Molecular characterization and expression analysis of S1 self-incompatibility locus-linked pollen 3.15 gene in Citrus reticulata.

Gametophytic self-incompatibility (GSI) is controlled by a highly polymorphic locus called the S-locus, which is an important factor that can result in seedless fruit in Citrus. The S1 self-incompatibility locus-linked pollen 3.15 gene (S1-3.15 ) belongs to a type of S locus gene. The role of S1-3.15 in the SI reaction of Citrus has not yet been reported. In this study, full-length sequences of cDNA and DNA encoding the S1-3.15 gene, referred to as CrS1-3.15 , were isolated from 'Wuzishatangju' (Self-incompatibility, SI) and 'Shatangju' (Self-compatibility, SC). The predicted amino acid sequences of CrS1-3.15 between 'Wuzishatangju' and 'Shatangju' differ by only three amino acids. Compared to 'Wuzishatangju', three bases were substituted in the genomic DNA of CrS1-3.15 from 'Shatangju'. Southern blot results showed that one copy of CrS1-3.15 existed in the genomic DNA of both 'Wuzishatangju' and 'Shatangju'. The expression level of the CrS1-3.15 gene in the ovaries of 'Shatangju' was approximately 60-fold higher than that in the ovaries of 'Wuzishatangju'. When 'Wuzishatangju' was cross-pollinated, the expression of CrS1-3.15 was upregulated in the ovaries at 3 d, and the highest expression levels were detected in the ovaries at 6 d after cross-pollination of 'Wuzishatangju' × 'Shatangju'. To obtain the CrS1-3.15 protein, the full-length cDNA of CrS1-3.15 genes from 'Wuzishatangju' and 'Shatangju' was successfully expressed in Pichia pastoris. Pollen germination frequency of 'Wuzishatangju' was inhibited significantly with increasing CrS1-3.15 protein concentrations from SI 'Wuzishatangju'.

Gut microbiota profiling revealed the regulating effects of salidroside on iron metabolism in diabetic mice.

Background: Diabetes is a common metabolic disease that is associated with gut microbiota dysbiosis and iron metabolism. Salidroside (SAL) is the main ingredient of the traditional Chinese herb Rhodiola, previous studies have shown that SAL could reshape the gut microbiota and limit iron accumulation. Therefore, it is possible that SAL can act as an alternative therapy for diabetes, and its underlying mechanism is worth exploring. Methods: SAL was used to treat diabetic db/db mice. Serum glucose and iron levels and the histopathology of myocardial fibres were evaluated. The gut microbiota composition was determined by 16S rRNA Illumina sequencing technology. Results: Treatment with SAL significantly reduced blood glucose and ameliorated diabetic cardiomyopathy in diabetic db/db mice, which was accompanied by inhibited ferroptosis and iron accumulation. Furthermore, the 16S rRNA sequencing results showed that SAL induced a change in the gut microbiota composition. Overall, SAL could increase the proportion of probiotic bacteria and decrease Lactobacillus to improve gut microbiota. Specifically, SAL increased the ratio of Bacteroidetes to Firmicutes in diabetic mice. The most significant biomarker was the genus Lactobacillus between the MD group and the SAL group. In addition, COG and KEGG analyses suggested that SAL mainly participated in nutrient metabolism, among them iron metabolism was associated with the abundance of Lactobacillus. Conclusions: SAL could reduce the glucose level and protect against diabetic cardiomyopathy in diabetic mice, which might be mediated by the change in the gut microbiota and the regulation of iron metabolism. The findings suggested that SAL was a promising complementary option for diabetes therapy.

Genome-Wide Analysis of Basic Helix-Loop-Helix Transcription Factors to Elucidate Candidate Genes Related to Fruit Ripening and Stress in Banana (Musa acuminata L. AAA Group, cv. Cavendish).

