Construction of cytomegalovirus promoter-driven gene expression system in Laodelphax striatellus.

The small brown planthopper (SBPH, Laodelphax striatellus) is a significant rice pest, responsible for transmitting rice stripe virus (RSV) in a persistent and propagative manner. RSV is one of the most detrimental rice viruses, causing rice stripe disease, which results in considerable loss of rice grain yield. While RNA interference and gene knockout techniques have enabled gene downregulation in SBPH, no system currently exists for the overexpression of endogenous or exogenous genes. Consequently, the development of a protein expression system for SBPH is imperative to serve as a technical foundation for pest control and gene function investigations. This study aimed to construct an expression vector using the promoter of the constitutive-expressed tubulin gene of SBPH, and promoter of human cytomegalovirus (CMV). Fluorescence experiments demonstrated that both tubulin and CMV promoter could drive green fluorescent protein (GFP) expression in SBPH, and could also facilitate the expression of a nucleocapsid protein (NP) -GFP fusion protein containing viral NP with comparable efficiency. Through expression vector optimization, we have identified that the 3 tandem CMV promoters display a significantly higher promoter activity compared with both the 2 tandem CMV promoters and the single CMV promoter. In addition, the incorporation of Star polycation nanoparticles significantly enhanced the expression efficiency in SBPH. These results provide a promising technical platform for investigating gene functions in SBPH.

Lead remediation by geological fluorapatite combined with Penicillium Oxalicum and Red yeast.

Phosphate solubilizing fungi Penicillium oxalicum (POX) and Red yeast Rhodotorula mucilaginosa (Rho) have been applied in Pb remediation with the combination of fluorapatite (FAp), respectively. The secretion of oxalic acid by POX and the production of extracellular polymers (EPS) by Rho dominate the Pb remediation. In this study, the potential of Pb remediation by the fungal combined system (POX and Rho) with FAp was investigated. After six days of incubation, the combination of POX and Rho showed the highest Pb remove ratio (99.7%) and the lowest TCLP-Pb concentration (2.9 mg/L). The EPS combined with POX also enhanced Pb remediation, which has a 99.3% Pb removal ratio and 5.5 mg/L TCLP-Pb concentration. Meanwhile, Rho and EPS can also stimulate POX to secrete more oxalic acid, which reached 1510.1 and 1450.6 mg/L in six days, respectively. The secreted oxalic acid can promote FAp dissolution and the formation of lead oxalate and pyromorphite. Meanwhile, the EPS produced by Rho can combine with Pb to form EPS-Pb. In the combined system of POX + Rho and POX + EPS, all of the lead oxalate, pyromorphite, and EPS-Pb were observed. Our findings suggest that the combined application of POX and Rho with FAp is an effective approach for enhancing Pb remediation.

GIS-based non-grain cultivated land susceptibility prediction using data mining methods.

The purpose of the present study is to predict and draw up non-grain cultivated land (NCL) susceptibility map based on optimized Extreme Gradient Boosting (XGBoost) model using the Particle Swarm Optimization (PSO) metaheuristic algorithm. In order to, a total of 184 NCL areas were identified based on historical records, and a total of 16 NCL susceptibility conditioning factors (NCLSCFs) were considered, based on both a systematic literature survey and local environmental conditions. The results showed that the XGBoost model optimized by PSO performed well in comparison to other machine learning algorithms; the values of sensitivity, specificity, PPV, NPV, and AUC are 0.93, 0.89, 0.88, 0.93, and 0.96, respectively. Slope, rainfall, fault density, distance from fault and drainage density are most important variables. According to the results of this study, the use of meta-innovative algorithms such as PSO can greatly enhance the ability of machine learning models.

Bacterial protoplast-derived nanovesicles carrying CRISPR-Cas9 tools re-educate tumor-associated macrophages for enhanced cancer immunotherapy.

