BAG1, which was first discovered, can be used as a new anti-apoptotic protein, which participates in the programmed death of cells and synergizes with Bcl-2 to improve cell survival. The C-terminus conserved BAG domain of BAG, which can be associated with the chaperone 70 kDa heat shock protein.
THE BAG FAMILY IS EVOLUTIONARILY CONSERVED AND IS FOUND IN ANIMALS, PLANTS, AND YEASTS. BAG proteins have been identified in plants such as Arabidopsis, rice, maize, tomato, banana, poplar and soybean.
Sugarcane is the world's main sugar crop and biofuel. At present, it is believed that modern sugarcane cultivars are crossed between tropical species and dense hands, and then backcrossed with tropical species.
For a long time, the complexity of the sugarcane genome has hindered the study of sugarcane molecular biology. Until now, the haploid sugarcane cutting hand dense AP85-441 genome sequence has been assembled, which provides convenience for sugarcane genetic research and molecular breeding.
Materials and methods of the experiment
In order to identify the members of the BAG gene family of sugarseed cutting hand-cutting denses, the Cryptomarkov model file of the BAG conserved domain was downloaded from the PFAM database, and the Arabidopsis thaliana, rice, and maize BAG proteins were used as reference sequences.
The HMMER3.1 program and BLASTP were used to search the sugarcane cutting hand dense protein database, and the E values of HMMER and BLAST were set to 1e-10 and 1e-5, respectively.
In order to study the evolutionary relationship of sugarcane BAG protein in plants, the ClustaW algorithm of MEGA-X software was used to compare the BAG protein sequences of sugarcane, Arabidopsis, rice, maize, tomato, banana and poplar.
The phylogenetic tree was constructed using adjacency, 1000 Bootstrap repeat tests were set, and then the phylogenetic tree was beautified using the Evolvie online website.
Through the MEME online website setting, 1~10 motifs with a width of 6~50 amino acids were found to analyze the conserved motif of sugarcane cutting hand-cut BAG protein.
Conserved motifs, conserved domains, and gene structures were visualized using the GeneStructureView tool in TBtools based on genomic annotation files.
Analysis of experimental results
A total of 24 BAG gene family members were identified in the AP85-441 genome of sugarcane cutting hand. According to the results of homologous gene alignment and clustering between sugarcane and rice, the sugarcane BAG gene was named with reference to the name of the homologous gene in rice.
On this basis, the alleles belonging to the corresponding homologous chromosomes A, B, C, and D in the circumcision are distinguished by the corresponding letters "a", "b", "c" and "d".
SsBAGs, on the other hand, which do not have homologous genes in rice, are named SsBAG7 and SsBAG8 respectively according to the similarity of their protein sequences, and although there are members clustered in the same branch, the numbers 1, 2, 3 and 4 are used instead because they are not all alleles on the homologous chromosome set.
Therefore, the name of SsBAGs contains information about the orthologous genes in rice, as well as the homologous chromosomes in the genome of the rice.
The results of physicochemical property analysis showed that the length of the encoded amino acid sequence of sugarcane BAG gene was 140~1318, and the theoretical isoelectric point was 4.70~10.19, including 9 acidic proteins and 15 basic proteins.
The molecular weight is 15.45~145.38kDa, and the protein instability coefficient is 37.03~71.96.
A total of 15 SsBAGs had cytoplasmic and nuclear localization signals, 6 SsBAGs only had nuclear localization signals, and SsBAG5b, SsBAG5c and SsBAG8.4 were predicted to be localized in chloroplasts.
In order to elucidate the evolutionary relationship of the BAG gene family in sugarcane, a total of 87 BAG proteins from sugarcane, Arabidopsis, rice, maize, tomato, banana and poplar were used to construct a phylogenetic tree by adjacency.
According to the phylogenetic relationship, the members of the BAG gene family were divided into three groups: GroupI., GroupII.-1 and GroupII.-2. SsBAGs have the highest number in GroupI., with 18 for Group II.-1 for 4 and Group II.-2 for 2.
Except for the high similarity between SsBAG8.1~8.4 and poplar PtrBAG3, the evolutionary distance between sugarcane SsBAGs and monocotyledon maize ZmBAGs and rice OsBAGs was closer than that of dicots Arabidopsis thaliana AtBAGs.
In plants, the BAG domain in the BAG protein can interact with the ATPase domain of the Hsp70 protein and regulate its activity. As a ubiquitin-like domain, UBQ plays an important role in pathways such as apoptosis and protein folding.
The IQ domain can regulate the activity of binding proteins by sensing changes in calcium levels, allowing BAG to act as a hub connecting calcium signaling and chaperone proteins.
In the study, all 24 sugarcane BAG proteins contained a conserved BAG domain. Most of the 15 members of GroupI contain both UBQ and BD domains, while Group II. members all contain BD and IQ domains.
Although SsBAG8.1, SsBAG8.2, and SsBAG8.3 belong to GroupI, they are supposed to be sequences in the UBQ coding region, but the mutation results in the deletion of the UBQ domain.
Therefore, these three proteins only predicted the BAG domain, which had never been reported in Arabidopsis thaliana and rice, suggesting that sugarcane polyploidy may have contributed to some degree of functional differentiation of BAG proteins.
The analysis of the conserved motif composition can further illustrate the subtle differences between the BAG proteins in sugarcane.
