Cloning and Sequence Analysis of Beta-actin Gene Full Length cDNA from Trachidermus fasciatus

[Objective] The purpose of this experiment was to reveal the sequence and characteristics of Trachidermus fasciatus beta-actin gene.[Method] Using total RNA of muscle in T.fasciatus as template,three cDNA fragments of beta-actin gene in T.fasciatus were amplified by RT-PCR,5＇-RACE and 3＇-RACE.[Result] A full-length of 1905 bp beta-actin cDNA sequence in T.fasciatus,which includes a 1128 bp length open reading frame encoding a 375-amino acid peptide,was obtained.Sequence alignment of nucleotide and amino acid sequence revealed that T.fasciatus beta-actin shared high homology with that of Epinephelus coioides,Rachycentron canadum,Chrysophrys auratus and of relatively low homology with mammalian and bird.The phylogenetic analysis showed that T.fasciatus beta-actin had closest relationship with Epinephelus coioides.And RT-PCR analysis suggested that the beta-actin gene expressed in four tissues,i.e.,muscle,liver,intestine and brain.[Conclusion] The full length sequence of beta-actin gene with high conservation in T.fasciatus was obtained for the first time.

RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

BACKGROUND Validation of the reference gene(RG)stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction(RT-qPCR)data normalisation.Commonly,in an unreliable way,several studies use genes involved in essential cellular functions[glyceraldehyde-3-phosphate dehydro-genase(GAPDH),18S rRNA,andβ-actin]without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes.Furthermore,such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recom-mend two or more genes.It impacts the credibility of these studies and causes dis-tortions in the gene expression findings.For tissue engineering,the accuracy of gene expression drives the best experimental or therapeutical approaches.We cultivated DPSCs under two conditions:Undifferentiated and osteogenic dif-ferentiation,both for 35 d.We evaluated the gene expression of 10 candidates for RGs[ribosomal protein,large,P0(RPLP0),TATA-binding protein(TBP),GAPDH,actin beta(ACTB),tubulin(TUB),aminolevulinic acid synthase 1(ALAS1),tyro-sine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,zeta(YWHAZ),eukaryotic translational elongation factor 1 alpha(EF1a),succinate dehydrogenase complex,subunit A,flavoprotein(SDHA),and beta-2-micro-globulin(B2M)]every 7 d(1,7,14,21,28,and 35 d)by RT-qPCR.The data were analysed by the four main algorithms,ΔCt method,geNorm,NormFinder,and BestKeeper and ranked by the RefFinder method.We subdivided the samples into eight subgroups.RESULTS All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm.The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs.Either theΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes.However,geNorm analysis showed RPLP0/EF1αin the first place.These algorithms’two least stable RGs were B2M/GAPDH.For BestKeeper,ALAS1 was ranked as the most stable RG,and SDHA as the least stable RG.The pair RPLP0/TBP was detected in most subgroups as the most stable RGs,following the RefFinfer ranking.CONCLUSION For the first time,we show that RPLP0/TBP are the most stable RGs,whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.

Genetic dissection and validation of a major QTL for grain weight on chromosome 3B in bread wheat(Triticum aestivum L.)

Grain weight is one of the key components of wheat(Triticum aestivum L.)yield.Genetic manipulation of grain weight is an efficient approach for improving yield potential in breeding programs.A recombinant inbred line(RIL)population derived from a cross between W7268 and Chuanyu 12(CY12)was employed to detect quantitative trait loci(QTLs)for thousand-grain weight(TGW),grain length(GL),grain width(GW),and the ratio of grain length to width(GLW)in six environments.Seven major QTLs,QGl.cib-2D,QGw.cib-2D,QGw.cib-3B,QGw.cib-4B.1,QGlw.cib-2D.1,QTgw.cib-2D.1 and QTgw.cib-3B.1,were consistently identified in at least four environments and the best linear unbiased estimation(BLUE)datasets,and they explained 2.61 to 34.85%of the phenotypic variance.Significant interactions were detected between the two major TGW QTLs and three major GW loci.In addition,QTgw.cib-3B.1 and QGw.cib-3B were co-located,and the improved TGW at this locus was contributed by GW.Unlike other loci,QTgw.cib-3B.1/QGw.cib-3B had no effect on grain number per spike(GNS).They were further validated in advanced lines using Kompetitive Allele Specific PCR(KASP)markers,and a comparison analysis indicated that QTgw.cib-3B.1/QGw.cib-3B is likely a novel locus.Six haplotypes were identified in the region of this QTL and their distribution frequencies varied between the landraces and cultivars.According to gene annotation,spatial expression patterns,ortholog analysis and sequence variation,the candidate gene of QTgw.cib-3B.1/QGw.cib-3B was predicted.Collectively,the major QTLs and KASP markers reported here provide valuable information for elucidating the genetic architecture of grain weight and for molecular marker-assisted breeding in grain yield improvement.