The CRISPR-Cas9 system offers substantial potential for cancer therapy by enabling precise manipulation of key genes involved in tumorigenesis and immune response. Despite its promise, the system faces critical challenges, including the preservation of cell viability post-editing and ensuring safe in vivo delivery. To address these issues, this study develops an in vivo CRISPR-Cas9 system targeting tumor-associated macrophages (TAMs). We employ bacterial protoplast-derived nanovesicles (NVs) modified with pH-responsive PEG-conjugated phospholipid derivatives and galactosamine-conjugated phospholipid derivatives tailored for TAM targeting. Utilizing plasmid-transformed E. coli protoplasts as production platforms, we successfully load NVs with two key components: a Cas9-sgRNA ribonucleoprotein targeting Pik3cg, a pivotal molecular switch of macrophage polarization, and bacterial CpG-rich DNA fragments, acting as potent TLR9 ligands. This NV-based, self-assembly approach shows promise for scalable clinical production. Our strategy remodels the tumor microenvironment by stabilizing an M1-like phenotype in TAMs, thus inhibiting tumor growth in female mice. This in vivo CRISPR-Cas9 technology opens avenues for cancer immunotherapy, overcoming challenges related to cell viability and safe, precise in vivo delivery.

Pathotype Characterization of Plasmodiophora brassicae by European Clubroot Differential and Williams Sets in China.

Clubroot disease caused by the soil-borne Plasmodiophora brassicae is devastating to Brassicaceae crops and spreading rapidly in China in recent years, resulting in great yield losses annually. Virulence of P. brassicae populations specializes and is in dynamic change in the fields. Information on the pathotypes and their distributions is crucial to control the clubroot disease. Presently, the pathotypes of P. brassicae prevalent in China, however, are not well determined. In this study, we used 16 Brassica hosts, including the European Clubroot Differential (ECD) and Williams sets, to designate the pathotypes of 33 P. brassicae populations from 13 provinces. The 33 P. brassicae populations could be divided into 26 pathotypes by the ECD set or seven pathotypes by the Williams set, revealing ECD16/15/31 and ECD16/31/31 or P4 and P2 as the predominant pathotypes. We found that the Brassica rapa differentials ECD01 to ECD04 showed stable and high levels of resistance to most pathotypes of P. brassicae in China, thereby providing valuable resources for clubroot-resistance breeding of Brassicaceae crops. The ECD set exhibited much higher discernibility and further divided the isolates that belonged to the P4 pathotype into 10 ECD pathotypes. Isolates of ECD16/23/31 and ECD16/15/31 were strongly virulent on Huashuang 5R, the first and widely used clubroot-resistant cultivar of oilseed rape in China. As we learn, 26 pathotypes are the most diverse populations of P. brassicae characterized until now in China. Our study provides new insights into virulence specialization of P. brassicae and their geographical distributions, contributing to exploitation of clubroot-resistant resources and the field layout of the present resistant Brassica crops in China.

The Regulation of Phosphorus Release by Penicillium chrysogenum in Different Phosphate via the TCA Cycle and Mycelial Morphology.

Phosphate-solubilizing fungi (PSF) efficiently dissolve insoluble phosphates through the production of organic acids. This study investigates the mechanisms of organic acid secretion by PSF, specifically Penicillium chrysogenum, under tricalcium phosphate (Ca3(PO4)2, Ca-P) and ferric phosphate (FePO4, Fe-P) conditions. Penicillium chrysogenum exhibited higher phosphorus (P) release efficiency from Ca-P (693.6 mg/L) than from Fe-P (162.6 mg/L). However, Fe-P significantly enhanced oxalic acid (1193.7 mg/L) and citric acid (227.7 mg/L) production by Penicillium chrysogenum compared with Ca-P (905.7 and 3.5 mg/L, respectively). The presence of Fe-P upregulated the expression of genes and activity of enzymes related to the tricarboxylic acid cycle, including pyruvate dehydrogenase and citrate synthase. Additionally, Fe-P upregulated the expression of chitinase and endoglucanase genes, inducing a transformation of Penicillium chrysogenum mycelial morphology from pellet to filamentous. The filamentous morphology exhibited higher efficiency in oxalic acid secretion and P release from Fe-P and Ca-P. Compared with pellet morphology, filamentous morphology enhanced P release capacity by > 40% and > 18% in Ca-P and Fe-P, respectively. This study explored the strategies employed by PSF to improve the dissolution of different insoluble phosphates.

