Approximately half of all cancers have p53 inactivating mutations,in addition to which most malignancies inactivate the p53 pathway by increasing p53 inhibitors,decreasing p53 activators,or inactivating p53 downstream...Approximately half of all cancers have p53 inactivating mutations,in addition to which most malignancies inactivate the p53 pathway by increasing p53 inhibitors,decreasing p53 activators,or inactivating p53 downstream targets.A growing number of researches have demonstrated that p53 can influence tumor progression through the tumor microenvironment(TME).TME is involved in the process of tumor development and metastasis and affects the clinical prognosis of patients.p53 participates in host immunity and engages in the immune landscape of the TME,but the specific mechanisms remain to be investigated.This review briefly explores the interactions between different states of p53 and TME components and their mechanisms,as well as their effects on tumor progression.To understand the progress of drug development and clinical studies related to p53 and tumor microenvironment.展开更多
The tea plant cultivar‘Zhonghuang 2'(ZH2)possesses albino-induced yellow leaves that contain low levels of catechins but high contents of amino acids.However,the molecular mechanism underlying the yellow leaf phe...The tea plant cultivar‘Zhonghuang 2'(ZH2)possesses albino-induced yellow leaves that contain low levels of catechins but high contents of amino acids.However,the molecular mechanism underlying the yellow leaf phenotype of ZH2 has not been elucidated clearly.In the current research,the yellow shoots(ZH2-Y)and naturally converted green shoots(ZH2-G)of ZH2 were studied using metabolic and proteomic profiling for a better understanding of the mechanism underlying phenotype formation.In total,107 differentially changed metabolites(DCMs)were identified from the GC-MS-based metabolomics,and 189 differentially accumulated proteins(DAPs)were identified from the tandem mass tag(TMT)-based quantitative proteomics.Subsequently,integrated analysis revealed that‘porphyrin and chlorophyll metabolism',‘carbon fixation in photosynthetic organisms',and‘phenylpropanoid biosynthesis'pathways were commonly enriched for DAPs and DCMs.We further found that the inhibition of chlorophyll biosynthesis,the deficiency of photosynthetic proteins and the imbalance of the ROS-scavenging system were the crucial reasons responsible for the chlorosis,chloroplast abnormality and photooxidative damage of ZH2 leaves.Altogether,our research combines metabolomics and proteomics approaches to uncover the molecular mechanism leading to the yellow leaf phenotype of tea plants.展开更多
Naturally occurring yellow leaf mutants are an important resource for studying pigment content and biosynthesis,as well as related gene expression.In our ongoing cultivation of Rehmannia chingii H.L.Li,we found an off...Naturally occurring yellow leaf mutants are an important resource for studying pigment content and biosynthesis,as well as related gene expression.In our ongoing cultivation of Rehmannia chingii H.L.Li,we found an off-type yellow plant.The yellowing started with the new leaves and gradually spread downward until the entire plant exhibited a stable shade of yellow.We studied the differences in the chlorophyll and carotenoid content,carotenoid profile,and transcriptome of this yellow-leaf mutant(P2).Compared to the wild-type R.chingii plant(P1),P2 leaves had significantly lower chlorophyll and carotenoid content.LC-MS/MS analysis revealed that P2 had higher quantities of severalmetabolites in the carotenoid biosynthesis pathway.Transcriptome sequencing results showed that genes involved in porphyrin metabolism,carbon fixation,photosynthesis and antenna proteins,terpenoid backbone biosynthesis,and carotenoid biosynthesis were differentially expressed between P1 and P2.Large-scale expression differences were observed in the phytohormone and MAPK signaling pathways,as well as in 15 transcription factor families.We discuss possible mechanisms responsible for the yellowleaf color in P2.These preliminary data are valuable for further exploring the molecular mechanisms of leaf color formation and associated pathways.展开更多
Magnaporthe oryzae,the causal agent of rice blast,induces significant upregulation of OsPR10b,a pathogenesis-related(PR)pollen allergen(BetV-1)family gene.To investigate its role in immunity,we generated OsPR10b knock...Magnaporthe oryzae,the causal agent of rice blast,induces significant upregulation of OsPR10b,a pathogenesis-related(PR)pollen allergen(BetV-1)family gene.To investigate its role in immunity,we generated OsPR10b knockout mutants in the Zhonghua 11(ZH11)background.OsPR10b was predominantly expressed in rice calli and strongly induced by M.oryzae infection.Knockout mutants(ospr10b-1 and ospr10b-2)exhibited heightened susceptibility to both M.oryzae and Xanthomonas oryzae pv.oryzae(Xoo),demonstrating that OsPR10b positively regulates resistance to blast and bacterial blight.Our findings elucidate OsPR10b’s role in rice immunity and provide genetic resources for disease-resistant breeding.展开更多
Leaf color mutants (LCMs) provide crucial insights into the regulatory mechanisms underlying chloroplast development,photo synthesis,and stre ss adaptation.In this study,we identified a temperature-sensitive albino mu...Leaf color mutants (LCMs) provide crucial insights into the regulatory mechanisms underlying chloroplast development,photo synthesis,and stre ss adaptation.In this study,we identified a temperature-sensitive albino mutant,tsa4,characterized by an albino phenotype at the seedling stage and abnormal chloroplast development at temperatures below 25℃.展开更多
Aerial organs in rice,including leaves,stems,and grains,are crucial for photosynthesis,lodging resistance,and yield.Therefore,an in-depth study on the development of these organs can lay a foundation for achieving hig...Aerial organs in rice,including leaves,stems,and grains,are crucial for photosynthesis,lodging resistance,and yield.Therefore,an in-depth study on the development of these organs can lay a foundation for achieving high and stable rice yields.In this study,we isolated a novel slender aerial organ mutant sao,which is characterized by a significant reduction in the width of leaves,stems,and grains.Histological analysis revealed that the slender phenotype of aerial organs in sao is caused by impaired cell proliferation and elongation.展开更多
Tiller number is a crucial agronomic trait for achieving high yield in rice.NAC proteins play critical roles in regulating plant growth and development.However,the role of NAC transcription factors in regulating tille...Tiller number is a crucial agronomic trait for achieving high yield in rice.NAC proteins play critical roles in regulating plant growth and development.However,the role of NAC transcription factors in regulating tiller number remains poorly understood.In this study,we isolated a rice NAC gene,OsNAC022,which is conserved in cereal crops and functions as a transcriptional activator.To investigate the role of this gene in rice,we used CRISPR/Cas9 technology to generate a homozygous mutant named CR-osnac022,which exhibited increased tiller number.展开更多