Genetic and epigenetic targets of natural dietary compounds as anti-Alzheimer's agents

Alzheimer’s disease is a progressive neurodegenerative disorder and the most common cause of dementia that principally affects older adults.Pathogenic factors,such as oxidative stress,an increase in acetylcholinesterase activity,mitochondrial dysfunction,genotoxicity,and neuroinflammation are present in this syndrome,which leads to neurodegeneration.Neurodegenerative pathologies such as Alzheimer’s disease are considered late-onset diseases caused by the complex combination of genetic,epigenetic,and environmental factors.There are two main types of Alzheimer’s disease,known as familial Alzheimer’s disease(onset<65 years)and late-onset or sporadic Alzheimer’s disease(onset≥65 years).Patients with familial Alzheimer’s disease inherit the disease due to rare mutations on the amyloid precursor protein(APP),presenilin 1 and 2(PSEN1 and PSEN2)genes in an autosomaldominantly fashion with closely 100%penetrance.In contrast,a different picture seems to emerge for sporadic Alzheimer’s disease,which exhibits numerous non-Mendelian anomalies suggesting an epigenetic component in its etiology.Importantly,the fundamental pathophysiological mechanisms driving Alzheimer’s disease are interfaced with epigenetic dysregulation.However,the dynamic nature of epigenetics seems to open up new avenues and hope in regenerative neurogenesis to improve brain repair in Alzheimer’s disease or following injury or stroke in humans.In recent years,there has been an increase in interest in using natural products for the treatment of neurodegenerative illnesses such as Alzheimer’s disease.Through epigenetic mechanisms,such as DNA methylation,non-coding RNAs,histone modification,and chromatin conformation regulation,natural compounds appear to exert neuroprotective effects.While we do not purport to cover every in this work,we do attempt to illustrate how various phytochemical compounds regulate the epigenetic effects of a few Alzheimer’s disease-related genes.

A review of the literature on the use of CRISPR/Cas9 gene therapy to treat hepatocellular carcinoma

Noncoding RNAs instruct the Cas9 nuclease to site speifillyl cleave DNA in the CRISPR/Cas9 system.Despite the high incidence of hepatocellular carcinoma(HCC),the patient's outcome is poor.As a result of the emergence of therapeutic resistance in HCC patients,dlinicians have faced difficulties in treating such tumor.In addition,CRISPR/Cas9 screens were used to identify genes that improve the dlinical response of HCC patients.It is the objective of this article to summarize the current understanding of the use of the CRISPR/Cas9 system for the treatment of cancer,with a particular emphasis on HCC as part of the current state of knowledge.Thus,in order to locate recent developments in oncology research,we examined both the Scopus database and the PubMed database.The ability to selectively interfere with gene expression in combinatorial CRISPR/Cas9 screening can lead to the discovery of new effective HCC treatment regimens by combining clinically approved drugs.Drug resistance can be overcome with the help of the CRISPR/Cas9 system.HCC signature genes and resistance to treatment have been uncovered by genome-scale CRISPR activation screening although this method is not without limitations.It has been extensively examined whether CRISPR can be used as a tool for disease research and gene therapy.CRISPR and its applications to tumor research,particularly in HCC,are examined in this study through a review of the literature.

Genetically modified non-human primate models for research on neurodegenerative diseases

Neurodegenerative diseases(NDs)are a group of debilitating neurological disorders that primarily affect elderly populations and include Alzheimer's disease(AD),Parkinson's disease(PD),Huntington's disease(HD),and amyotrophic lateral sclerosis(ALS).Currently,there are no therapies available that can delay,stop,or reverse the pathological progression of NDs in clinical settings.As the population ages,NDs are imposing a huge burden on public health systems and affected families.Animal models are important tools for preclinical investigations to understand disease pathogenesis and test potential treatments.While numerous rodent models of NDs have been developed to enhance our understanding of disease mechanisms,the limited success of translating findings from animal models to clinical practice suggests that there is still a need to bridge this translation gap.Old World nonhuman primates(NHPs),such as rhesus,cynomolgus,and vervet monkeys,are phylogenetically,physiologically,biochemically,and behaviorally most relevant to humans.This is particularly evident in the similarity of the structure and function of their central nervous systems,rendering such species uniquely valuable for neuroscience research.Recently,the development of several genetically modified NHP models of NDs has successfully recapitulated key pathologies and revealed novel mechanisms.This review focuses on the efficacy of NHPs in modeling NDs and the novel pathological insights gained,as well as the challenges associated with the generation of such models and the complexities involved in their subsequent analysis.