Characterization and Potential Function Analysis of the SRS Gene Family in Brassica napus.

SRS (SHI-related sequence) transcription factors play a crucial role in plant growth, development, and abiotic stress response. Although Brassica napus (B. napus) is one of the most important oil crops in the world, the role of SRS genes in B. napus (BnSRS) has not been well investigated. Therefore, we employed a bioinformatics approach to identify BnSRS genes from genomic data and investigated their characteristics, functions, and expression patterns, to gain a better understanding of how this gene family is involved in plant development and growth. The results revealed that there were 34 BnSRS gene family members in the genomic sequence of B. napus, unevenly distributed throughout the sequence. Based on the phylogenetic analysis, these BnSRS genes could be divided into four subgroups, with each group sharing comparable conserved motifs and gene structure. Analysis of the upstream promoter region showed that BnSRS genes may regulate hormone responses, biotic and abiotic stress response, growth, and development in B. napus. The protein-protein interaction analysis revealed the involvement of BnSRS genes in various biological processes and metabolic pathways. Our analysis of BnSRS gene expression showed that 23 BnSRS genes in the callus tissue exhibited a dominant expression pattern, suggesting their critical involvement in cell dedifferentiation, cell division, and tissue development. In addition, association analysis between genotype and agronomic traits revealed that BnSRS genes may be linked to some important agronomic traits in B. napus, suggesting that BnSRS genes were widely involved in the regulation of important agronomic traits (including C16.0, C18.0, C18.1, C18.2 C18.3, C20.1, C22.1, GLU, protein, TSW, and FFT). In this study, we predicted the evolutionary relationships and potential functions of BnSRS gene family members, providing a basis for the development of BnSRS gene functions which could facilitate targeted functional studies and genetic improvement for elite breeding in B. napus.

Functional and evolutionary study of MLO gene family in the regulation of Sclerotinia stem rot resistance in Brassica napus L.

BACKGROUND: Oilseed rape (Brassica napus L.) is known as one of the most important oilseed crops cultivated around the world. However, its production continuously faces a huge challenge of Sclerotinia stem rot (SSR), a destructive disease caused by the fungus Sclerotinia sclerotiorum, resulting in huge yield loss annually. The SSR resistance in B. napus is quantitative and controlled by a set of minor genes. Identification of these genes and pyramiding them into a variety are a major strategy for SSR resistance breeding in B. napus. RESULTS: Here, we performed a genome-wide association study (GWAS) using a natural population of B. napus consisting of 222 accessions to identify BnaA08g25340D (BnMLO2_2) as a candidate gene that regulates the SSR resistance. BnMLO2_2 was a member of seven homolog genes of Arabidopsis Mildew Locus O 2 (MLO2) and the significantly SNPs were mainly distributed in the promoter of BnMLO2_2, suggesting a role of BnMLO2_2 expression level in the regulation of SSR resistance. We expressed BnMLO2_2 in Arabidopsis and the transgenic plants displayed an enhanced SSR resistance. Transcriptome profiling of different tissues of B. napus revealed that BnMLO2_2 had the most expression level in leaf and silique tissues among all the 7 BnMLO2 members and also expressed higher in the SSR resistant accession than in the susceptible accession. In Arabidopsis, mlo2 plants displayed reduced resistance to SSR, whereas overexpression of MLO2 conferred plants an enhanced SSR resistance. Moreover, a higher expression level of MLO2 showed a stronger SSR resistance in the transgenic plants. The regulation of MLO2 in SSR resistance may be associated with the cell death. Collinearity and phylogenetic analysis revealed a large expansion of MLO family in Brassica crops. CONCLUSION: Our study revealed an important role of BnMLO2 in the regulation of SSR resistance and provided a new gene candidate for future improvement of SSR resistance in B. napus and also new insights into understanding of MLO family evolution in Brassica crops.