CRISPR-Cas9 has emerged as a powerful tool for gene editing,and it has been widely used in plant functional genomics research and crop genetic breeding(Chen et al.2019).The target specificity of CRISPR-Cas9 relies on ...CRISPR-Cas9 has emerged as a powerful tool for gene editing,and it has been widely used in plant functional genomics research and crop genetic breeding(Chen et al.2019).The target specificity of CRISPR-Cas9 relies on the 20-base-pair single guide RNA(sgRNA),which makes creating plant-specific mutant libraries through large-scale synthesis of sgRNAs targeting multiple genes or even the whole genome relatively quick and straightforward.展开更多
Background:Innovative oral targeted therapies are warranted for patients with human epidermal growth factor receptor 2(HER2)-mutant non-small-cell lung cancer(NSCLC).Zongertinib is an oral,irreversible,HER2-selective ...Background:Innovative oral targeted therapies are warranted for patients with human epidermal growth factor receptor 2(HER2)-mutant non-small-cell lung cancer(NSCLC).Zongertinib is an oral,irreversible,HER2-selective tyrosine kinase inhibitor that has been shown to have efficacy in persons with advanced or metastatic solid tumors with HER2 alterations in a phase 1 study.展开更多
Chloroplasts are essential for normal plant growth and development.In plants,pentatricopeptide repeat(PPR)proteins mediate RNA processing in chloroplasts.Here,we characterized a rice albino leaf 5(al5)mutant which exh...Chloroplasts are essential for normal plant growth and development.In plants,pentatricopeptide repeat(PPR)proteins mediate RNA processing in chloroplasts.Here,we characterized a rice albino leaf 5(al5)mutant which exhibits albinism during early leaf development.The MutMap+analysis and transformation experiments revealed that AL5 encodes a chloroplast-localized P-type PPR protein.The AL5 mutation resulted in the defective splicing of ribosomal protein L2(rpl2)and ribosomal protein S12(rps12),which are involved in the synthesis of chloroplast 50S and 30S ribosomal subunits,respectively.The RNA-electrophoretic mobility shift assay(REMSA)further demonstrated that AL5 directly binds to rpl2 transcripts.Finally,disruption of AL5 led to reduced expression of plastid-encoded polymerase(PEP)-dependent plastid genes and nuclear-encoded photosynthetic genes.Notably,the albino al5 mutant phenotype was regulated by low temperature.These results suggest that AL5 participates in plastid RNA splicing and plays an important role in chloroplast development in rice.展开更多
Lesion mimic often exhibits leaf disease-like symptoms even in the absence of pathogen infection,and is characterized by a hypersensitive-response(HR)that closely linked to plant disease resistance.Despite this,only a...Lesion mimic often exhibits leaf disease-like symptoms even in the absence of pathogen infection,and is characterized by a hypersensitive-response(HR)that closely linked to plant disease resistance.Despite this,only a few lesion mimic genes have been identified in wheat.In this investigation,a lesion mimic wheat mutant named je0297 was discovered,showing no alteration in yield components when compared to the wild type(WT).Segregation ratio analysis of the F_(2)individuals resulting from the cross between the WT and the mutant revealed that the lesion mimic was governed by a single recessive gene in je0297.Using Bulked segregant analysis(BSA)and exome capture sequencing,we mapped the lesion mimic gene designated as lm6 to chromosome 6BL.Further gene fine mapping using 3315 F_(2)individuals delimited the lm6 within a 1.18 Mb region.Within this region,we identified 16 high-confidence genes,with only two displaying mutations in je0297.Notably,one of the two genes,responsible for encoding flavonol synthase,exhibited altered expression levels.Subsequent phenotype analysis of TILLING mutants confirmed that the gene encoding flavonol synthase was indeed the causal gene for lm6.Transcriptome sequencing analysis revealed that the DEGs between the WT and mutant were significantly enriched in KEGG pathways related to flavonoid biosynthesis,including flavone and flavonol biosynthesis,isoflavonoid biosynthesis,and flavonoid biosynthesis pathways.Furthermore,more than 30 pathogen infection-related(PR)genes exhibited upregulation in the mutant.Corresponding to this expression pattern,the flavonoid content in je0297 showed a significant decrease in the 4^(th)leaf,accompanied by a notable accumulation of reactive oxygen,which likely contributed to the development of lesion mimic in the mutant.This investigation enhances our comprehension of cell death signaling pathways and provides a valuable gene resource for the breeding of disease-resistant wheat.展开更多
Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.I...Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.In this study,we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa,a variety widely cultivated in Northeast China.Subsequently,we created a single transcript CRISPR/Cas9(CRISPR_2.0)toolkit,incorporating multiplex gRNAs,designed for gene editing in Gongnong 1.Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter,a widely employed polymeraseⅡconstitutive promoter known for strong transgene expression in dicots.To assess the toolkit’s efficiency,we targeted PALM1,a gene associated with a recognizable multifoliate phenotype.Utilizing the CRISPR_2.0 toolkit,we directed PALM1 editing at two sites in the wild-type Gongnong 1.Results indicated a 35.1%occurrence of editing events all in target 2 alleles,while no mutations were detected at target 1 in the transgenic-positive lines.To explore more efficient sgRNAs,we developed a rapid,reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation,incorporating the visible reporter MtLAP1.This screening system demonstrated that most purple visible hairy roots underwent gene editing.Notably,sgRNA3,with an 83.0%editing efficiency,was selected using the visible hairy root system.As anticipated,tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype.These palm1 lines demonstrated an average crude protein yield increase of 21.5%compared to trifoliolate alfalfa.Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.展开更多