Wilm′s tumor gene1肽疫苗Galinpepimut-S在肿瘤免疫治疗中的应用

目的为Wilm′s tumor gene1(WT1)肽疫苗Galinpepimut-S(GPS)用于肿瘤免疫治疗的后续研究提供参考。方法采用计算机检索中国知网、PubMed等数据库自建库起至2022年12月的肿瘤免疫治疗相关文献,总结GPS在肿瘤免疫治疗中的应用现状。结果GPS能激发自身免疫系统,对WT1抗原产生强烈免疫反应而发挥抗肿瘤作用,在卵巢癌、恶性胸膜间皮瘤、急性髓系白血病、多发性骨髓瘤的治疗中均显示出较好的疗效。结论以GPS为代表的肿瘤疫苗是未来肿瘤治疗的重要方向,需进一步进行临床研究,以获取更多数据。

Pangenome and multi-tissue gene atlas provide new insights into the domestication and highland adaptation of yaks

Background The genetic diversity of yak,a key domestic animal on the Qinghai-Tibetan Plateau(QTP),is a vital resource for domestication and breeding efforts.This study presents the first yak pangenome obtained through the de novo assembly of 16 yak genomes.Results We discovered 290 Mb of nonreference sequences and 504 new genes.Our pangenome-wide presence and absence variation(PAV)analysis revealed 5,120 PAV-related genes,highlighting a wide range of variety-specific genes and genes with varying frequencies across yak populations.Principal component analysis(PCA)based on binary gene PAV data classified yaks into three new groups:wild,domestic,and Jinchuan.Moreover,we pro-posed a‘two-haplotype genomic hybridization model'for understanding the hybridization patterns among breeds by integrating gene frequency,heterozygosity,and gene PAV data.A gene PAV-GWAS identified a novel gene(Bos-Gru3G009179)that may be associated with the multirib trait in Jinchuan yaks.Furthermore,an integrated transcrip-tome and pangenome analysis highlighted the significant differences in the expression of core genes and the muta-tional burden of differentially expressed genes between yaks from high and low altitudes.Transcriptome analysis across multiple species revealed that yaks have the most unique differentially expressed m RNAs and lnc RNAs(between high-and low-altitude regions),especially in the heart and lungs,when comparing high-and low-altitude adaptations.Conclusions The yak pangenome offers a comprehensive resource and new insights for functional genomic studies,supporting future biological research and breeding strategies.

AMME chromosomal region gene 1基因变异矮小相关综合征一例及文献复习

目的探讨1例身材矮小、面中部发育不全患儿的病因,以提高临床医师对特殊矮小综合征的认识。方法收集1例身材矮小、面中部发育不全患儿的临床资料,对患儿及父母行基因检测,并给予患儿常规治疗、随访。结果结合患儿特殊面容及基因检测,诊断为AMMECR1基因变异矮小相关综合征,结合文献复习总结AMMECR1基因变异矮小相关综合征特点。结论AMMECR1基因变异矮小相关综合征是一种罕见的X连锁遗传性疾病,临床主要表现为身材矮小、运动语言落后、肌张力减低、听力损失、面中部发育不全,部分存在心脏改变、腭裂、骨骼改变及椭圆形红细胞增多症、智力落后和肾钙质沉着症。该文报道1例AMMECR1基因新变异引起身材矮小、面中部发育不全患儿的病例资料,结合特殊面容及基因检测,诊断为AMMECR1基因变异矮小相关综合征。AMMECR1基因变异矮小相关综合征是一种罕见的X连锁遗传性疾病,本文初步概括其特点,并结合文献进行分析,以提高临床医师对AMMECR1基因变异矮小相关综合征的诊治。

Identification of hub genes associated with Helicobacter pylori infection and type 2 diabetes mellitus:A pilot bioinformatics study