Genome-wide analysis of hyperosmolality-gated calcium-permeable channel (OSCA) family members and their involvement in various osmotic stresses in Brassica napus.

Plant hyperosmolality-gated calcium-permeable channel (OSCA) is a calcium permeable cation channel that responds to hyperosmotic stress and plays a pivotal role in plant growth, development and stress response. Through a genome-wide survey, 41 OSCA genes were identified from the genome of Brassica napus. The OSCA family genes were unevenly distributed over 14 chromosomes of B. napus and phylogenetic analysis separated the OSCA family into four clades. Motif analyses indicated that OSCA proteins in the same clade were highly conserved and the protein conserved motifs shared similar composition patterns. The OSCA promoter regions contained many hormone-related elements and stress response elements. Gene duplication analysis elucidated that WGD/segmental duplication was the main driving force for the expansion of OSCA genes during evolution and these genes mainly underwent purifying selection. RNA-seq and qRT-PCR analysis of different tissues showed that OSCA genes are expressed and function mainly in the root. Among these genes, BnOSCA3.1a and BnOSCA3.1c had relatively high expression levels under osmotic stresses and cold stress and were highly expressed in different tissues. Protein interaction network analysis showed that a total of 5802 proteins might interact with OSCAs in B. napus, while KEGG/GO enrichment analysis indicated that OSCAs and their interacting proteins were mainly involved in plant response to abiotic stress. This systematic analysis of the OSCAs in B. napus identified gene structures, evolutionary features, expression patterns and related biological processes. These findings will facilitate further functional and evolutionary analysis of OSCAs in B. napus for breeding of osmotic-stress-resistant plants.

The plant trans-Golgi network component ECHIDNA regulates defense, cell death, and endoplasmic reticulum stress.

The trans-Golgi network (TGN) acts as a central platform for sorting and secreting various cargoes to the cell surface, thus being essential for the full execution of plant immunity. However, the fine-tuned regulation of TGN components in plant defense and stress response has been not fully elucidated. Our study revealed that despite largely compromising penetration resistance, the loss-of-function mutation of the TGN component protein ECHIDNA (ECH) induced enhanced postinvasion resistance to powdery mildew in Arabidopsis thaliana. Genetic and transcriptome analyses and hormone profiling demonstrated that ECH loss resulted in salicylic acid (SA) hyperaccumulation via the ISOCHORISMATE SYNTHASE 1 biosynthesis pathway, thereby constitutively activating SA-dependent innate immunity that was largely responsible for the enhanced postinvasion resistance. Furthermore, the ech mutant displayed accelerated SA-independent spontaneous cell death and constitutive POWDERY MILDEW RESISTANCE 4-mediated callose depositions. In addition, ECH loss led to a chronically prolonged endoplasmic reticulum stress in the ech mutant. These results provide insights into understanding the role of TGN components in the regulation of plant immunity and stress responses.

Genome-Wide Characterization of Trehalose-6-Phosphate Synthase Gene Family of Brassica napus and Potential Links with Agronomic Traits.

Trehalose and trehalose-6 phosphate played important roles in floral organ development, embryonic development, cell morphogenesis, and signal transduction under abiotic stress. However, little is known about the trehalose-6-phosphate synthase (TPS) gene family in Brassica napus. In this study, in total, 26 TPS genes in B. napus (BnTPS genes) were identified and classified into two groups. In each group, the BnTPS genes showed relatively conserved gene structures. The protein-protein interaction (PPI) network and enrichment analysis indicated that BnTPS genes were involved in the glycolysis/gluconeogenesis, fructose and mannose metabolism, galactose metabolism, pentose phosphate pathway, carbohydrate transmembrane transport, trehalose-phosphatase activity, etc. The expression of BnTPS genes varied greatly across different tissues, while most of the BnTPS genes showed a considerable improvement in expression under different abiotic stresses, indicating that BnTPS genes were significantly responsive to the abiotic treatments. In addition, the association mapping analysis revealed that eight BnTPS genes were potential regulators of particular agronomic traits. Among them, the gene BnTPS23 was significantly associated with the primary flowering time (PFT), full flowering time (FFT1), and final flowering time (FFT2), suggesting that BnTPS genes may play an important role in regulating key agronomic traits in B. napus. In summary, our research provides a better understanding of BnTPS genes, facilitates the breeding of superior B. napus varieties, and paves the way for future functional studies.

