The Potential Role of Genic-SSRs in Driving Ecological Adaptation Diversity in Caragana Plants.

Caragana, a xerophytic shrub genus widely distributed in northern China, exhibits distinctive geographical substitution patterns and ecological adaptation diversity. This study employed transcriptome sequencing technology to investigate 12 Caragana species, aiming to explore genic-SSR variations in the Caragana transcriptome and identify their role as a driving force for environmental adaptation within the genus. A total of 3666 polymorphic genic-SSRs were identified across different species. The impact of these variations on the expression of related genes was analyzed, revealing a significant linear correlation (p < 0.05) between the length variation of 264 polymorphic genic-SSRs and the expression of associated genes. Additionally, 2424 polymorphic genic-SSRs were located in differentially expressed genes among Caragana species. Through weighted gene co-expression network analysis, the expressions of these genes were correlated with 19 climatic factors and 16 plant functional traits in various habitats. This approach facilitated the identification of biological processes associated with habitat adaptations in the studied Caragana species. Fifty-five core genes related to functional traits and climatic factors were identified, including various transcription factors such as MYB, TCP, ARF, and structural proteins like HSP90, elongation factor TS, and HECT. The roles of these genes in the ecological adaptation diversity of Caragana were discussed. Our study identified specific genomic components and genes in Caragana plants responsive to heterogeneous habitats. The results contribute to advancements in the molecular understanding of their ecological adaptation, lay a foundation for the conservation and development of Caragana germplasm resources, and provide a scientific basis for plant adaptation to global climate change.

RADseq-based population genomic analysis and environmental adaptation of rare and endangered recretohalophyte Reaumuria trigyna.

Genetic diversity reflects the survival potential, history, and population dynamics of an organism. It underlies the adaptive potential of populations and their response to environmental change. Reaumuria trigyna is an endemic species in the Eastern Alxa and West Ordos desert regions in China. The species has been considered a good candidate to explore the unique survival strategies of plants that inhabit this area. In this study, we performed population genomic analyses based on restriction-site associated DNA sequencing to understand the genetic diversity, population genetic structure, and differentiation of the species. Analyses of 92,719 high-quality single-nucleotide polymorphisms (SNPs) indicated that overall genetic diversity of R. trigyna was low (HO = 0.249 and HE = 0.208). No significant genetic differentiation was observed among the investigated populations. However, a subtle population genetic structure was detected. We suggest that this might be explained by adaptive diversification reinforced by the geographical isolation of populations. Overall, 3513 outlier SNPs were located in 243 gene-coding sequences in the R. trigyna transcriptome. Potential sites under diversifying selection occurred in genes (e.g., AP2/EREBP, E3 ubiquitin-protein ligase, FLS, and 4CL) related to phytohormone regulation and synthesis of secondary metabolites which have roles in adaptation of species. Our genetic analyses provide scientific criteria for evaluating the evolutionary capacity of R. trigyna and the discovery of unique adaptions. Our findings extend knowledge of refugia, environmental adaption, and evolution of germplasm resources that survive in the Ordos area.

Distinct enzyme activities of serine protease p37k in silkworm midgut and molting fluid.

Serine proteases possess various biological functions. The serine protease p37k exhibits gelatinolytic activity in the silkworm midgut and degrades cuticular proteins in the molting fluid. In this study, we analyzed the activity changes of recombinant p37k (re-p37k) and p37k in the midgut and molting fluid of Bombyx mori. Firstly, in vitro-expressed re-p37k was activated when a 22 kDa band was observed by western blot. Re-p37k exhibits strong gelatinolytic activity, with the highest activity observed at pH 7.0-9.0 and 45 °C. Compared to p37k in the midgut, re-p37k loses thermal stability but can be restored by midgut extract or ions. E64, AEBSF, and an inhibitor cocktail inhibited the hydrolytic activity of re-p37k on epidermal proteins but did not inhibit the gelatinolytic activity. Subsequently, zymography showed that the positions of gelatinolytic band produced by p37k in the midgut and molting fluid were different, 35 kDa and 40 kDa, respectively. Finally, when heated midgut extract was added to re-p37k or molting fluid, the gelatinolytic band shifted from 40 kDa to 35 kDa, and the proteolytic activity of p37k in the molting fluid was inhibited. Collectively, our results demonstrate that p37k exhibits different activities in various tissues, suggesting its distinct tissue-specific functions during insect metamorphosis.