BACKGROUND Helicobacter pylori(H.pylori)infection is related to various extragastric diseases including type 2 diabetes mellitus(T2DM).However,the possible mechanisms connecting H.pylori infection and T2DM remain unknown.AIM To explore potential molecular connections between H.pylori infection and T2DM.METHODS We extracted gene expression arrays from three online datasets(GSE60427,GSE27411 and GSE115601).Differentially expressed genes(DEGs)commonly present in patients with H.pylori infection and T2DM were identified.Hub genes were validated using human gastric biopsy samples.Correlations between hub genes and immune cell infiltration,miRNAs,and transcription factors(TFs)were further analyzed.RESULTS A total of 67 DEGs were commonly presented in patients with H.pylori infection and T2DM.Five significantly upregulated hub genes,including TLR4,ITGAM,C5AR1,FCER1G,and FCGR2A,were finally identified,all of which are closely related to immune cell infiltration.The gene-miRNA analysis detected 13 miRNAs with at least two gene cross-links.TF-gene interaction networks showed that TLR4 was coregulated by 26 TFs,the largest number of TFs among the 5 hub genes.CONCLUSION We identified five hub genes that may have molecular connections between H.pylori infection and T2DM.This study provides new insights into the pathogenesis of H.pylori-induced onset of T2DM.

RNA sequencing of exosomes secreted by fibroblast and Schwann cells elucidates mechanisms underlying peripheral nerve regeneration

Exosomes exhibit complex biological functions and mediate a variety of biological processes,such as promoting axonal regeneration and functional recove ry after injury.Long non-coding RNAs(IncRNAs)have been reported to play a crucial role in axonal regeneration.Howeve r,the role of the IncRNA-microRNAmessenger RNA(mRNA)-competitive endogenous RNA(ceRNA)network in exosome-mediated axonal regeneration remains unclear.In this study,we performed RNA transcriptome sequencing analysis to assess mRNA expression patterns in exosomes produced by cultured fibroblasts(FC-EXOs)and Schwann cells(SCEXOs).Diffe rential gene expression analysis,Gene Ontology analysis,Kyoto Encyclopedia of Genes and Genomes analysis,and protein-protein intera ction network analysis were used to explo re the functions and related pathways of RNAs isolated from FC-EXOs and SC-EXOs.We found that the ribosome-related central gene Rps5 was enriched in FC-EXOs and SC-EXOs,which suggests that it may promote axonal regeneration.In addition,using the miRWalk and Starbase prediction databases,we constructed a regulatory network of ceRNAs targeting Rps5,including 27 microRNAs and five IncRNAs.The ceRNA regulatory network,which included Ftx and Miat,revealed that exsosome-derived Rps5 inhibits scar formation and promotes axonal regeneration and functional recovery after nerve injury.Our findings suggest that exosomes derived from fibro blast and Schwann cells could be used to treat injuries of peripheral nervous system.

Mining elite loci and candidate genes for root morphology-related traits at the seedling stage by genome-wide association studies in upland cotton(Gossypium hirsutum L.)

Root system architecture plays an essential role in water and nutrient acquisition in plants,and it is significantly involved in plant adaptations to various environmental stresses.In this study,a panel of 242 cotton accessions was collected to investigate six root morphological traits at the seedling stage,including main root length(MRL),root fresh weight(RFW),total root length(TRL),root surface area(RSA),root volume(RV),and root average diameter(AvgD).The correlation analysis of the six root morphological traits revealed strong positive correlations of TRL with RSA,as well as RV with RSA and AvgD,whereas a significant negative correlation was found between TRL and AvgD.Subsequently,a genome-wide association study(GWAS)was performed using the root phenotypic and genotypic data reported previously for the 242 accessions using 56,010 single nucleotide polymorphisms(SNPs)from the CottonSNP80K array.A total of 41 quantitative trait loci(QTLs)were identified,including nine for MRL,six for RFW,nine for TRL,12 for RSA,12 for RV and two for AvgD.Among them,eight QTLs were repeatedly detected in two or more traits.Integrating these results with a transcriptome analysis,we identified 17 candidate genes with high transcript values of transcripts per million(TPM)≥30 in the roots.Furthermore,we functionally verified the candidate gene GH_D05G2106,which encodes a WPP domain protein 2in root development.A virus-induced gene silencing(VIGS)assay showed that knocking down GH_D05G2106significantly inhibited root development in cotton,indicating its positive role in root system architecture formation.Collectively,these results provide a theoretical basis and candidate genes for future studies on cotton root developmental biology and root-related cotton breeding.