Gliomas are primary brain tumors derived from glial cells of the central nervous system,afflicting both adults and children with distinct characteristics and therapeutic challenges.Recent developments have ushered in ...Gliomas are primary brain tumors derived from glial cells of the central nervous system,afflicting both adults and children with distinct characteristics and therapeutic challenges.Recent developments have ushered in novel clinical and molecular prognostic factors,reshaping treatment paradigms based on classi-fication and grading,determined by histological attributes and cellular lineage.This review article delves into the diverse treatment modalities tailored to the specific grades and molecular classifications of gliomas that are currently being discussed and used clinically in the year 2023.For adults,the therapeutic triad typically consists of surgical resection,chemotherapy,and radiotherapy.In contrast,pediatric gliomas,due to their diversity,require a more tailored approach.Although complete tumor excision can be curative based on the location and grade of the glioma,certain non-resectable cases demand a chemotherapy approach usually involving,vincristine and carboplatin.Addi-tionally,if surgery or chemotherapy strategies are unsuccessful,Vinblastine can be used.Despite recent advancements in treatment methodologies,there remains a need of exploration in the literature,particularly concerning the efficacy of treatment regimens for isocitrate dehydrogenase type mutant astrocytomas and fine-tuned therapeutic approaches tailored for pediatric cohorts.This review article explores into the therapeutic modalities employed for both adult and pediatric gliomas in the context of their molecular classification.展开更多
Gliomas originate from glial cells in the central nervous system.Approximately 80%-85%of malignant brain tumors in adults are gliomas.The most common central nervous system tumor in children is low-grade pediatric gli...Gliomas originate from glial cells in the central nervous system.Approximately 80%-85%of malignant brain tumors in adults are gliomas.The most common central nervous system tumor in children is low-grade pediatric glioma.Diagnosis was determined by histological features until 2016 when the World Health Organization classification integrated molecular data with anatomopathological information to achieve a more integral diagnosis.Molecular characterization has led to better diagnostic and prognostic staging,which in turn has increased the precision of treatment.Current efforts are focused on more effective therapies to prolong survival and improve the quality of life of adult and pediatric patients with glioma.However,improvements in survival have been modest.Currently,clinical guidelines,as well as the article by Mohamed et al accompanying this editorial piece,are adapting treatment recommendations(surgery,chemotherapy,and radiotherapy)according to diagnosis and prognosis guided by molecular biomarkers.Furthermore,this paves the way for the design of clinical trials with new therapies,which is especially important in pediatric gliomas.展开更多
The strawberry species Fragaria nilgerrensis Schlechtendal ex J.Gay,renowned for its distinctive white,fragrant peach-like fruits and strong disease resistance,is an exceptional research material.In a previous study,a...The strawberry species Fragaria nilgerrensis Schlechtendal ex J.Gay,renowned for its distinctive white,fragrant peach-like fruits and strong disease resistance,is an exceptional research material.In a previous study,an ethyl methane sulfonate(EMS)mutant library was established for this species,resulting in various yellow leaf mutants.Leaf yellowing materials are not only the ideal materials for basic studies on photosynthesis mechanism,chloroplast development,and molecular regulation of various pigments,but also have important utilization value in ornamental plants breeding.The present study focused on four distinct yellow leaf mutants:mottled yellow leaf(MO),yellow green leaf(YG),light green leaf(LG),and buddha light leaf(BU).The results revealed that the flavonoid content and carotenoid-to-chlorophyll ratio exhibited a significant increase among these mutants,while experiencing a significant decrease in chlorophyll and carotenoid contents compared to the wild type(WT).To clarify the regulatory mechanisms and network relationships underlying these mutants,the RNA-seq and weighted gene coexpression network(WGCNA)analyses were employed.The results showed flavonoid metabolism pathway was enriched both in MO and YG mutants,while the chlorophyll biosynthesis pathway and carotenoid degradation pathway were only enriched in MO and YG mutants,respectively.Subsequently,key structural genes and transcription factors were identified on metabolic pathways of three pigments through correlation analyses and quantitative experiments.Furthermore,a R2R3-MYB transcription factor,FnMYB4,was confirmed to be positively correlated with flavonoid synthesis through transient overexpression,virus-induced gene silencing(VIGS),and RNA interference(RNAi),accompanying by reoccurrence and attenuation of mutant phenotype.Finally,dual-luciferase(LUC)and yeast one-hybrid assays confirmed the binding of FnMYB4 to the FnFLS and FnF3H promoters,indicating that FnMYB4 positively regulates flavonoid synthesis.In addition,correlation analyses suggested that FnMYB4 also might be involved in chlorophyll and carotenoid metabolisms.These findings demonstrated the pivotal regulatory role of FnMYB4 in strawberry leaf coloration.展开更多
Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-st...Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.展开更多
Glass catfish(Kryptopterus vitreolus)are notable in the aquarium trade for their highly transparent body pattern.This transparency is due to the loss of most reflective iridophores and light-absorbing melanophores in ...Glass catfish(Kryptopterus vitreolus)are notable in the aquarium trade for their highly transparent body pattern.This transparency is due to the loss of most reflective iridophores and light-absorbing melanophores in the main body,although certain black and silver pigments remain in the face and head.To date,however,the molecular mechanisms underlying this transparent phenotype remain largely unknown.To explore the genetic basis of this transparency,we constructed a chromosome-level haplotypic genome assembly for the glass catfish,encompassing 32 chromosomes and 23344 protein-coding genes,using PacBio and Hi-C sequencing technologies and standard assembly and annotation pipelines.Analysis revealed a premature stop codon in the putative albinism-related tyrp1b gene,encoding tyrosinase-related protein 1,rendering it a nonfunctional pseudogene.Notably,a synteny comparison with over 30 other fish species identified the loss of the endothelin-3(edn3b)gene in the glass catfish genome.To investigate the role of edn3b,we generated edn3b^(−/−)mutant zebrafish,which exhibited a remarkable reduction in black pigments in body surface stripes compared to wild-type zebrafish.These findings indicate that edn3b loss contributes to the transparent phenotype of the glass catfish.Our high-quality chromosome-scale genome assembly and identification of key genes provide important molecular insights into the transparent phenotype of glass catfish.These findings not only enhance our understanding of the molecular mechanisms underlying transparency in glass catfish,but also offer a valuable genetic resource for further research on pigmentation in various animal species.展开更多
