GhHAM regulates GoPGF-dependent gland development and contributes to broad-spectrum pest resistance in cotton.

Cotton is a globally cultivated crop, producing 87% of the natural fiber used in the global textile industry. The pigment glands, unique to cotton and its relatives, serve as a defense structure against pests and pathogens. However, the molecular mechanism underlying gland formation and the specific role of pigment glands in cotton's pest defense are still not well understood. In this study, we cloned a gland-related transcription factor GhHAM and generated the GhHAM knockout mutant using CRISPR/Cas9. Phenotypic observations, transcriptome analysis, and promoter-binding experiments revealed that GhHAM binds to the promoter of GoPGF, regulating pigment gland formation in cotton's multiple organs via the GoPGF-GhJUB1 module. The knockout of GhHAM significantly reduced gossypol production and increased cotton's susceptibility to pests in the field. Feeding assays demonstrated that more than 80% of the cotton bollworm larvae preferred ghham over the wild type. Furthermore, the ghham mutants displayed shorter cell length and decreased gibberellins (GA) production in the stem. Exogenous application of GA3 restored stem cell elongation but not gland formation, thereby indicating that GhHAM controls gland morphogenesis independently of GA. Our study sheds light on the functional differentiation of HAM proteins among plant species, highlights the significant role of pigment glands in influencing pest feeding preference, and provides a theoretical basis for breeding pest-resistant cotton varieties to address the challenges posed by frequent outbreaks of pests.

CONSTITUTIVE EXPRESSER OF PATHOGENESIS-RELATED GENES 5 is an RNA-binding protein controlling plant immunity via an RNA processing complex.

Plant innate immunity is capable of combating diverse and ever evolving pathogens. The plasticity of innate immunity could be boosted by RNA processing. Arabidopsis thaliana CONSTITUTIVE EXPRESSER OF PATHOGENESIS-RELATED GENES 5 (CPR5), a key negative immune regulator, is a component of the nuclear pore complex. Here we further identified CPR5 as a component of RNA processing complexes. Through genetic screening, we found that RNA splicing activator NineTeen Complex and RNA polyadenylation factor CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR, coordinately function downstream of CPR5 to activate plant immunity. CPR5 and these two regulators form a complex that is localized in nuclear speckles, an RNA processing organelle. Intriguingly, we found that CPR5 is an RNA-binding protein belonging to the Transformer 2 (Tra2) subfamily of the serine/arginine-rich family. The RNA recognition motif of CPR5 protein binds the Tra2-targeted RNA sequence in vitro and is functionally replaceable by those of Tra2 subfamily proteins. In planta, it binds RNAs of CPR5-regulated alternatively spliced genes (ASGs) identified by RNA-seq. ARGONAUTE 1 (AGO1) is one of the ASGs and, consistent with this, the ago1 mutant suppresses the cpr5 phenotype. These findings reveal that CPR5 is an RNA-binding protein linking RNA processing with plant immunity.

Strigolactones positively regulate Verticillium wilt resistance in cotton via crosstalk with other hormones.

Verticillium wilt caused by Verticillium dahliae is a serious vascular disease in cotton (Gossypium spp.). V. dahliae induces the expression of the CAROTENOID CLEAVAGE DIOXYGENASE 7 (GauCCD7) gene involved in strigolactone (SL) biosynthesis in Gossypium australe, suggesting a role for SLs in Verticillium wilt resistance. We found that the SL analog rac-GR24 enhanced while the SL biosynthesis inhibitor TIS108 decreased cotton resistance to Verticillium wilt. Knock-down of GbCCD7 and GbCCD8b genes in island cotton (Gossypium barbadense) decreased resistance, whereas overexpression of GbCCD8b in upland cotton (Gossypium hirsutum) increased resistance to Verticillium wilt. Additionally, Arabidopsis (Arabidopsis thaliana) SL mutants defective in CCD7 and CCD8 putative orthologs were susceptible, whereas both Arabidopsis GbCCD7- and GbCCD8b-overexpressing plants were more resistant to Verticillium wilt than wild-type (WT) plants. Transcriptome analyses showed that several genes related to the jasmonic acid (JA)- and abscisic acid (ABA)-signaling pathways, such as MYELOCYTOMATOSIS 2 (GbMYC2) and ABA-INSENSITIVE 5, respectively, were upregulated in the roots of WT cotton plants in responses to rac-GR24 and V. dahliae infection but downregulated in the roots of both GbCCD7- and GbCCD8b-silenced cotton plants. Furthermore, GbMYC2 suppressed the expression of GbCCD7 and GbCCD8b by binding to their promoters, which might regulate the homeostasis of SLs in cotton through a negative feedback loop. We also found that GbCCD7- and GbCCD8b-silenced cotton plants were impaired in V. dahliae-induced reactive oxygen species (ROS) accumulation. Taken together, our results suggest that SLs positively regulate cotton resistance to Verticillium wilt through crosstalk with the JA- and ABA-signaling pathways and by inducing ROS accumulation.