The results showed that all SsBAGs contained motif3, and the distribution order and number of BAG proteins in the same cluster branch were similar in the phylogenetic tree, indicating that the motifs of sugarcane BAGs were conserved to a certain extent.
GroupI. has a total of 8 conserved motifs, and the members encoding intact BDs and having UBQs all contain 5 common motifs, indicating the importance of these motifs for maintaining normal protein structure and function.
GroupII.-1 and GroupII.-2 have common motif3 and motif9, and motif7 also appears in some members of these two groups.
GroupI.与GroupII.-1的motif分布顺序和数量差异很大,而GroupII.-2与GroupII.-1拥有相同的motif9,以及与GroupI.拥有相同的motif2,这表明GroupII.-2为中间类型。
The results of gene structure analysis showed that SsBAGs contained 1~8 exons. Except for SsBAG6b and SsBAG6c, which only had one exon without an intron, the rest of the family members had at least one intron.
Among them, 13 SsBAGs contained 4 exons, and 6 SsBAGs contained 3 exons. Members in Group II do not have UTRs, while 7 members in Group I. have UTRs at both ends of the 5' and 3'.
The gene sequences of SsBAG8.2 and SsBAG8.3 are longer than those of other BAG genes, but these sequences are mainly introns, so the final encoded protein is normal in size and has no additional domains.
SsBAG7.1 and SsBAG7.2 have the largest number of exons and the longest protein sequences, but no additional domains are predicted in these proteins.
The 24 sugarcane BAG genes were distributed on 19 chromosomes, and there were 2 BAG genes on 5 chromosomes of Chr3A, Chr4B, Chr5B, Chr6B and Chr6C, and only 1 BAG gene on the remaining 14 chromosomes.
However, the BAG gene was not included on the two homologous chromosome sets of Chr1 and Chr7, and a possible tandem repeat gene pair was detected on Chr5B.
Among the BAG members, only 2 BAG members had scattered replications, and the remaining 22 were genome-wide or fragment replications.
It can be seen that genome-wide replication or fragment replication is the main way of the expansion of the BAG family of sugarcape, which is consistent with the characteristics of gene family amplification of polyploid plant species.
Discussion of experimental results
BAG protein is a multifunctional chaperone regulatory protein, which is related to the regulation of plant growth and development, and also plays a role in the response of plants to adversity stresses such as drought, salinity, high temperature and low temperature.
Compared with animals, the functional research of plant BAG protein is not deep enough. The identification and expression pattern analysis of BAG protein in sugarcane have not been reported.
This study comprehensively analyzed the phylogeny and transcription profile of sugarcape BAG gene, which provided useful clues for revealing the biological function of BAG.
It should be noted that, unlike the naming method based on chromosome position, the naming of SsBAGs in this experiment is based on the sequence similarity of its homologous members in rice, referring to the existing BAG naming in rice.
Thus, sugarseed BAG proteins have similar sequences and conserved domains to the BAG members of their corresponding names in rice. For example, SsBAG1/2/3/4 and their rice homologous proteins contain BD and UBQ, while SsBAG5 and SsBAG6 also have BD and IQ domains as expected.
Moreover, the same group of members of the BAG family has similar motif composition, gene structure, cDNA length, and protein isoelectric points. The expression pattern of SsBAGs in sugarcane was very similar to that of its homologous genes in rice.
Like their homologous genes in rice, SsBAG1/2/3/4 in sugarcane exhibits high levels of expression in many tissues at different stages of development, while other genes exhibit low or moderate expression in specific regions or stages.
The results showed that the transcription level of SsBAG1/2 and its homologous genes in rice was higher in mature stems, while the transcription level of SsBAG3/4 and its homologous genes in rice was higher in young stems.
In addition, the expression of sugarcane BAG gene under drought treatment was similar to that of its homologous gene in rice. After drought treatment, the expression of BAG1/3 gene in rice and sugarcane was significantly down-regulated in tolerant varieties, while the expression of BAG4/6 gene was not significantly changed.
Therefore, it can be inferred that the information of the sugarcane SsBAG gene and the corresponding rice OsBAG gene can be cross-referenced.
For example, OsBAG5/6 was clearly expressed in the tissues and organs of rice during the reproductive growth period, and it can be expected that SsBAG5/6 in sugarcane could be expressed during the reproductive growth period.
In turn, the expression pattern of SsBAG2/5 in sugarcane under drought stress could provide information for the expression of OsBAG2/5 in rice.
In this study, the expression of the sugarcane BAG family and the previously reported sugarcane AP2/ERF family was basically preserved and doubled after gene duplication and amplification, and it is speculated that the function of related genes was enhanced after sugarcane polyploidy.
This trait may be related to the stress resistance, disease resistance and regenerative properties of sugarcane as a perennial crop, which has evolutionary positive significance.
Based on the high-quality genome of AP85-441, 24 members of the BAG gene family, including alleles, were identified at the genome-wide level, and their evolutionary relationships, sequence characteristics and expression patterns were systematically analyzed.
At the same time, RT-qPCR experiments showed that the expressions of ScBAG1a, ScBAG3a and ScBAG4a were significantly down-regulated after drought treatment, suggesting that they may play a role in the response of sugarcane to drought stress.
Subcellular localization experiments showed that both ScBAG1a and ScBAG2a were localized in the cytoplasm and nucleus. The results of this study lay a foundation for further research on the function of sugarcane BAG gene family, and provide candidate target genes and new ideas for sugarcane resistance breeding.