Resistance to the necrotrophic pathogen Rhizoctonia solani is a major bottleneck in cotton breeding, hindered by the lack of resistance germplasm, accurate phenotyping methods, and defined resistance genes. Here, we established a precise phenotyping system and performed a genome-wide association study (GWAS) on a natural population of Upland cotton (Gossypium hirsutum). A major resistance locus on chromosome D03, which explains 19.65% of the phenotypic variation, was identified. GhGLR4.8, encoding a glutamate receptor-like protein, was pinpointed as the causal gene. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated knockout significantly compromised resistance, whereas overexpression of the elite haplotype GhGLR4.8Hap3 conferred robust protection. Functional characterization indicated that GhGLR4.8 promotes lignin deposition and strengthens cell wall structure while maintaining reactive oxygen species (ROS) balance. Loss-of-function mutants displayed excessive ROS accumulation accompanied by pronounced tissue maceration, suggesting that GhGLR4.8 restricts fungal invasion through coordinated regulation of ROS homeostasis. This study reports the first major resistance gene against R. solani in cotton, providing key insights into the balance between ROS signaling and structural defense, as well as a valuable genetic resource for molecular breeding to stabilize cotton yield.

Cotton (Gossypium hirsutum L.) plants and seeds contain gossypol, a toxic compound that acts as a defence mechanism against pests and diseases but also limits the use of cottonseeds. This study aimed to investigate the mechanisms involved in the synthesis of gossypol in cotton and to create a promising germplasm material to conform to the Chinese cottonseed food safety standard (gossypol concent below 0.02 %) in cottonseed. Gl(2) or Gl(3) was overexpressed in Gossypium hirsutum acc. Jin668 via the 35S promoter using the Agrobacterium-mediated method, and the gossypol content in different tissues of cotton decreased by 86.52 similar to 99.99 %. This glandless phenotype resulted from the transcriptional repression of endogenous Gl(2) or Gl(3) due to the overexpression of exogenous Gl(2) or Gl(3). Additionally, four genes involved in pigment gland development were identified, and silencing these genes resulted in a significant (P < 0.05) decrease in gossypol content. Furthermore, the alpha GP seed-specific promoter was used to overexpress Gl(2) or Gl(3) in cottonseed plants, and a promising germplasm material was created in which the gossypol content, specifically in the seeds, decreased by 87 %. These results demonstrate that the overexpression of Gl(2) or Gl(3) in cotton represses the transcription of endogenous Gl(2) or Gl(3), resulting in a glandless phenotype in cotton plants via effects on the genes involved in pigment gland development. Moreover, a new cotton germplasm line with reduced gossypol levels only in seeds was developed for potential utilization in the food industry.

Leukemia stem cells (LSCs) play a critical role in the initiation, recurrence, and resistance to treatment of leukemia. Eradicating LSCs is crucial for the complete elimination of the disease. CD45RA is identified as an important marker for LSC subsets in acute myeloid leukemia (AML), providing a strategic target for therapy. In this report, we introduce Hu3A4, an innovative humanized CD45RA antibody devised to target LSCs expressing this antigen. Hu3A4 retains the antigen-recognition ability of its parental antibody while removing sequences from the variable region that could elicit human anti-mouse immune reactions. The modified variable regions of the heavy and light chains were intricately fused with the constant regions of human IgG1 heavy and light chains, respectively, producing a humanized antibody that emulates the structure of natural IgG. Hu3A4 was produced through recombinant expression in Chinese Hamster Ovary (CHO) cells, which ensured stable gene integration. In vitro tests revealed that Hu3A4 could effectively target and lyse the cells. Further, in vivo studies highlighted Hu3A4 ' s substantial anti-leukemic activity, significantly prolonging survival times in treated animal models compared to controls (P < 0.01). To summarize, Hu3A4 exhibits remarkable bioactivity and offers a promising therapeutic potential for the treatment of leukemia patients. Progressing Hu3A4 through additional preclinical and clinical studies is crucial to validate its efficacy as a therapeutic agent for leukemia.