The emergence of antiretroviral resistance mutations represents a major threat to the achievement of national and global goals for the elimination of HIV-1 infection. The global strategy in 2019 in Cte d'Ivoire is...The emergence of antiretroviral resistance mutations represents a major threat to the achievement of national and global goals for the elimination of HIV-1 infection. The global strategy in 2019 in Cte d'Ivoire is a new national policy for the management of people living with HIV with the administration of dolutegravir (DTG)-based fixed-dose combination. The aim of our study was to evaluate HIV-1 resistance to antiretrovirals (ARVs) in infected adult subjects in Cte d’Ivoire in the context of a systematic switch to a DTG-based combination. Between February 2022 and October 2023, a cross-sectional survey with random sampling was conducted in 06 services caring for people living with HIV. A total of 139 participants were included in the study. Adults with a viral load ≥ 1000 copies/mL were tested for HIV-1 ARV resistance mutations. Molecular analyses were performed using protocol of ANRS-MIE (National Agency for Research on AIDS and emerging infectious diseases). The interpretation is performed by HIVGRAD (https://www.hiv-grade.de/cms/grade/). The frequencies of HIV-1 resistance to non-nucleotide reverse transcriptase inhibitors (NNRTIs), nucleotide reverse transcriptase inhibitors (NRTIs), integrase inhibitors (IINTs) and protease inhibitors (PIs) were 82%, 73%, 19% and 11% respectively. The main mutations observed in the different classes were K103N (45%), M184V (64%), E157Q (19%) and L10V/M46I/A71V/I54V (6%) respectively. This study reveals the emergence of resistance to DTG-based fixed-dose combinations, favored by high rates of resistance to NRTIs and NNRTIs. This finding underlines the need for enhanced viral load monitoring and HIV-1 genotyping tests to guide the choice of NRTIs for combination therapy. In addition, monitoring for mutations to second-generation NRTIs is essential, given the scale-up of DTG-based regimens currently underway in Cte d’Ivoire.展开更多
New clinical approaches are imperative beyond the widely adopted National Comprehensive Cancer Network (NCCN) guidelines, utilized by prominent cancer institutions. Cancer is the leading cause of death among individua...New clinical approaches are imperative beyond the widely adopted National Comprehensive Cancer Network (NCCN) guidelines, utilized by prominent cancer institutions. Cancer is the leading cause of death among individuals younger than 85 years within the United States. Despite significant technological advances, including the expenditure of hundreds of billions, treatment outcomes and overall survival have not notably improved for most types of advanced cancer over the last several decades. Over the past 24 years, Envita Medical Centers has pioneered a unique form of personalized treatment approach for late-stage and refractory cancer patients, introducing groundbreaking innovations in the field. Our integrated algorithm utilizes advanced genomics, transcriptomics, and highly tailored immunotherapy, resulting in remarkable outcome improvements. This study presents Envita’s innovative personalized treatment algorithms and examines the response outcomes of 199 late-stage cancer patients treated at Envita Medical Centers over a two-year period. Compared to standard of care and palliative chemotherapy, Envita’s treatment demonstrated a remarkable 35-fold improvement in overall response rates (Figure 1). Moreover, 88% of the patients, the majority presenting with Stage 3 or 4 cancer, experienced a 43-fold improvement in quality of life with minimal side effects, as compared to standard of care chemotherapy and palliative care. This revolutionary success is attributed to Envita’s personalized therapeutic algorithms, which incorporate customized immunotherapy. Envita’s precision care approach has also achieved a 100% better response rate compared to over 65 global chemotherapy clinical trials with more than 2700 patients. The results from this study suggest that a wider utilization of Envita’s personalized approach can significantly benefit patients with late-stage and refractory cancer.展开更多
Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides,which are advantageous for plant immunity,animal nutrition and health.However,thermophilic endo-chitinases are scar...Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides,which are advantageous for plant immunity,animal nutrition and health.However,thermophilic endo-chitinases are scarce and the transformation from exo-to endo-activity of chitinases is still a challenging problem.In this study,to enhance the endo-activity of the thermophilic chitinase Chi304,we proposed two approaches for rational design based on comprehensive structural and evolutionary analyses.Four effective single-point mutants were identified among 28 designed mutations.The ratio of(GlcNAc)3 to(GlcNAc)2 quantity(DP3/2)in the hydrolysates of the four single-point mutants undertaking colloidal chitin degradation were 1.89,1.65,1.24,and 1.38 times that of Chi304,respectively.When combining to double-point mutants,the DP3/2 proportions produced by F79A/W140R,F79A/M264L,F79A/W272R,and M264L/W272R were 2.06,1.67,1.82,and 1.86 times that of Chi304 and all four double-point mutants exhibited enhanced endo-activity.When applied to produce chitooligosaccharides(DP≥3),F79A/W140R accumulated the most(GlcNAc)4,while M264L/W272R was the best to produce(GlcNAc)3,which was 2.28 times that of Chi304.The two mutants had exposed shallower substrate-binding pockets and stronger binding abilities to shape the substrate.Overall,this research offers a practical approach to altering the cutting pattern of a chitinase to generate functional chitooligosaccharides.展开更多
基金supported by the Natural Science Foundation of Hunan Province(No.2021JJ30589)Hunan Provincial Natural Science Foundation Sector Joint Fund(No.2023JJ60049).
文摘Approximately half of all cancers have p53 inactivating mutations,in addition to which most malignancies inactivate the p53 pathway by increasing p53 inhibitors,decreasing p53 activators,or inactivating p53 downstream targets.A growing number of researches have demonstrated that p53 can influence tumor progression through the tumor microenvironment(TME).TME is involved in the process of tumor development and metastasis and affects the clinical prognosis of patients.p53 participates in host immunity and engages in the immune landscape of the TME,but the specific mechanisms remain to be investigated.This review briefly explores the interactions between different states of p53 and TME components and their mechanisms,as well as their effects on tumor progression.To understand the progress of drug development and clinical studies related to p53 and tumor microenvironment.