Genome-wide association study reveals a GLYCOGEN SYNTHASE KINASE 3 gene regulating plant height in Brassica napus.

Rapeseed (Brassica napus) is an allotetraploid crop that is the main source of edible oils and feed proteins in the world. The ideal plant architecture breeding is a major objective of rapeseed breeding and determining the appropriate plant height is a key element of the ideal plant architecture. Therefore, this study aims to improve the understanding of the genetic controls underlying plant height. The plant heights of 230 rapeseed accessions collected worldwide were investigated in field experiments over two consecutive years in Wuhan, China. Whole-genome resequencing of these accessions yielded a total of 1,707,194 informative single nucleotide polymorphisms (SNPs) that were used for genome-wide association analysis (GWAS). GWAS and haplotype analysis showed that BnaA01g09530D, which encodes BRASSINOSTEROID-INSENSITIVE 2 and belongs to the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family, was significantly associated with plant height in B. napus. Moreover, a total of 31 BnGSK3s with complete domains were identified from B. napus genome and clustered into four groups according to phylogenetic analysis, gene structure, and motif distribution. The expression patterns showed that BnGSK3s exhibited significant differences in 13 developmental tissues in B. napus, suggesting that BnGSK3s may be involved in tissue-specific development. Sixteen BnGSK3 genes were highly expressed the in shoot apical meristem, which may be related to plant height or architecture development. These results are important for providing new haplotypes of plant height in B. napus and for extending valuable genetic information for rapeseed genetic improvement of plant architecture.

Differential alternative splicing genes and isoform co-expression networks of Brassica napus under multiple abiotic stresses.

Alternative splicing (AS) is an important regulatory process that affects plant development and stress responses by greatly increasing the complexity of transcriptome and proteome. To understand how the AS landscape of B. napus changes in response to abiotic stresses, we investigated 26 RNA-seq libraries, including control and treatments with cold, dehydration, salt, and abscisic acid (ABA) at two different time points, to perform comparative alternative splicing analysis. Apparently, AS events increased under all stresses except dehydration for 1 h, and intron retention was the most common AS mode. In addition, a total of 357 differential alternative splicing (DAS) genes were identified under four abiotic stresses, among which 81 DAS genes existed in at least two stresses, and 276 DAS genes were presented under only one stress. A weighted gene co-expression network analysis (WGCNA) based on the splicing isoforms, rather than the genes, pinpointed out 23 co-expression modules associated with different abiotic stresses. Among them, a number of significant hub genes were also found to be DAS genes, which encode key isoforms involved in responses to single stress or multiple stresses, including RNA-binding proteins, transcription factors, and other important genes, such as RBP45C, LHY, MYB59, SCL30A, RS40, MAJ23.10, and DWF4. The splicing isoforms of candidate genes identified in this study could be a valuable resource for improving tolerance of B. napus against multiple abiotic stresses.

Alternative splicing reprogramming in fungal pathogen Sclerotinia sclerotiorum at different infection stages on Brassica napus.