lncRNA7 and lncRNA2 modulate cell wall defense genes to regulate cotton resistance to Verticillium wilt.

In plants, long noncoding RNAs (lncRNAs) regulate disease resistance against fungi and other pathogens. However, the specific mechanism behind this regulation remains unclear. In this study, we identified disease resistance-related lncRNAs as well as their regulating genes and assessed their functions by infection of cotton (Gossypium) chromosome segment substitution lines with Verticillium dahliae. Our results demonstrated that lncRNA7 and its regulating gene Pectin methylesterase inhibitor 13 (GbPMEI13) positively regulated disease resistance via the silencing approach, while ectopic overexpression of GbPMEI13 in Arabidopsis (Arabidopsis thaliana) promoted growth and enhanced resistance to V. dahliae. In contrast, lncRNA2 and its regulating gene Polygalacturonase 12 (GbPG12) negatively regulated resistance to V. dahliae. We further found that fungal disease-related agents, including the pectin-derived oligogalacturonide (OG), could downregulate the expression of lncRNA2 and GbPG12, leading to pectin accumulation. Conversely, OG upregulated the expression of lncRNA7, which encodes a plant peptide phytosulfokine (PSK-α), which was confirmed by lncRNA7 overexpression and Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS) experiments. We showed that PSK-α promoted 3-Indoleacetic acid (IAA) accumulation and activated GbPMEI13 expression through Auxin Response Factor 5. Since it is an inhibitor of pectin methylesterase (PME), GbPMEI13 promotes pectin methylation and therefore increases the resistance to V. dahliae. Consistently, we also demonstrated that GbPMEI13 inhibits the mycelial growth and spore germination of V. dahliae in vitro. In this study, we demonstrated that lncRNA7, lncRNA2, and their regulating genes modulate cell wall defense against V. dahliae via auxin-mediated signaling, providing a strategy for cotton breeding.

The use of ribosome-nascent chain complex-seq to reveal the translated mRNA profile and the role of ASN1 in resistance to Verticillium wilt in cotton.

We combined traditional mRNA-seq and RNC-seq together to reveal post-transcriptional regulation events impacting gene expression and interactions between the serious fungal pathogen Verticillium dahliae and a susceptible host, Gossypium hirsutum TM-1. After screening the differentially expressed and translated genes, V. dahliae infection was observed to influence gene transcription and translation in its host. Interestingly, the asparagine synthase (ASN1) gene transcripts increased significantly with the increase of infection time, while the rate of ASN1 protein accumulation in host TM-1 was distinctly lower than that in resistant hosts. We knocked down the ASN1 gene in resistant plants (ZZM2), and found that Verticillium-resistance was significantly reduced upon knockdown of ASN1. Our study revealed both transcriptional and post-transcriptional regulation of gene expression in TM-1 cotton plants infected by V. dahliae, and showed that ASN1 functions in the V. dahliae resistance process. These insights support breeding of disease resistance in cotton.

Large-fragment insertion activates gene GaFZ (Ga08G0121) and is associated with the fuzz and trichome reduction in cotton (Gossypium arboreum).