基金supported by the Project for Collaborative Promotion of Agricultural Major Technology of Zhejiang Province(Grant No.2022XTTGCY01-02)the National Natural Science Foundation of China(Grant Nos.31700615,32172633)+1 种基金the China Agriculture Research System of MOF and MARA(Grant No.CARS19-01A)the Special Project of Zhejiang Province(Grant No.2020R52036)。
文摘The tea plant cultivar‘Zhonghuang 2'(ZH2)possesses albino-induced yellow leaves that contain low levels of catechins but high contents of amino acids.However,the molecular mechanism underlying the yellow leaf phenotype of ZH2 has not been elucidated clearly.In the current research,the yellow shoots(ZH2-Y)and naturally converted green shoots(ZH2-G)of ZH2 were studied using metabolic and proteomic profiling for a better understanding of the mechanism underlying phenotype formation.In total,107 differentially changed metabolites(DCMs)were identified from the GC-MS-based metabolomics,and 189 differentially accumulated proteins(DAPs)were identified from the tandem mass tag(TMT)-based quantitative proteomics.Subsequently,integrated analysis revealed that‘porphyrin and chlorophyll metabolism',‘carbon fixation in photosynthetic organisms',and‘phenylpropanoid biosynthesis'pathways were commonly enriched for DAPs and DCMs.We further found that the inhibition of chlorophyll biosynthesis,the deficiency of photosynthetic proteins and the imbalance of the ROS-scavenging system were the crucial reasons responsible for the chlorosis,chloroplast abnormality and photooxidative damage of ZH2 leaves.Altogether,our research combines metabolomics and proteomics approaches to uncover the molecular mechanism leading to the yellow leaf phenotype of tea plants.
基金funded by the Beijing Gardening andGreeningYouth InnovationTalent Support Program(kjcx202336)theKey R&D Project of theOpen Subject of the Beijing Key Laboratory for Greening Plant Breeding(YZZD202403).
文摘Naturally occurring yellow leaf mutants are an important resource for studying pigment content and biosynthesis,as well as related gene expression.In our ongoing cultivation of Rehmannia chingii H.L.Li,we found an off-type yellow plant.The yellowing started with the new leaves and gradually spread downward until the entire plant exhibited a stable shade of yellow.We studied the differences in the chlorophyll and carotenoid content,carotenoid profile,and transcriptome of this yellow-leaf mutant(P2).Compared to the wild-type R.chingii plant(P1),P2 leaves had significantly lower chlorophyll and carotenoid content.LC-MS/MS analysis revealed that P2 had higher quantities of severalmetabolites in the carotenoid biosynthesis pathway.Transcriptome sequencing results showed that genes involved in porphyrin metabolism,carbon fixation,photosynthesis and antenna proteins,terpenoid backbone biosynthesis,and carotenoid biosynthesis were differentially expressed between P1 and P2.Large-scale expression differences were observed in the phytohormone and MAPK signaling pathways,as well as in 15 transcription factor families.We discuss possible mechanisms responsible for the yellowleaf color in P2.These preliminary data are valuable for further exploring the molecular mechanisms of leaf color formation and associated pathways.
基金supported by the Special Fund for Agro-scientific Research in the Public Interest of Fujian Province,China(Grant No.2023R1021006)the National Natural Science Foundation of China(Grant No.32402387)+1 种基金the extended research project of the National Natural Science Foundation of China(Grant No.GJYS202511)the 5511 Collaborative Engineering Project,China(Grant No.XTCXGC2021001).
文摘Magnaporthe oryzae,the causal agent of rice blast,induces significant upregulation of OsPR10b,a pathogenesis-related(PR)pollen allergen(BetV-1)family gene.To investigate its role in immunity,we generated OsPR10b knockout mutants in the Zhonghua 11(ZH11)background.OsPR10b was predominantly expressed in rice calli and strongly induced by M.oryzae infection.Knockout mutants(ospr10b-1 and ospr10b-2)exhibited heightened susceptibility to both M.oryzae and Xanthomonas oryzae pv.oryzae(Xoo),demonstrating that OsPR10b positively regulates resistance to blast and bacterial blight.Our findings elucidate OsPR10b’s role in rice immunity and provide genetic resources for disease-resistant breeding.
基金financially supported by the National Natural Science Foundation of China(Grant Nos.32341026 and 32171998)the Hunan Provincial Science and Technology Innovation Program,China(Grant No.2023NK1010)the Changsha Natural Science Foundation,China(Grant No.20209001).
文摘Leaf color mutants (LCMs) provide crucial insights into the regulatory mechanisms underlying chloroplast development,photo synthesis,and stre ss adaptation.In this study,we identified a temperature-sensitive albino mutant,tsa4,characterized by an albino phenotype at the seedling stage and abnormal chloroplast development at temperatures below 25℃.
基金supported by the Sichuan Science and Technology Program,China(Grant No.2024JDRC0064)the Chongqing Talent Program Foundation,China(Grant No.cstc2024ycjh-bgzxm0063)+1 种基金the National Natural Science Foundation of China(Grant No.32470354)the Sichuan Science and Technology Innovation and Entrepreneurship Seedling Foundation,China(Grant No.2024JDRC0064).
文摘Aerial organs in rice,including leaves,stems,and grains,are crucial for photosynthesis,lodging resistance,and yield.Therefore,an in-depth study on the development of these organs can lay a foundation for achieving high and stable rice yields.In this study,we isolated a novel slender aerial organ mutant sao,which is characterized by a significant reduction in the width of leaves,stems,and grains.Histological analysis revealed that the slender phenotype of aerial organs in sao is caused by impaired cell proliferation and elongation.
基金supported by the National Natural Science Foundation of China(Grant No.32372083)the Fundamental Research Funds for Central Public Welfare Research Institutes of Chinese Rice Research Institute(Grant No.CPSIBRF-CNRRI-202401)+1 种基金the Strategic Cooperation Project between Chongqing Municipal People’s Government and Chinese Academy of Agricultural Sciences‘Introduction,Excavation and Innovative Utilization of High-quality Rice Resources’,the China Agriculture Research System(Grant No.CARS-01-14)the Agricultural Science and Technology Innovation Program,China(Grant No.CAAS‐ASTIP‐2013‐CNRRI).