Alternative splicing (AS) is an important post-transcriptional mechanism promoting the diversity of transcripts and proteins to regulate various life processes in eukaryotes. Sclerotinia stem rot is a major disease of Brassica napus caused by Sclerotinia sclerotiorum, which causes severe yield loss in B. napus production worldwide. Although many transcriptome studies have been carried out on the growth, development, and infection of S. sclerotiorum, the genome-wide AS events of S. sclerotiorum remain poorly understood, particularly at the infection stage. In this study, transcriptome sequencing was performed to systematically explore the genome-scale AS events of S. sclerotiorum at five important infection stages on a susceptible oilseed rape cultivar. A total of 130 genes were predicted to be involved in AS from the S. sclerotiorum genome, among which 98 genes were differentially expressed and may be responsible for AS reprogramming for its successful infection. In addition, 641 differential alternative splicing genes (DASGs) were identified during S. sclerotiorum infection, accounting for 5.76% of all annotated S. sclerotiorum genes, and 71 DASGs were commonly found at all the five infection stages. The most dominant AS type of S. sclerotiorum was found to be retained introns or alternative 3' splice sites. Furthermore, the resultant AS isoforms of 21 DASGs became pseudogenes, and 60 DASGs encoded different putative proteins with different domains. More importantly, 16 DASGs of S. sclerotiorum were found to have signal peptides and possibly encode putative effectors to facilitate the infection of S. sclerotiorum. Finally, about 69.27% of DASGs were found to be non-differentially expressed genes, indicating that AS serves as another important way to regulate the infection of S. sclerotiorum on plants besides the gene expression level. Taken together, this study provides a genome-wide landscape for the AS of S. sclerotiorum during infection as well as an important resource for further elucidating the pathogenic mechanisms of S. sclerotiorum.

Genome-wide characterization of ovate family protein gene family associated with number of seeds per silique in Brassica napus.

Ovate family proteins (OFPs) were firstly identified in tomato as proteins controlling the pear shape of the fruit. Subsequent studies have successively proved that OFPs are a class of negative regulators of plant development, and are involved in the regulation of complex traits in different plants. However, there has been no report about the functions of OFPs in rapeseed growth to date. Here, we identified the OFPs in rapeseed at the genomic level. As a result, a total of 67 members were obtained. We then analyzed the evolution from Arabidopsis thaliana to Brassica napus, illustrated their phylogenetic and syntenic relationships, and compared the gene structure and conserved domains between different copies. We also analyzed their expression patterns in rapeseed, and found significant differences in the expression of different members and in different tissues. Additionally, we performed a GWAS for the number of seeds per silique (NSPS) in a rapeseed population consisting of 204 natural accessions, and identified a new gene BnOFP13_2 significantly associated with NSPS, which was identified as a novel function of OFPs. Haplotype analysis revealed that the accessions with haplotype 3 had a higher NSPS than other accessions, suggesting that BnOFP13_2 is associated with NSPS. Transcript profiling during the five stages of silique development demonstrated that BnOFP13_2 negatively regulates NSPS. These findings provide evidence for functional diversity of OFP gene family and important implications for oilseed rape breeding.

Genome-wide identification and functional exploration of the legume lectin genes in Brassica napus and their roles in Sclerotinia disease resistance.

As one of the largest classes of lectins, legume lectins have a variety of desirable features such as antibacterial and insecticidal activities as well as anti-abiotic stress ability. The Sclerotinia disease (SD) caused by the soil-borne fungus Sclerotinia sclerotiorum is a devastating disease affecting most oil crops such as Brassica napus. Here, we identified 130 legume lectin (LegLu) genes in B. napus, which could be phylogenetically classified into seven clusters. The BnLegLu gene family has been significantly expanded since the whole-genome duplication (WGD) or segmental duplication. Gene structure and conserved motif analysis suggested that the BnLegLu genes were well conserved in each cluster. Moreover, relative to those genes only containing the legume lectin domain in cluster VI-VII, the genes in cluster I-V harbored a transmembrane domain and a kinase domain linked to the legume lectin domain in the C terminus. The expression of most BnLegLu genes was relatively low in various tissues. Thirty-five BnLegLu genes were responsive to abiotic stress, and 40 BnLegLu genes were strongly induced by S. sclerotiorum, with a most significant up-regulation of 715-fold, indicating their functional roles in SD resistance. Four BnLegLu genes were located in the candidate regions of genome-wide association analysis (GWAS) results which resulted from a worldwide rapeseed population consisting of 324 accessions associated with SD. Among them, the positive role of BnLegLus-16 in SD resistance was validated by transient expression in tobacco leaves. This study provides important information on BnLegLu genes, particularly about their roles in SD resistance, which may help targeted functional research and genetic improvement in the breeding of B. napus.