To address inconsistent feature distributions caused by multiple working conditions in industrial processes and the lack of fault type labels, this paper proposes a Multi-source semi-supervised conditional constraint domain adaptation (MSSCCDA) fault classification method. Data from multiple working conditions are divided into multiple source domains and a target domain. A Convolutional neural network (CNN) extracts features from the multi-source domain data, and a triplet loss method reduces the feature distance between the same categories in different source domains. A semi-supervised conditional constraint domain adaptation method is proposed, which combines adversarial domain adaptation with a limited amount of labeled target domain data to pre-train the feature extractor. During adversarial training, center loss regularization is introduced as a conditional constraint for class feature alignment. The process of optimizing the feature extractor involves jointly leveraging adversarial loss to reduce inter-domain discrepancies between the source and target domains and minimizing class differences within target domain. Experiments conducted on the Tennessee-Eisman (TE) process and industrial three-phase flow (TFF) show that the average accuracy improves to 93% and 93%, respectively. The effectiveness of the proposed method is further validated through a comparison with two traditional domain adaptation approaches and five multi-source domain adaptation techniques.

Edible oils with high unsaturated fatty acids, particularly oleic acid, are beneficial to human health. Cotton is one of the top five oil crops in the world, but the mechanism of high-quality oil synthesis and regulatory networks in cotton are largely unclear. Here, we identified Leafy cotyledon1-like 1 (GhL1L1), a NF-YB subfamily gene that is specifically expressed during somatic embryogenesis and seed maturation in cotton. Overexpression of GhL1L1 regulates the contents of unsaturated fatty acids in cotton, especially in the seeds, which is associated with altered expression of the cotton fatty acid biosynthesis-related genes. GhL1L1 synergistically enhanced the expression of GhFAD2-1A by binding to the G-box in its promoter, leading to an increase in the content of linoleic acid. Furthermore, this activation could be enhanced by GhNF-YC2 and GhNF-YA1 by form a transcriptional complex. Collectively, these results contribute to provide new insights into the molecular mechanism of oil biosynthesis in cotton and can facilitate genetic manipulation of cotton varieties with enhanced oil content.

Although conventional hybrid breeding has paved the way for improving cotton production and other properties, it is undoubtedly time and labor consuming, while the cultivation of male sterile line can fix the problem. Here, we induced male sterile mutants by simultaneously editing three cotton EXCESS MICROSPOROCYTES1 ( GhEMS1 ) genes by CRISPR/Cas9. Notably, the GhEMS1 genes are homologous to AtEMS1 genes, which inhibit the production of middle layer and tapetum cells as well, leading to male sterility in cotton. Interestingly, there are necrosis-like dark spots on the surface of the anthers of GhEMS1s mutants, which is different from AtEMS1 mutant whose anther surface is clean and smooth, suggesting that the function of EMS1 gene has not been uncovered yet. Moreover, we have detected mutations in GhEMS1 genes from T 0 to T 3 mutant plants, which had necrosis-like dark spots as well, indicating that the mutation of the three GhEMS1 genes could be stably inherited. Dynamic transcriptomes showed plant hormone pathway and anther development genetic network were differential expression in mutant and wild-type anthers. And the lower level of IAA content in the mutant anthers than that in the wild type at four anther developmental stages may be the reason for the male sterility. This study not only facilitates the exploration of the basic research of cotton male sterile lines, but also provides germplasms for accelerating the hybrid breeding in cotton.

The double-recessive genic male-sterile (ms) line ms5 ms6 has been used to develop cotton (Gossypium hirsutum) hybrids for many years, but its molecular-genetic basis has remained unclear. Here, we identified the Ms5 and Ms6 loci through map-based cloning and confirmed their function in male sterility through CRISPR/Cas9 gene editing. Ms5 and Ms6 are highly expressed in stages 7-9 anthers and encode the cytochrome P450 mono-oxygenases CYP703A2-A and CYP703A2-D. The ms5 mutant carries a single-nucleotide C-to-T nonsense mutation leading to premature chain termination at amino acid 312 (GhCYP703A2-A312aa ), and ms6 carries three nonsynonymous substitutions (D98E, E168K, and G198R) and a synonymous mutation (L11L). Enzyme assays showed that GhCYP703A2 proteins hydroxylate fatty acids, and the ms5 (GhCYP703A2-A312aa ) and ms6 (GhCYP703A2-DD98E,E168K,G198R ) mutant proteins have decreased enzyme activities. Biochemical and lipidomic analyses showed that in ms5 ms6 plants, C12-C18 free fatty acid and phospholipid levels are significantly elevated in stages 7-9 anthers, while stages 8-10 anthers lack sporopollenin fluorescence around the pollen, causing microspore degradation and male sterility. Overall, our characterization uncovered functions of GhCYP703A2 in sporopollenin formation and fertility, providing guidance for creating male-sterile lines to facilitate hybrid cotton production and therefore exploit heterosis for improvement of cotton.