文摘Tiller number is a crucial agronomic trait for achieving high yield in rice.NAC proteins play critical roles in regulating plant growth and development.However,the role of NAC transcription factors in regulating tiller number remains poorly understood.In this study,we isolated a rice NAC gene,OsNAC022,which is conserved in cereal crops and functions as a transcriptional activator.To investigate the role of this gene in rice,we used CRISPR/Cas9 technology to generate a homozygous mutant named CR-osnac022,which exhibited increased tiller number.
基金supported by the National Natural Science Foundation of China(32272670 and 31972986)the Key Research and Development Program of Shaanxi Province,China(2023-YBNY-059)。
文摘CRISPR-Cas9 has emerged as a powerful tool for gene editing,and it has been widely used in plant functional genomics research and crop genetic breeding(Chen et al.2019).The target specificity of CRISPR-Cas9 relies on the 20-base-pair single guide RNA(sgRNA),which makes creating plant-specific mutant libraries through large-scale synthesis of sgRNAs targeting multiple genes or even the whole genome relatively quick and straightforward.
基金Funded by Boehringer IngelheimBeamion LUNG-1 ClinicalTrials.gov number,NCT04886804.
文摘Background:Innovative oral targeted therapies are warranted for patients with human epidermal growth factor receptor 2(HER2)-mutant non-small-cell lung cancer(NSCLC).Zongertinib is an oral,irreversible,HER2-selective tyrosine kinase inhibitor that has been shown to have efficacy in persons with advanced or metastatic solid tumors with HER2 alterations in a phase 1 study.
基金supported by the Open Competition Program of Top Ten Critical Priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan of Guangdong Province(2022SDZG05)the Guangdong Province Rural Revitalization Strategy Special Fund Seed Industry Revitalization Project(2022-NJS-15-001)the Key-Area Research and Development Program of Guangdong Province(2022B0202060005).
文摘Chloroplasts are essential for normal plant growth and development.In plants,pentatricopeptide repeat(PPR)proteins mediate RNA processing in chloroplasts.Here,we characterized a rice albino leaf 5(al5)mutant which exhibits albinism during early leaf development.The MutMap+analysis and transformation experiments revealed that AL5 encodes a chloroplast-localized P-type PPR protein.The AL5 mutation resulted in the defective splicing of ribosomal protein L2(rpl2)and ribosomal protein S12(rps12),which are involved in the synthesis of chloroplast 50S and 30S ribosomal subunits,respectively.The RNA-electrophoretic mobility shift assay(REMSA)further demonstrated that AL5 directly binds to rpl2 transcripts.Finally,disruption of AL5 led to reduced expression of plastid-encoded polymerase(PEP)-dependent plastid genes and nuclear-encoded photosynthetic genes.Notably,the albino al5 mutant phenotype was regulated by low temperature.These results suggest that AL5 participates in plastid RNA splicing and plays an important role in chloroplast development in rice.
基金supported by the National Key Research and Development Program of China(2022YFD1200700)the Nuclear Energy Development Research Program of the State Administration of Science,Technology,and Industry for National Defense(Crop Varietal Improvement and Insect Pests Control by Nuclear Radiation)the China Agriculture Research System of MOF and MARA(CARS-03)。
文摘Lesion mimic often exhibits leaf disease-like symptoms even in the absence of pathogen infection,and is characterized by a hypersensitive-response(HR)that closely linked to plant disease resistance.Despite this,only a few lesion mimic genes have been identified in wheat.In this investigation,a lesion mimic wheat mutant named je0297 was discovered,showing no alteration in yield components when compared to the wild type(WT).Segregation ratio analysis of the F_(2)individuals resulting from the cross between the WT and the mutant revealed that the lesion mimic was governed by a single recessive gene in je0297.Using Bulked segregant analysis(BSA)and exome capture sequencing,we mapped the lesion mimic gene designated as lm6 to chromosome 6BL.Further gene fine mapping using 3315 F_(2)individuals delimited the lm6 within a 1.18 Mb region.Within this region,we identified 16 high-confidence genes,with only two displaying mutations in je0297.Notably,one of the two genes,responsible for encoding flavonol synthase,exhibited altered expression levels.Subsequent phenotype analysis of TILLING mutants confirmed that the gene encoding flavonol synthase was indeed the causal gene for lm6.Transcriptome sequencing analysis revealed that the DEGs between the WT and mutant were significantly enriched in KEGG pathways related to flavonoid biosynthesis,including flavone and flavonol biosynthesis,isoflavonoid biosynthesis,and flavonoid biosynthesis pathways.Furthermore,more than 30 pathogen infection-related(PR)genes exhibited upregulation in the mutant.Corresponding to this expression pattern,the flavonoid content in je0297 showed a significant decrease in the 4^(th)leaf,accompanied by a notable accumulation of reactive oxygen,which likely contributed to the development of lesion mimic in the mutant.This investigation enhances our comprehension of cell death signaling pathways and provides a valuable gene resource for the breeding of disease-resistant wheat.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA26030301)Hohhot Key R&D Project(2023-JBGSS-1),the National Natural Science Foundation of China(U23A200206,32071864,32325035)+1 种基金the Taishan Scholar Program of Shandong(to Chunxiang Fu)the Shandong Provincial Natural Science Foundation(ZR202210270038)。
文摘Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.In this study,we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa,a variety widely cultivated in Northeast China.Subsequently,we created a single transcript CRISPR/Cas9(CRISPR_2.0)toolkit,incorporating multiplex gRNAs,designed for gene editing in Gongnong 1.Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter,a widely employed polymeraseⅡconstitutive promoter known for strong transgene expression in dicots.To assess the toolkit’s efficiency,we targeted PALM1,a gene associated with a recognizable multifoliate phenotype.Utilizing the CRISPR_2.0 toolkit,we directed PALM1 editing at two sites in the wild-type Gongnong 1.Results indicated a 35.1%occurrence of editing events all in target 2 alleles,while no mutations were detected at target 1 in the transgenic-positive lines.To explore more efficient sgRNAs,we developed a rapid,reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation,incorporating the visible reporter MtLAP1.This screening system demonstrated that most purple visible hairy roots underwent gene editing.Notably,sgRNA3,with an 83.0%editing efficiency,was selected using the visible hairy root system.As anticipated,tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype.These palm1 lines demonstrated an average crude protein yield increase of 21.5%compared to trifoliolate alfalfa.Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.