Analysis of Tissue-Specific Defense Responses to Sclerotinia sclerotiorum in Brassica napus.

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (S. sclerotiorum) is the main disease threat of oilseed rape (Brassica napus), resulting in huge economic losses every year. SSR resistance manifests as quantitative disease resistance (QDR), and no gene with complete SSR resistance has been cloned or reported so far. Transcriptome analysis has revealed a large number of defense-related genes and response processes. However, the similarities and differences in the defense responses of different tissues are rarely reported. In this study, we analyzed the similarities and differences of different tissues in response to S. sclerotiorum at 24 h post inoculation (hpi) by using the published transcriptome data for respective leaf and stem inoculation. At 24 hpi, large differences in gene expression exist in leaf and stem, and there are more differentially expressed genes and larger expression differences in leaf. The leaf is more sensitive to S. sclerotiorum and shows a stronger response than stem. Different defense responses appear in the leaf and stem, and the biosynthesis of lignin, callose, lectin, chitinase, PGIP, and PR protein is activated in leaf. In the stem, lipid metabolism-mediated defense responses are obviously enhanced. For the common defense responses in both leaf and stem, the chain reactions resulting from signal transduction and biological process take the primary responsibility. This research will be beneficial to exploit the potential of different tissues in plant defense and find higher resistance levels of genotypic variability in different environments. Our results are significant in the identification of resistance genes and analysis of defense mechanisms.

Genome-wide identification and functional analysis of cupin_1 domain-containing members involved in the responses to Sclerotinia sclerotiorum and abiotic stress in Brassica napus.

Cupin_1 domain-containing proteins (CDPs) are ubiquitously present in higher plants, which are known to play essential roles in various biological processes. In this study, we carried out genome-wide characterization and systematic investigation of the CDP genes in Brassica napus. A total of 96 BnCDPs, including 71 germin-like proteins (GLPs; proteins with a single cupin_1 domain) and 25 CDP bicupins (proteins with two cupin_1 domains), were identified and clustered into six distinct subfamilies (I-VI) based on the phylogenic analysis, gene structure and motif distribution. Further analysis indicated that whole-genome duplication (WGD) and segmental duplication are main contributors to the species-specific expansion of the BnCDP gene family, and all the duplicated genes subsequently underwent strong purification selection. The promoter region of BnCDPs showed enrichment of cis-regulatory elements associated with development, hormone and stress, as well as transcription factor binding sites, which validates the prediction that BnCDPs are widely involved in plant growth and biotic and abiotic stress responses. The BnCDPs in different subfamilies exhibited obvious differences in expression among 30 developmental tissues/stages of B. napus, implying that BnCDPs may be involved in tissue- and stage-specific developmental processes. Similar trends in expression of most BnCDPs were observed under Sclerotinia sclerotiorum inoculation and four abiotic stresses (dehydration, cold, ABA and salinity), particularly the BnGLPs in subfamily I and III with single cupin_1 domain, revealing that BnCDPs are of great importance in the environmental adaption of B. napus. We then performed a genome-wide association study (GWAS) of 274 B. napus core germplasms on S. sclerotiorum resistance and identified four significantly associated loci harboring five BnGLPs. The expression levels of two candidate genes, BnGLP1.A08 and BnGLP1.C08, were significantly correlated with S. sclerotiorum resistance. Their functional responses to multiple stages of S. sclerotiorum inoculation and four abiotic stresses were further examined through qPCR. Overall, this study provides rich resources for research on the function and evolutionary playground of CDP genes.