High temperature (HT) causes male sterility and decreases crop yields. Our previous works have demonstrated that sugar and auxin signaling pathways, Gossypium hirsutum Casein kinase I (GhCKI), and DNA methylation are all involved in HT-induced male sterility in cotton. However, the signaling mechanisms leading to distinct GhCKI expression patterns induced by HT between HT-tolerant and HT-sensitive cotton anthers remain largely unknown. Here, we identified a GhCKI promoter (ProGhCKI) region that functions in response to HT in anthers and found the transcription factor GhMYB4 binds to this region to act as an upstream positive regulator of GhCKI. In the tapetum of early-stage cotton anthers, upregulated expression of GhMYB4 under HT and overexpressed GhMYB4 under normal temperature both led to severe male sterility phenotypes, coupled with enhanced expression of GhCKI. We also found that GhMYB4 interacts with GhMYB66 to form a heterodimer to enhance its binding to ProGhCKI. However, GhMYB66 showed an expression pattern similar to GhMYB4 under HT but did not directly bind to ProGhCKI. Furthermore, HT reduced siRNA-mediated CHH DNA methylations in the GhMYB4 promoter, which enhanced the expression of GhMYB4 in tetrad stage anthers and promoted the formation of the GhMYB4/GhMYB66 heterodimer, which in turn elevated the transcription of GhCKI in the tapetum, leading to male sterility. Overall, we shed light on the GhMYB66-GhMYB4-GhCKI regulatory pathway in response to HT in cotton anthers.

The pollen wall exine provides a protective layer for the male gametophyte and is largely composed of sporopollenin, which comprises fatty acid derivatives and phenolics. However, the biochemical nature of the external exine is poorly understood. Here, we show that the male sterile line 1355A of cotton mutated in NO SPINE POLLEN (GhNSP) leads to defective exine formation. The GhNSP locus was identified through map-based cloning and confirmed by genetic analysis (co-segregation test and allele prediction using the CRISPR/Cas9 system). In situ hybridization showed that GhNSP is highly expressed in tapetum. GhNSP encodes a polygalacturonase protein homologous to AtQRT3, which suggests a function for polygalacturonase in pollen exine formation. These results indicate that GhNSP is functionally different from AtQRT3, the latter has the function of microspore separation. Biochemical analysis showed that the percentage of de-esterified pectin was significantly increased in the 1355A anthers at developmental stage 8. Furthermore, immunofluorescence studies using antibodies to the de-esterified and esterified homogalacturonan (JIM5 and JIM7) showed that the Ghnsp mutant exhibits abundant of de-esterified homogalacturonan in the tapetum and exine, coupled with defective exine formation. The characterization of GhNSP provides new understanding of the role of polygalacturonase and de-esterified homogalacturonan in pollen exine formation.

The potassium transporter family HAK/KUP/KT is a large group of proteins that are important in plant potassium transport and play a crucial role in plant growth and development. The members of the family play an important role in the response of plants to abiotic stress by maintaining osmotic balance. However, the function of the family in cotton is unclear. In this study, whole genome identification and characterization of the HAK/KUP/KT family from upland cotton (Gossypium hirsutum) were carried out. Bioinformatics methods were used to identify HAK/KUP/KT family members from the G. hirsutum genome and to analyse the physical and chemical properties, basic characteristics, phylogeny, chromosome location and expression of HAK/KUP/KT family members. A total of 41 HAK/KUP/KT family members were identified in the G. hirsutum genome. Phylogenetic analysis grouped these genes into four clusters (I, II, III, IV), containing 6, 10, 3 and 22 genes, respectively. Chromosomal distribution, gene structure and conserved motif analyses of the 41 GhHAK genes were subsequently performed. The RNA-seq data and qRT-PCR results showed that the family had a wide range of tissue expression patterns, and they responded to certain drought stresses. Through expression analysis, seven HAK/KUP/KT genes involved in drought stress were screened, and four genes with obvious phenotypes under drought stress were obtained by VIGS verification, which laid a theoretical foundation for the function of the cotton HAK/KUP/KT family.