Cotton seeds are typically covered by lint and fuzz fibres. Natural 'fuzzless' mutants are an ideal model system for identifying genes that regulate cell initiation and elongation. Here, using a genome-wide association study (GWAS), we identified a ~ 6.2 kb insertion, larINDELFZ , located at the end of chromosome 8, composed of a ~ 5.0 kb repetitive sequence and a ~ 1.2 kb fragment translocated from chromosome 12 in fuzzless Gossypium arboreum. The presence of larINDELFZ was associated with a fuzzless seed and reduced trichome phenotypes in G. arboreum. This distant insertion was predicted to be an enhancer, located ~ 18 kb upstream of the dominant-repressor GaFZ (Ga08G0121). Ectopic overexpression of GaFZ in Arabidopsis thaliana and G. hirsutum suggested that GaFZ negatively modulates fuzz and trichome development. Co-expression and interaction analyses demonstrated that GaFZ might impact fuzz fibre/trichome development by repressing the expression of genes in the very-long-chain fatty acid elongation pathway. Thus, we identified a novel regulator of fibre/trichome development while providing insights into the importance of noncoding sequences in cotton.

A copy number variant at the HPDA-D12 locus confers compact plant architecture in cotton.

Improving yield is a primary mission for cotton (Gossypium hirsutum) breeders; development of cultivars with suitable architecture for high planting density (HPDA) can increase yield per unit area. We characterized a natural cotton mutant, AiSheng98 (AS98), which exhibits shorter height, shorter branch length, and more acute branch angle than wild-type. A copy number variant at the HPDA locus on Chromosome D12 (HPDA-D12), encoding a dehydration-responsive element-binding (DREB) transcription factor, GhDREB1B, strongly affects plant architecture in the AS98 mutant. We found an association between a tandem duplication of a c. 13.5 kb segment in HPDA-D12 and elevated GhDREB1B expression resulting in the AS98 mutant phenotype. GhDREB1B overexpression confers a significant decrease in plant height and branch length, and reduced branch angle. Our results suggest that fine-tuning GhDREB1B expression may be a viable engineering strategy for modification of plant architecture favorable to high planting density in cotton.

Knockdown of Gh_A05G1554 (GhDHN_03) and Gh_D05G1729 (GhDHN_04) Dehydrin genes, Reveals their potential role in enhancing osmotic and salt tolerance in cotton.

In this investigation, whole-genome identification and functional characterization of the cotton dehydrin genes was carried out. A total of 16, 7, and 7 dehydrin proteins were identified in G. hirsutum, G. arboreum and G. raimondii, respectively. Through RNA sequence data and RT-qPCR validation, Gh_A05G1554 (GhDHN_03) and Gh_D05G1729 (GhDHN_04) were highly upregulated, and knockdown of the two genes, significantly reduced the ability of the plants to tolerate the effects of osmotic and salt stress. The VIGS-plants recorded significantly higher concentration levels of oxidants, hydrogen peroxide (H2O2) and malondialdehyde (MDA), furthermore, the four stress responsive genes GhLEA2, Gh_D12G2017 (CDKF4), Gh_A07G0747 (GPCR) and a transcription factor, trihelix, Gh_A05G2067, were significantly downregulated in VIGS-plants, but upregulated in wild types under osmotic and salt stress condition. The result indicated that dehydrin proteins are vital for plants and can be exploited in developing a more osmotic and salt stress-resilient germplasm to boost and improve cotton production.

Genome sequencing of the Australian wild diploid species Gossypium australe highlights disease resistance and delayed gland morphogenesis.

The diploid wild cotton species Gossypium australe possesses excellent traits including resistance to disease and delayed gland morphogenesis, and has been successfully used for distant breeding programmes to incorporate disease resistance traits into domesticated cotton. Here, we sequenced the G. australe genome by integrating PacBio, Illumina short read, BioNano (DLS) and Hi-C technologies, and acquired a high-quality reference genome with a contig N50 of 1.83 Mb and a scaffold N50 of 143.60 Mb. We found that 73.5% of the G. australe genome is composed of various repeat sequences, differing from those of G. arboreum (85.39%), G. hirsutum (69.86%) and G. barbadense (69.83%). The G. australe genome showed closer collinear relationships with the genome of G. arboreum than G. raimondii and has undergone less extensive genome reorganization than the G. arboreum genome. Selection signature and transcriptomics analyses implicated multiple genes in disease resistance responses, including GauCCD7 and GauCBP1, and experiments revealed induction of both genes by Verticillium dahliae and by the plant hormones strigolactone (GR24), salicylic acid (SA) and methyl jasmonate (MeJA). Experiments using a Verticillium-resistant domesticated G. barbadense cultivar confirmed that knockdown of the homologues of these genes caused a significant reduction in resistance against Verticillium dahliae. Moreover, knockdown of a newly identified gland-associated gene GauGRAS1 caused a glandless phenotype in partial tissues using G. australe. The G. australe genome represents a valuable resource for cotton research and distant relative breeding as well as for understanding the evolutionary history of crop genomes.