[Characterization and immunofluorescence localization analysis of carboxypeptidase A in molt fluid of silkworm].

Molting is an important physiological phenomenon of many metamorphosis insects, during which the old and new epidermis are separated by enzymes present in the molting fluid. Various proteomic studies have discovered the presence of Bombyx mori carboxypeptidase A (Bm-CPA) in the molting fluid of silkworm, but its function remains unclear. In order to better understand the role of Bm-CPA in the molting process of silkworm, Bm-CPA was analyzed by bioinformatics analysis, real-time fluorescence quantitative PCR, antibody preparation, immunofluorescence staining, and expression in Pichia pastoris. The results showed that Bm-CPA had a conserved M14 zinc carboxypeptidase domain and glycosylation site. Its expression was regulated by ecdysone 20E, and large expression was observed in the epidermis of the upper cluster stage. Immunofluorescence staining showed that Bm-CPA was enriched in the epidermis during the molting stage, and the inhibitor of Bm-CPA led to the larval death due to the inability to molt. We also successfully obtained a large number of recombinant Bm-CPA proteins by Pichia pastoris expression in vitro. These results may facilitate further understanding the molting development process of silkworm.

[Genome-wide identification of the BmAKR gene family in the silkworm (Bombyx mori) and their expression analysis in diapause eggs and nondiapause eggs].

The aldo-keto reductase super family (AKRs) has a wide range of substrate specificity. However, the systematic identification of insect AKR gene family members has not been reported. In this study, bioinformatics methods were used to predict the phylogenetic evolution, physical and chemical properties, chromosome location, conserved motifs, and gene structure of AKR family members in Bombyx mori (BmAKR). Transcriptome data or quantitative real time polymerase chain reaction (qRT-PCR) were used to analyze the expression level of BmAKR genes during different organizational periods and silkworm eggs in different developmental states. Moreover, Western blotting was used to detect the expression level of the BmAKR in silkworm eggs. The results showed that 11 BmAKR genes were identified. These genes were distributed on 4 chromosomes of the silkworm genome, all of which had the (α/ß) 8-barrel motif, and their physical and chemical characteristics were relatively similar. Phylogenetic analysis showed that the BmAKR genes could be divided into 2 subgroups (AKR1 and AKR2). Transcriptome data analysis showed that the expression of BmAKR genes were quite different in different tissues and periods. Moreover, the expression analysis of BmAKR genes in silkworm eggs showed that some genes were expressed significantly higher in nondiapause eggs than in diapause eggs; but another gene, BmAKR1-1, was expressed significantly higher in diapause eggs than in nondiapause eggs. The detection of protein level found that the difference trend of BmAKR1-1 in diapause eggs and non-diapause eggs was consistent with the results of qRT-PCR. In conclusion, BmAKR1-1 was screened out as candidates through the identification and analysis of the BmAKR genes in silkworm, which may regulate silkworm egg development is worthy of further investigation.

[Gene expression and immunolocalization of chitin deacetylase BmCDA2 in silkworm].

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.

Proteomic Analysis of the Midgut Contents of Silkworm in the Pupal Stage.

The silkworm Bombyx mori, a lepidopteran insect, possesses an 8-10-day pupal stage, during which significant changes occur in the midgut, where it first condenses into the yellow body, and then undergoes decomposition. To gain insights into this transformation process, proteomics was performed on Bombyx mori midgut contents on day 2 and day 7 after pupation. The results revealed the identification of 771 proteins with more than one unique peptide. An analysis using AgriGO demonstrated that these proteins were predominantly associated with catalytic activity. Among the identified proteins, a considerable number were found to be involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, nucleic acid degradation, and energy support. Additionally, variations in the levels of certain proteases were observed between the midgut contents on day 2 and day 7 after pupation. An in-depth analysis of the two-dimensional electrophoresis of the midgut contents on day 7 after pupation led to the identification of twelve protein spots with potential gelatinolytic activity. Among these, six proteases were identified through mass spectrometry, including the p37k protease, vitellin-degrading protease, chymotrypsin-2, etc. These proteases may be responsible for the digestion of the yellow body during the later stages of pupal development.