文摘Gliomas are primary brain tumors derived from glial cells of the central nervous system,afflicting both adults and children with distinct characteristics and therapeutic challenges.Recent developments have ushered in novel clinical and molecular prognostic factors,reshaping treatment paradigms based on classi-fication and grading,determined by histological attributes and cellular lineage.This review article delves into the diverse treatment modalities tailored to the specific grades and molecular classifications of gliomas that are currently being discussed and used clinically in the year 2023.For adults,the therapeutic triad typically consists of surgical resection,chemotherapy,and radiotherapy.In contrast,pediatric gliomas,due to their diversity,require a more tailored approach.Although complete tumor excision can be curative based on the location and grade of the glioma,certain non-resectable cases demand a chemotherapy approach usually involving,vincristine and carboplatin.Addi-tionally,if surgery or chemotherapy strategies are unsuccessful,Vinblastine can be used.Despite recent advancements in treatment methodologies,there remains a need of exploration in the literature,particularly concerning the efficacy of treatment regimens for isocitrate dehydrogenase type mutant astrocytomas and fine-tuned therapeutic approaches tailored for pediatric cohorts.This review article explores into the therapeutic modalities employed for both adult and pediatric gliomas in the context of their molecular classification.
文摘Gliomas originate from glial cells in the central nervous system.Approximately 80%-85%of malignant brain tumors in adults are gliomas.The most common central nervous system tumor in children is low-grade pediatric glioma.Diagnosis was determined by histological features until 2016 when the World Health Organization classification integrated molecular data with anatomopathological information to achieve a more integral diagnosis.Molecular characterization has led to better diagnostic and prognostic staging,which in turn has increased the precision of treatment.Current efforts are focused on more effective therapies to prolong survival and improve the quality of life of adult and pediatric patients with glioma.However,improvements in survival have been modest.Currently,clinical guidelines,as well as the article by Mohamed et al accompanying this editorial piece,are adapting treatment recommendations(surgery,chemotherapy,and radiotherapy)according to diagnosis and prognosis guided by molecular biomarkers.Furthermore,this paves the way for the design of clinical trials with new therapies,which is especially important in pediatric gliomas.
基金the National Natural Science Foundation of China(Grant No.32372652)the Liaoning Provincial Science and Technology Project of‘Jiebangguashuai’(Grant No.2022JH1/10400016)the Shenyang Academician and Expert Workstation Project(Grant No.2022-15).
文摘The strawberry species Fragaria nilgerrensis Schlechtendal ex J.Gay,renowned for its distinctive white,fragrant peach-like fruits and strong disease resistance,is an exceptional research material.In a previous study,an ethyl methane sulfonate(EMS)mutant library was established for this species,resulting in various yellow leaf mutants.Leaf yellowing materials are not only the ideal materials for basic studies on photosynthesis mechanism,chloroplast development,and molecular regulation of various pigments,but also have important utilization value in ornamental plants breeding.The present study focused on four distinct yellow leaf mutants:mottled yellow leaf(MO),yellow green leaf(YG),light green leaf(LG),and buddha light leaf(BU).The results revealed that the flavonoid content and carotenoid-to-chlorophyll ratio exhibited a significant increase among these mutants,while experiencing a significant decrease in chlorophyll and carotenoid contents compared to the wild type(WT).To clarify the regulatory mechanisms and network relationships underlying these mutants,the RNA-seq and weighted gene coexpression network(WGCNA)analyses were employed.The results showed flavonoid metabolism pathway was enriched both in MO and YG mutants,while the chlorophyll biosynthesis pathway and carotenoid degradation pathway were only enriched in MO and YG mutants,respectively.Subsequently,key structural genes and transcription factors were identified on metabolic pathways of three pigments through correlation analyses and quantitative experiments.Furthermore,a R2R3-MYB transcription factor,FnMYB4,was confirmed to be positively correlated with flavonoid synthesis through transient overexpression,virus-induced gene silencing(VIGS),and RNA interference(RNAi),accompanying by reoccurrence and attenuation of mutant phenotype.Finally,dual-luciferase(LUC)and yeast one-hybrid assays confirmed the binding of FnMYB4 to the FnFLS and FnF3H promoters,indicating that FnMYB4 positively regulates flavonoid synthesis.In addition,correlation analyses suggested that FnMYB4 also might be involved in chlorophyll and carotenoid metabolisms.These findings demonstrated the pivotal regulatory role of FnMYB4 in strawberry leaf coloration.
基金the National Key R&D Program of China(Nos.2018YFD0901506,2018YFD0900305)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(No.2018 SDKJ0406-3)。
文摘Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.