Identifying favorable alleles for improving key agronomic traits in upland cotton.

BACKGROUND: Gossypium hirsutum L. is grown worldwide and is the largest source of natural fiber crop. We focus on exploring the favorable alleles (FAs) for upland cotton varieties improvement, and further understanding the history of accessions selection and acumination of favorable allele during breeding. RESULTS: The genetic basis of phenotypic variation has been studied. But the accumulation of favorable alleles in cotton breeding history in unknown, and potential favorable alleles to enhance key agronomic traits in the future cotton varieties have not yet been identified. Therefore, 419 upland cotton accessions were screened, representing a diversity of phenotypic variations of 7362 G. hirsutum, and 15 major traits were investigated in 6 environments. These accessions were categorized into 3 periods (early, medium, and modern) according to breeding history. All accessions were divided into two major groups using 299 polymorphic microsatellite markers: G1 (high fiber yield and quality, late maturity) and G2 (low fiber yield and quality, early maturity). The proportion of G1 genotype gradually increased from early to modern breeding periods. Furthermore, 21 markers (71 alleles) were significantly associated (-log P > 4) with 15 agronomic traits in multiple environments. Seventeen alleles were identified as FAs; these alleles accumulated more in the modern period than in other periods, consistent with their phenotypic variation trends in breeding history. Our results demonstrate that the favorable alleles accumulated through breeding effects, especially for common favorable alleles. However, the potential elite accessions could be rapidly screened by rare favorable alleles. CONCLUSION: In our study, genetic variation and genome-wide associations for 419 upland cotton accessions were analyzed. Two favorable allele types were identified during three breeding periods, providing important information for yield/quality improvement of upland cotton germplasm.

Genome-wide comparative analysis of NBS-encoding genes in four Gossypium species.

BACKGROUND: Nucleotide binding site (NBS) genes encode a large family of disease resistance (R) proteins in plants. The availability of genomic data of the two diploid cotton species, Gossypium arboreum and Gossypium raimondii, and the two allotetraploid cotton species, Gossypium hirsutum (TM-1) and Gossypium barbadense allow for a more comprehensive and systematic comparative study of NBS-encoding genes to elucidate the mechanisms of cotton disease resistance. RESULTS: Based on the genome assembly data, 246, 365, 588 and 682 NBS-encoding genes were identified in G. arboreum, G. raimondii, G. hirsutum and G. barbadense, respectively. The distribution of NBS-encoding genes among the chromosomes was nonrandom and uneven, and was tended to form clusters. Gene structure analysis showed that G. arboreum and G. hirsutum possessed a greater proportion of CN, CNL, and N genes and a lower proportion of NL, TN and TNL genes compared to that of G. raimondii and G. barbadense, while the percentages of RN and RNL genes remained relatively unchanged. The percentage changes among them were largest for TNL genes, about 7 times. Exon statistics showed that the average exon numbers per NBS gene in G. raimondii and G. barbadense were all greater than that in G. arboretum and G. hirsutum. Phylogenetic analysis revealed that the TIR-NBS genes of G. barbadense were closely related with that of G. raimondii. Sequence similarity analysis showed that diploid cotton G. arboreum possessed a larger proportion of NBS-encoding genes similar to that of allotetraploid cotton G. hirsutum, while diploid G. raimondii possessed a larger proportion of NBS-encoding genes similar to that of allotetraploid cotton G. barbadense. The synteny analysis showed that more NBS genes in G. raimondii and G. arboreum were syntenic with that in G. barbadense and G. hirsutum, respectively. CONCLUSIONS: The structural architectures, amino acid sequence similarities and synteny of NBS-encoding genes between G. arboreum and G. hirsutum, and between G. raimondii and G. barbadense were the highest among comparisons between the diploid and allotetraploid genomes, indicating that G. hirsutum inherited more NBS-encoding genes from G. arboreum, while G. barbadense inherited more NBS-encoding genes from G. raimondii. This asymmetric evolution of NBS-encoding genes may help to explain why G. raimondii and G. barbadense are more resistant to Verticillium wilt, whereas G. arboreum and G. hirsutum are more susceptible to Verticillium wilt. The disease resistances of the allotetraploid cotton were related to their NBS-encoding genes especially in regard from which diploid progenitor they were derived, and the TNL genes may have a significant role in disease resistance to Verticillium wilt in G. raimondii and G. barbadense.