The floral nectary, first recognized and described by Carl Linnaeus, is a remarkable organ that serves to provide carbohydrate-rich nectar to visiting pollinators in return for gamete transfer between flowers. Therefore, the nectary has indispensable biological significance in plant reproduction and even in evolution. Only two genes, CRC and STY, have been reported to regulate floral nectary development. However, it is still unknown what genes contribute to extrafloral nectary development. Here, we report that a nectary development gene in Gossypium (GoNe), annotated as an APETALA 2/ethylene-responsive factor (AP2/ERF), is responsible for the formation of both floral and extrafloral nectaries. GoNe plants that are silenced via virus-induced gene silencing technology and/or knocked out by Cas9 produce a nectariless phenotype. Point mutation and gene truncation simultaneously in duplicated genes Ne1 Ne2 lead to impaired nectary development in tetraploid cotton. There is no difference in the expression of the CRC and STY genes between the nectary TM-1 and the nectariless MD90ne in cotton. Therefore, the GoNe gene responsible for the formation of floral and extrafloral nectaries may be independent of CRC and STY. A complex mechanism might exist that restricts the nectary to a specific position with different genetic factors. Characterization of these target genes regulating nectary production has provided insights into the development, evolution, and function of nectaries and insect-resistant breeding.

As the main byproduct of cotton fiber, the cotton seed yields about 1.6 times that of fiber, with its oil rich in unsaturated fatty acids, mainly linoleic acid. It is desirable for breeders to increase the oil content of cottonseed without affecting the yield and quality of cotton fiber. In this study, a seed-specific promoter- (alpha-globulin gene promoter-) driven GhDGAT1 overexpression vector (PαGlob-GhDGAT1) was constructed and used to transform an upland cotton line YZ1 (Gossypium hirsutum). Overexpression of the cotton gene GhDGAT1 in cotton seeds increased its total oil content from 4.7% to 13.9% in different transgenic lines and different generations. With the increase of oil content, the composition and contents of the main fatty acids in cotton seed also changed, as reflected by the contents of the main saturated fatty acids and unsaturated oleic acid. GhDGAT1 could be used to increase oil content and improve oil composition in cottonseed.

棉酚作为棉花的主要植保素之一,抵抗病虫害侵害的同时也对单胃动物和人具有毒害性.本研究利用RNAi技术结合种子特异启动子,降低杜松烯合酶GhCAD1-A基因在种子中的表达,可用于选育棉花种子低酚而植株其它组织棉酚含量正常的材料.此类棉花既可以控制病虫害的侵害,又可以消除棉酚对棉籽的抗营养作用,对充分利用棉籽副产品、增加人类植物营养来源具有积极意义.

[目的]在棉花纤维产量和品质不受影响的前提下,创造高油酸、低亚油酸棉花新种质.[方法]棉花FAD2-1基因是催化单不饱和脂肪酸的油酸形成多不饱和脂肪酸的亚油酸的关键基因,采用信息分析学方法分析其蛋白质性质、结构、功能;克隆GhFAD2-1的保守片段381 bp,构建RNA干涉载体,采用农杆菌介导的下胚轴侵染方法将干涉载体转入棉花.利用气相色谱-质谱联用仪(Gas chromatography-mass spectrometer,GC-MS)测定转基因T2～T4植株种子的脂肪酸成分及含量.对T4转基因株系进行拷贝数鉴定、目的基因表达量检测、农艺性状及纤维品质考察.[结果]成功构建GhFAD2-1的RNA干涉载体并转入棉花,转基因株系目的基因GhFAD2-1的表达量显著低于对照,具有高油酸、低亚油酸的表型并且能稳定遗传给后代;转基因株系可将棉花种子油酸提高224.1％,将亚油酸降低237.5％.与对照材料相比,转基因株系的农艺性状、纤维品质没有明显差异.[结论]验证了棉花GhFA D2-1基因的功能并且为培育高油酸棉花品种提供了帮助.