The basic helix-loop-helix (bHLH) proteins are a superfamily of transcription factors (TFs) that can bind to specific DNA target sites, playing a central role in a wide range of metabolic, physiological, and developmental processes in higher organisms. However, no systemic analysis of bHLH TFs has been reported in banana, a typical climacteric fruit in tropical and subtropical regions. In our study, 259 MabHLH TF genes were identified in the genome of Musa acuminata (A genome), and phylogenetic analysis indicated that these MabHLHs could be classified into 23 subfamilies with the bHLHs from rice and Arabidopsis. The amino acid sequences of the bHLH domain in all MabHLH protein sequences were quite conserved, especially Arg-12, Arg-13, Leu-23, and Leu-79. Distribution mapping results showed that 258 MabHLHs were localized on the 11 chromosomes in the M. acuminata genome. The results indicated that 40.7% of gene duplication events were located in collinear fragments, and segmental duplications might have played a key role in the expansion of MabHLHs. Moreover, the expression profiles of MabHLHs in different fruit development and ripening stages and under various abiotic and biotic stresses were investigated using available RNA-sequencing data to obtain fruit development, ripening-specific, and stress-responsive candidate genes. Finally, a co-expression network of MabHLHs was constructed by weighted gene co-expression network analysis to elucidate the MabHLHs that might participate in important metabolic biosynthesis pathways in banana during development and the response to stress. A total of 259 MabHLHs were identified, and their sequence features, conserved domains, phylogenetic relationships, chromosomal distributions, gene duplications, expression profiles, and co-expression networks were investigated. This study systematically identified the MabHLHs in the M. acuminata genome at the genome-wide level, providing important candidate genes for further functional analysis. These findings improve our understanding of the molecular basis of developmental and stress tolerance in an important banana cultivar.

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata.

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from 'Wuzishatangju' (self-incompatible, SI) and 'Shatangju' (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between 'Wuzishatangju' and 'Shatangju', respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of 'Wuzishatangju' and 'Shatangju'. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of 'Shatangju' was approximately 10-fold higher than in anthers of 'Wuzishatangju'. The highest expression level of CrUBE1 was detected in pistils at 7days after self-pollination of 'Wuzishatangju', which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from 'Wuzishatangju' and 'Shatangju' were successfully expressed in Pichia pastoris. Pollen germination frequency of 'Wuzishatangju' was significantly inhibited with increasing of CrUBE1 protein concentrations from 'Wuzishatangju'.

Cloning and expression analysis of S-RNase homologous gene in Citrus reticulata Blanco cv. Wuzishatangju.

S-RNase-based self-incompatibility is the most widespread form of genetically controlled mate selection in plants and that S-RNase controls pollination specificity in the pistils. 'Wuzishatangju' (Citrus reticulata Blanco), a nature bud mutant from a self-compatible (SC) cultivar 'Shatangju', displays gametophytic self-incompatibility (GSI). In this study, full-length sequences of cDNA and DNA of the S-RNase homologous gene were obtained from 'Wuzishatangju' and 'Shatangju'. There was no difference in ORF sequences of the S-RNase cDNA between 'Wuzishatangju' and 'Shatangju'. However, 13, 9 and 6 consecutive bases were missing in 'Wuzishatangju' cDNA 5' UTR, 3' UTR and genomic DNA, respectively. Tissue-specific expression of the S-RNase gene was detected using semi-quantitative RT-PCR and quantitative real-time PCR. The expression level of the S-RNase gene in styles of 'Wuzishatangju' was approximately 10- and 5-fold higher than that in leaves and pollen, respectively. When 'Wuzishatangju' was self-pollinated, the expression of S-RNase in pistils peaked at 3 days, which was approximately 10-fold higher than that at 4h and 7 days, while in cross-pollination of 'Wuzishatangju' x 'Shatangju' the expression was very weak at 3 days. Results from a Southern blot showed that two copies of the S-RNase gene existed in genomic DNA of both 'Wuzishatangju' and 'Shatangju'.