Arabidopsis phytochrome B promotes SPA1 nuclear accumulation to repress photomorphogenesis under far-red light.

Phytochrome A (phyA) is the primary photoreceptor mediating deetiolation under far-red (FR) light, whereas phyB predominantly regulates light responses in red light. SUPPRESSOR OF PHYA-105 (SPA1) forms an E3 ubiquitin ligase complex with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), which is responsible for the degradation of various photomorphogenesis-promoting factors, resulting in desensitization to light signaling. However, the role of phyB in FR light signaling and the regulatory pathway from light-activated phytochromes to the COP1-SPA1 complex are largely unknown. Here, we confirm that PHYB overexpression causes an etiolation response with reduced ELONGATED HYPOCOTYL5 (HY5) accumulation under FR light. Notably, phyB exerts its nuclear activities and promotes seedling etiolation in both the presence and absence of phyA in response to FR light. PhyB acts upstream of SPA1 and is functionally dependent on it in FR light signaling. PhyB interacts and forms a protein complex with SPA1, enhancing its nuclear accumulation under FR light. During the dark-to-FR transition, phyB is rapidly imported into the nucleus and facilitates nuclear SPA1 accumulation. These findings support the notion that phyB plays a role in repressing FR light signaling. Activity modulation of the COP1-SPA E3 complex by light-activated phytochromes is an effective and pivotal regulatory step in light signaling.

Advances in phylogenomics.

Phylogenomics is a new phylogenetic field that aims to rebuild phylogenetic relationship of organisms using whole genome data. It can effectively eliminate the impact of horizontal gene transfer and variant evolutionary rates on phylogeny. According to the genome data type they are based on, these methods can be classified into five groups: multi-gene based, gene content based, gene order based, K-string based, and metabolic pathway based. The mechanism, speed, accuracy, applicable range and their application of these methods are summarized. The prospects of phylogenomics and challenges that it is faced with are also discussed.

Positive roles of the Ca2+ sensors GbCML45 and GbCML50 in improving cotton Verticillium wilt resistance.

As a universal second messenger, cytosolic calcium (Ca2+) functions in multifaceted intracellular processes, including growth, development and responses to biotic/abiotic stresses in plant. The plant-specific Ca2+ sensors, calmodulin and calmodulin-like (CML) proteins, function as members of the second-messenger system to transfer Ca2+ signal into downstream responses. However, the functions of CMLs in the responses of cotton (Gossypium spp.) after Verticillium dahliae infection, which causes the serious vascular disease Verticillium wilt, remain elusive. Here, we discovered that the expression level of GbCML45 was promoted after V. dahliae infection in roots of cotton, suggesting its potential role in Verticillium wilt resistance. We found that knockdown of GbCML45 in cotton plants decreased resistance while overexpression of GbCML45 in Arabidopsis thaliana plants enhanced resistance to V. dahliae infection. Furthermore, there was physiological interaction between GbCML45 and its close homologue GbCML50 by using yeast two-hybrid and bimolecular fluorescence assays, and both proteins enhanced cotton resistance to V. dahliae infection in a Ca2+-dependent way in a knockdown study. Detailed investigations indicated that several defence-related pathways, including salicylic acid, ethylene, reactive oxygen species and nitric oxide signalling pathways, as well as accumulations of lignin and callose, are responsible for GbCML45- and GbCML50-modulated V. dahliae resistance in cotton. These results collectively indicated that GbCML45 and GbCML50 act as positive regulators to improve cotton Verticillium wilt resistance, providing potential targets for exploitation of improved Verticillium wilt-tolerant cotton cultivars by genetic engineering and molecular breeding.

Analysis of sea-island cotton and upland cotton in response to Verticillium dahliae infection by RNA sequencing.