Cotton (Gossypium spp.) is one of the most important economic crops and exhibits yield-improving heterosis in specific hybrid combinations. The genic male-sterility system is the main strategy used for producing heterosis in cotton. To better understand the mechanisms of male sterility in cotton, we carried out two-dimensional electrophoresis (2-DE) and label-free quantitative proteomics analysis in the anthers of two near-isogenic lines, the male-sterile line 1355A and the male-fertile line 1355B. We identified 39 and 124 proteins that were significantly differentially expressed between these two lines in the anthers at the tetrad stage (stage 7) and uninucleate pollen stage (stage 8), respectively. Gene ontology-based analysis revealed that these differentially expressed proteins were mainly associated with pyruvate, carbohydrate, and fatty acid metabolism. Biochemical analysis revealed that in the anthers of line 1355A, glycolysis was activated, which was caused by a reduction in fructose, glucose, and other soluble sugars, and that accumulation of acetyl-CoA was increased along with a significant increase in C14:0 and C18:1 free fatty acids. However, the activities of pyruvate dehydrogenase and fatty acid biosynthesis were inhibited and fatty acid β-oxidation was activated at the translational level in 1355A. We speculate that in the 1355A anther, high rates of glucose metabolism may promote fatty acid synthesis to enable anther growth. These results provide new insights into the molecular mechanism of genic male sterility in upland cotton.

Antibody-targeting therapy has drawn great interests to the hematologists and oncologists. 3A4, a novel antibody recognizing human CD45RA antigen, is a new target molecule for leukemias and holds a therapeutic potential for myeloid lineage leukemias. However, murine antibodies cannot be safely used in patients because of their strong immune reaction, humanization of the antibodies interested will be an important development step for therapeutic purpose. The aim of this study was to engineer the mouse 3A4 and to investigate the biological activity of its chimeric form. The humanized antibody composed of the 3A4 single-chain fragment of variable region and the human IgG1 Fc region, which was named human-mouse chimeric antibody 3A4 (Hm3A4). The function and biological activities of Hm3A4 were characterized using a variety of biological approaches. The results showed that Hm3A4 retained a strong binding activity to its antigen and could significantly block the binding of parental 3A4 to the antigen. In vitro experiments revealed that Hm3A4 could kill the target cells through complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity function. In vivo, Hm3A4 showed efficient antileukemia activity outperforming the nontreated mice. In conclusion, the chimeric antibody has an excellent biological activity after humanization and holds targeting therapeutic potential for myeloid leukemia, which warrants further development of this agent.

Background How to develop new cotton varieties possessing high yield traits of Upland cotton and superior fiber quality traits of Sea Island cotton remains a key task for cotton breeders and researchers. While multiple attempts bring in little significant progresses, the development of Chromosome Segment Substitution Lines (CSSLs) from Gossypium barbadense in G. hirsutum background provided ideal materials for aforementioned breeding purposes in upland cotton improvement. Based on the excellent fiber performance and relatively clear chromosome substitution segments information identified by Simple Sequence Repeat (SSR) markers, two CSSLs, MBI9915 and MBI9749, together with the recurrent parent CCRI36 were chosen to conduct transcriptome sequencing during the development stages of fiber elongation and Secondary Cell Wall (SCW) synthesis (from 10DPA and 28DPA), aiming at revealing the mechanism of fiber development and the potential contribution of chromosome substitution segments from Sea Island cotton to fiber development of Upland cotton. Results In total, 15 RNA-seq libraries were constructed and sequenced separately, generating 705.433 million clean reads with mean GC content of 45.13% and average Q30 of 90.26%. Through multiple comparisons between libraries, 1801 differentially expressed genes (DEGs) were identified, of which the 902 up-regulated DEGs were mainly involved in cell wall organization and response to oxidative stress and auxin, while the 898 down-regulated ones participated in translation, regulation of transcription, DNA-templated and cytoplasmic translation based on GO annotation and KEGG enrichment analysis. Subsequently, STEM software was performed to explicate the temporal expression pattern of DEGs. Two peroxidases and four flavonoid pathway-related genes were identified in the “oxidation-reduction process”, which could play a role in fiber development and quality formation. Finally, the reliability of RNA-seq data was validated by quantitative real-time PCR of randomly selected 20 genes. Conclusions The present report focuses on the similarities and differences of transcriptome profiles between the two CSSLs and the recurrent parent CCRI36 and provides novel insights into the molecular mechanism of fiber development, and into further exploration of the feasible contribution of G. barbadense substitution segments to fiber quality formation, which will lay solid foundation for simultaneously improving fiber yield and quality of upland cotton through CSSLs.