基金supported by the National Key Research and Development Program of China(2022YFE0139700,2023YFE0205100)Key Laboratory of Tropical and Subtropical Fishery Resources Application and Cultivation,Ministry of Agriculture and Rural Affairs,Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences(20220202)+3 种基金Guangdong Provincial Special Fund for Modern Agriculture Industry Technology Innovation Team(2023KJ150)China-ASEAN Maritime Cooperation Fund(CAMC-2018F)National Freshwater Genetic Resource Center(FGRC18537)Guangdong Rural Revitalization Strategy Special Provincial Organization and Implementation Project Funds(2022-SBH-00-001)。
文摘Glass catfish(Kryptopterus vitreolus)are notable in the aquarium trade for their highly transparent body pattern.This transparency is due to the loss of most reflective iridophores and light-absorbing melanophores in the main body,although certain black and silver pigments remain in the face and head.To date,however,the molecular mechanisms underlying this transparent phenotype remain largely unknown.To explore the genetic basis of this transparency,we constructed a chromosome-level haplotypic genome assembly for the glass catfish,encompassing 32 chromosomes and 23344 protein-coding genes,using PacBio and Hi-C sequencing technologies and standard assembly and annotation pipelines.Analysis revealed a premature stop codon in the putative albinism-related tyrp1b gene,encoding tyrosinase-related protein 1,rendering it a nonfunctional pseudogene.Notably,a synteny comparison with over 30 other fish species identified the loss of the endothelin-3(edn3b)gene in the glass catfish genome.To investigate the role of edn3b,we generated edn3b^(−/−)mutant zebrafish,which exhibited a remarkable reduction in black pigments in body surface stripes compared to wild-type zebrafish.These findings indicate that edn3b loss contributes to the transparent phenotype of the glass catfish.Our high-quality chromosome-scale genome assembly and identification of key genes provide important molecular insights into the transparent phenotype of glass catfish.These findings not only enhance our understanding of the molecular mechanisms underlying transparency in glass catfish,but also offer a valuable genetic resource for further research on pigmentation in various animal species.
文摘The emergence of antiretroviral resistance mutations represents a major threat to the achievement of national and global goals for the elimination of HIV-1 infection. The global strategy in 2019 in Cte d'Ivoire is a new national policy for the management of people living with HIV with the administration of dolutegravir (DTG)-based fixed-dose combination. The aim of our study was to evaluate HIV-1 resistance to antiretrovirals (ARVs) in infected adult subjects in Cte d’Ivoire in the context of a systematic switch to a DTG-based combination. Between February 2022 and October 2023, a cross-sectional survey with random sampling was conducted in 06 services caring for people living with HIV. A total of 139 participants were included in the study. Adults with a viral load ≥ 1000 copies/mL were tested for HIV-1 ARV resistance mutations. Molecular analyses were performed using protocol of ANRS-MIE (National Agency for Research on AIDS and emerging infectious diseases). The interpretation is performed by HIVGRAD (https://www.hiv-grade.de/cms/grade/). The frequencies of HIV-1 resistance to non-nucleotide reverse transcriptase inhibitors (NNRTIs), nucleotide reverse transcriptase inhibitors (NRTIs), integrase inhibitors (IINTs) and protease inhibitors (PIs) were 82%, 73%, 19% and 11% respectively. The main mutations observed in the different classes were K103N (45%), M184V (64%), E157Q (19%) and L10V/M46I/A71V/I54V (6%) respectively. This study reveals the emergence of resistance to DTG-based fixed-dose combinations, favored by high rates of resistance to NRTIs and NNRTIs. This finding underlines the need for enhanced viral load monitoring and HIV-1 genotyping tests to guide the choice of NRTIs for combination therapy. In addition, monitoring for mutations to second-generation NRTIs is essential, given the scale-up of DTG-based regimens currently underway in Cte d’Ivoire.
文摘New clinical approaches are imperative beyond the widely adopted National Comprehensive Cancer Network (NCCN) guidelines, utilized by prominent cancer institutions. Cancer is the leading cause of death among individuals younger than 85 years within the United States. Despite significant technological advances, including the expenditure of hundreds of billions, treatment outcomes and overall survival have not notably improved for most types of advanced cancer over the last several decades. Over the past 24 years, Envita Medical Centers has pioneered a unique form of personalized treatment approach for late-stage and refractory cancer patients, introducing groundbreaking innovations in the field. Our integrated algorithm utilizes advanced genomics, transcriptomics, and highly tailored immunotherapy, resulting in remarkable outcome improvements. This study presents Envita’s innovative personalized treatment algorithms and examines the response outcomes of 199 late-stage cancer patients treated at Envita Medical Centers over a two-year period. Compared to standard of care and palliative chemotherapy, Envita’s treatment demonstrated a remarkable 35-fold improvement in overall response rates (Figure 1). Moreover, 88% of the patients, the majority presenting with Stage 3 or 4 cancer, experienced a 43-fold improvement in quality of life with minimal side effects, as compared to standard of care chemotherapy and palliative care. This revolutionary success is attributed to Envita’s personalized therapeutic algorithms, which incorporate customized immunotherapy. Envita’s precision care approach has also achieved a 100% better response rate compared to over 65 global chemotherapy clinical trials with more than 2700 patients. The results from this study suggest that a wider utilization of Envita’s personalized approach can significantly benefit patients with late-stage and refractory cancer.
基金supported by the National Key R&D Program of China[Grant No.2021YFC2103002]the National Natural Science Foundation of China[Grant No.32202720]+1 种基金The Agricultural Science and Technology Innovation Program(CAAS-ZDRW202304)the China Agriculture Research System of MOF and MARA(CARS-41).
文摘Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides,which are advantageous for plant immunity,animal nutrition and health.However,thermophilic endo-chitinases are scarce and the transformation from exo-to endo-activity of chitinases is still a challenging problem.In this study,to enhance the endo-activity of the thermophilic chitinase Chi304,we proposed two approaches for rational design based on comprehensive structural and evolutionary analyses.Four effective single-point mutants were identified among 28 designed mutations.The ratio of(GlcNAc)3 to(GlcNAc)2 quantity(DP3/2)in the hydrolysates of the four single-point mutants undertaking colloidal chitin degradation were 1.89,1.65,1.24,and 1.38 times that of Chi304,respectively.When combining to double-point mutants,the DP3/2 proportions produced by F79A/W140R,F79A/M264L,F79A/W272R,and M264L/W272R were 2.06,1.67,1.82,and 1.86 times that of Chi304 and all four double-point mutants exhibited enhanced endo-activity.When applied to produce chitooligosaccharides(DP≥3),F79A/W140R accumulated the most(GlcNAc)4,while M264L/W272R was the best to produce(GlcNAc)3,which was 2.28 times that of Chi304.The two mutants had exposed shallower substrate-binding pockets and stronger binding abilities to shape the substrate.Overall,this research offers a practical approach to altering the cutting pattern of a chitinase to generate functional chitooligosaccharides.