BACKGROUND: Cotton Verticillium wilt is a serious soil-borne vascular disease that causes great economic loss each year. However, due to the lack of resistant varieties of upland cotton, the molecular mechanisms of resistance to this disease, especially to the pathogen Verticillium dahliae, remain unclear. RESULTS: We used the RNA-seq method to research the molecular mechanisms of cotton defence responses to different races of Verticillium dahliae by comparing infected sea-island cotton and upland cotton. A total of 77,212 unigenes were obtained, and the unigenes were subjected to BLAST searching and annotated using the GO and KO databases. Six sets of digital gene expression data were mapped to the reference transcriptome. The gene expression profiles of cotton infected with Verticillium dahliae were compared to those of uninfected cotton; 44 differentially expressed genes were identified. Regarding genes involved in the phenylalanine metabolism pathway, the hydroxycinnamoyl transferase gene (HCT) was upregulated in upland cotton whereas PAL, 4CL, CAD, CCoAOMT, and COMT were upregulated in sea-island cotton. Almost no differentially expressed genes in this pathway were identified in sea-island cotton and upland cotton when they were infected with V. dahliae V991 and V. dahliae D07038, respectively. CONCLUSIONS: Our comprehensive gene expression data at the transcription level will help elucidate the molecular mechanisms of the cotton defence response to V. dahliae. By identifying the genes involved in the defence response of each type of cotton to V. dahliae, our data not only provide novel molecular information for researchers, but also help accelerate research on genes involved in defences in cotton.

Transcriptome analysis of stem development in the tumourous stem mustard Brassica juncea var. tumida Tsen et Lee by RNA sequencing.

BACKGROUND: Tumourous stem mustard (Brassica juncea var. tumida Tsen et Lee) is an economically and nutritionally important vegetable crop of the Cruciferae family that also provides the raw material for Fuling mustard. The genetics breeding, physiology, biochemistry and classification of mustards have been extensively studied, but little information is available on tumourous stem mustard at the molecular level. To gain greater insight into the molecular mechanisms underlying stem swelling in this vegetable and to provide additional information for molecular research and breeding, we sequenced the transcriptome of tumourous stem mustard at various stem developmental stages and compared it with that of a mutant variety lacking swollen stems. RESULTS: Using Illumina short-read technology with a tag-based digital gene expression (DGE) system, we performed de novo transcriptome assembly and gene expression analysis. In our analysis, we assembled genetic information for tumourous stem mustard at various stem developmental stages. In addition, we constructed five DGE libraries, which covered the strains Yong'an and Dayejie at various development stages. Illumina sequencing identified 146,265 unigenes, including 11,245 clusters and 135,020 singletons. The unigenes were subjected to a BLAST search and annotated using the GO and KO databases. We also compared the gene expression profiles of three swollen stem samples with those of two non-swollen stem samples. A total of 1,042 genes with significantly different expression levels occurring simultaneously in the six comparison groups were screened out. Finally, the altered expression levels of a number of randomly selected genes were confirmed by quantitative real-time PCR. CONCLUSIONS: Our data provide comprehensive gene expression information at the transcriptional level and the first insight into the understanding of the molecular mechanisms and regulatory pathways of stem swelling and development in this plant, and will help define new mechanisms of stem development in non-model plant organisms.

Gene expression profiling during gland morphogenesis of a mutant and a glandless upland cotton.

To identify genes involved in pigment gland morphogenesis in cotton, gene expression was profiled using genechip (Affymetrix) during pigment gland morphogenesis in cotton variety Xiangmian-18, which has glandless seeds but glanded plants, and a glandless line, N5. The results showed that 303 genes were differentially expressed by a factor greater than two during gland morphogenesis; 59% (180) of these genes shared similarity with known genes in GenBank. These genes play roles in defense response, response to oxidative stress, peroxidase activity, and other metabolic pathways. KOBAS (KEGG Orthology-Based Annotation System) indicate that these genes are involved in 68 biochemical pathways. These findings suggest that the related defense response, gossypol biosynthesis pathway and other complex regulation may be associated with pigment gland morphogenesis in cotton. The results may provide a basis for further study and serve as a guide for related research.

Yak (Bos grunniens) stomach lysozyme: molecular cloning, expression and its antibacterial activities.