The ETS-related gene (ERG) has been demonstrated to be associated with overall survival in cytogenetically normal acute myeloid leukemia and acute T cell-lymphoblastic leukemia (T-ALL) in adult patients. However, there are no data available regarding the impact of ERG expression on childhood ALL. In the present study, ERG expression levels were analyzed in bone marrow samples from 119 ALL pediatric patients. ALL patients demonstrated higher ERG expression compared with the controls (P<0.0001). In addition, low ERG expression identified a group of patients with higher white blood cell counts (P=0.011), higher percentages of T-ALL immunophenotype (P=0.027), and higher relapse rates (P=0.009). Survival analyses demonstrated that low ERG expression was associated with inferior relapse-free survival (RFS) in childhood ALL (P=0.036) and was an independent prognostic factor in multivariable analyses for RFS. In conclusion, low ERG expression is associated with poor outcomes and may be used to serve as a molecular prognostic marker to identify patients with a high risk of relapse in childhood ALL.

Cottonseed oil is recognized as an important oil in food industry for its unique characters: low flavor reversion and the high level of antioxidants (VitaminE) as well as unsaturated fatty acid. However, the cottonseed oil content of cultivated cotton ( Gossypium hirsutum ) is only around 20%. In this study, we modified the accumulation of oils by the down-regulation of phosphoenolpyruvate carboxylase 1 ( GhPEPC1 ) via RNA interference in transgenic cotton plants. The qRT-PCR and enzyme activity assay revealed that the transcription and expression of GhPEPC1 was dramatically down-regulated in transgenic lines. Consequently, the cottonseed oil content in several transgenic lines showed a significant (P < 0.01) increase (up to 16.7%) without obvious phenotypic changes under filed condition when compared to the control plants. In order to elucidate the molecular mechanism of GhPEPC1 in the regulation of seed oil content, we quantified the expression of the carbon metabolism related genes of transgenic GhPEPC1 RNAi lines by transcriptome analysis. This analysis revealed the decrease of GhPEPC1 expression led to the increase expression of triacylglycerol biosynthesis-related genes, which eventually contributed to the lipid biosynthesis in cotton. This result provides a valuable information for cottonseed oil biosynthesis pathway and shows the potential of creating high cottonseed oil germplasm by RNAi strategy for cotton breeding.

Owing to the difficulty in obtaining mammary gland tissue from lactating animals, it is difficult to test the expression levels of genes in mammary gland. The aim of the current study was to identify if milk fat globule (MFG) in buffalo milk was an alternative to mammary gland (MG) and milk somatic cell (MSC) for gene expression analysis. Six buffalos in late lactation were selected to collect MFG and MSC, and then MG was obtained by surgery. MFG was stained with acridine orange to successfully visualise RNA and several cytoplasmic crescents in MFG. The total RNA in MFG was successfully isolated and the integrity was assessed by agarose gel electrophoresis. We analysed the cellular components in MFG, MG and MSC through testing the expression of cell-specific genes by qRT-PCR. The results showed that adipocyte-specific gene (AdipoQ) and leucocyte-specific genes (CD43, CSF1 and IL1α) in MFG were not detected, whereas epithelial cell marker genes (Keratin 8 and Keratin 18) in MFG were higher than in MSC and lower than in MG, fibroblast marker gene (vimentin) in MFG was significantly lower than in MG and MSC, milk protein genes (LALBA, BLG and CSN2) and milk fat synthesis-related genes (ACC, BTN1A1, FABP3 and FAS) in MFG were higher than in MG and MSC. In conclusion, the total RNA in MFG mainly derives from mammary epithelial cells and can be used to study the functional gene expression of mammary epithelial cells.