The cDNA coding for stomach lysozyme in yak was cloned. The cloned cDNA contains a 432 bp open reading frame and encodes 143 amino acids (16.24 KDa) with a signal peptide of 18 amino acids. Further analysis revealed that its amino acid sequence shares many common properties with cow milk lysozyme. Expression of this gene was also detected in mammary gland tissue by RT-PCR. Phylogenetic relationships among yak stomach lysozyme and 8 cow lysozymes indicated that the yak enzyme is more closely related to both cow milk lysozyme and the pseudogene PsiNS4 than cow stomach lysozyme. Recombinant yak lysozyme purified by Ni(2+)-column showed a molecular weight of 33.78 kDa and exhibited lytic activity against Staphylococcus aureus, providing evidence of its antibacterial activities.

Comprehensive Analysis of Respiratory Burst Oxidase Homologs (Rboh) Gene Family and Function of GbRboh5/18 on Verticillium Wilt Resistance in Gossypium barbadense.

Respiratory burst oxidase homologs (Rbohs) play a predominant role in reactive oxygen species (ROS) production, which is crucial in plant growth, differentiation, as well as their responses to biotic and abiotic stresses. To date, however, there is little knowledge about the function of cotton Rboh genes. Here, we identified a total of 87 Rbohs from five sequenced Gossypium species (the diploids Gossypium arboreum, Gossypium raimondii, and Gossypium australe, and the allotetraploids Gossypium hirsutum and Gossypium barbadense) via BLAST searching their genomes. Phylogenetic analysis of the putative 87 cotton Rbohs revealed that they were divided into seven clades. All members within the same clade are generally similar to each other in terms of gene structure and conserved domain arrangement. In G. barbadense, the expression levels of GbRbohs in the CladeD were induced in response to a fungal pathogen and to hormones (i.e., jasmonic acid and abscisic acid), based upon which the main functional member in CladeD was discerned to be GbRboh5/18. Further functional and physiological analyses showed that the knock-down of GbRboh5/18 expression attenuates plant resistance to Verticillium dahliae infection. Combined with the molecular and biochemical analyses, we found less ROS accumulation in GbRboh5/18-VIGS plants than in control plants after V. dahliae infection. Overexpression of GbRboh5/18 in G. barbadense resulted in more ROS accumulation than in control plants. These results suggest that GbRboh5/18 enhances the cotton plants' resistance against V. dahliae by elevating the levels of ROS accumulation. By integrating phylogenetic, molecular, and biochemical approaches, this comprehensive study provides a detailed overview of the number, phylogeny, and evolution of the Rboh gene family from five sequenced Gossypium species, as well as elucidating the function of GbRboh5/18 for plant resistance against V. dahliae. This study sheds fresh light on the molecular evolutionary properties and function of Rboh genes in cotton, and provides a reference for improving cotton's responses to the pathogen V. dahliae.

[Analysis of genetic difference among Scrophularia ningpoensis cultivars by SRAP].

OBJECTIVE: The genetic difference among Scrophularia ningpoensis cultivars were analyzed in molecular level. METHOD: Ninety-two individuls of three S. ningpoensis cultivars were employed to be analyzed by the approach of Sequence-related Amplified Polymorphism (SRAP).The parameters were calculated by POPGENE1.31 and the relationship was constructed based on UPGMA method. RESULT: 1) A total of 227 bands were scored and 199 bands of them were polymorphic. 2) The result is showed that there is a medium level of genetic diversity among three cultivars. At species level: percentage of polymorphic loci PPB=52.42%, effective number of alleles N(e)=1.2812, Nei's gene diversity H=0.1671 and Shannon's information index H(sp)= 0.2526; At cultivar level: PPB=21.44%, N(e)=1.1216, Nei's gene diversity H=0.0725 and Shannon's information index H(pop)= 0.1083. 3) The Nei's coefficient of genetic differentiation was 0.5625, which was consistent with the Shannon's coefficient of genetic differentiation (0.5713). Most of the genetic variation existed among cultivars. 4) The gene flow (N(m)=0.3889) was less among cultivars, indicating that the degree of genetic differentiation was higher. 5) Genetic similarity coefficient were changed from 0.8082 to 0.9133. By clustering analysis, the classified result of SRAP marker between traditional modal character was almost same. CONCLUSION: The genetic diversity of samples of S. ningpoensis is medium. The genetic difference among cultivar is higher than that within cultivar.