Screening biomarkers for spinal cord injury using weighted gene co-expression network analysis and machine learning

Immune changes and inflammatory responses have been identified as central events in the pathological process of spinal co rd injury.They can greatly affect nerve regeneration and functional recovery.However,there is still limited understanding of the peripheral immune inflammato ry response in spinal cord inju ry.In this study.we obtained microRNA expression profiles from the peripheral blood of patients with spinal co rd injury using high-throughput sequencing.We also obtained the mRNA expression profile of spinal cord injury patients from the Gene Expression Omnibus(GEO)database(GSE151371).We identified 54 differentially expressed microRNAs and 1656 diffe rentially expressed genes using bioinformatics approaches.Functional enrichment analysis revealed that various common immune and inflammation-related signaling pathways,such as neutrophil extracellular trap formation pathway,T cell receptor signaling pathway,and nuclear factor-κB signal pathway,we re abnormally activated or inhibited in spinal cord inju ry patient samples.We applied an integrated strategy that combines weighted gene co-expression network analysis,LASSO logistic regression,and SVM-RFE algorithm and identified three biomarke rs associated with spinal cord injury:ANO10,BST1,and ZFP36L2.We verified the expression levels and diagnostic perfo rmance of these three genes in the original training dataset and clinical samples through the receiver operating characteristic curve.Quantitative polymerase chain reaction results showed that ANO20 and BST1 mRNA levels were increased and ZFP36L2 mRNA was decreased in the peripheral blood of spinal cord injury patients.We also constructed a small RNA-mRNA interaction network using Cytoscape.Additionally,we evaluated the proportion of 22 types of immune cells in the peripheral blood of spinal co rd injury patients using the CIBERSORT tool.The proportions of naive B cells,plasma cells,monocytes,and neutrophils were increased while the proportions of memory B cells,CD8^(+)T cells,resting natural killer cells,resting dendritic cells,and eosinophils were markedly decreased in spinal cord injury patients increased compared with healthy subjects,and ANO10,BST1 and ZFP26L2we re closely related to the proportion of certain immune cell types.The findings from this study provide new directions for the development of treatment strategies related to immune inflammation in spinal co rd inju ry and suggest that ANO10,BST2,and ZFP36L2 are potential biomarkers for spinal cord injury.The study was registe red in the Chinese Clinical Trial Registry(registration No.ChiCTR2200066985,December 12,2022).

A rare missense PAX6 mutation causes atypical aniridia in a three-generation Chinese family

●AIM:To investigate the molecular diagnosis of a threegeneration Chinese family affected with aniridia,and further to identify clinically a PAX6 missense mutation in members with atypical aniridia.●METHODS:Eleven family members with and without atypical aniridia were recruited.All family members underwent comprehensive ophthalmic examinations.A combination of whole exome sequencing(WES)and direct Sanger sequencing were performed to uncover the causative mutation.●RESULTS:Among the 11 family members,8 were clinically diagnosed with congenital aniridia(atypical aniridia phenotype).A rare heterozygous mutation c.622C>T(p.Arg208Trp)in exon 8 of PAX6 was identified in all affected family members but not in the unaffected members or in healthy control subjects.●CONCLUSION:A rare missense mutation in the PAX6 gene is found in members of a three-generation Chinese family with congenital atypical aniridia.This result contributes to an increase in the phenotypic spectrum caused by PAX6 missense heterozygous variants and provides useful information for the clinical diagnosis of atypical aniridia,which may also contribute to genetic counselling and family planning.

Fine mapping and characterization of stripe rust resistance gene YrAYH in near-isogenic lines derived from a cross involving wheat landrace Anyuehong

Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is a devastating disease in wheat worldwide.Discovering and characterizing new resistance genes/QTL is crucial for wheat breeding programs.In this study,we fine-mapped and characterized a stripe rust resistance gene,YRAYH,on chromosome arm 5BL in the Chinese wheat landrace Anyuehong(AYH).Evaluations of stripe rust response to prevalent Chinese Pst races in near-isogenic lines derived from a cross of Anyuehong and Taichung 29 showed that YrAYH conferred a high level of resistance at all growth stages.Fine mapping using a large segregating population of 9748 plants,narrowed the YRAYH locus to a 3.7 Mb interval on chromosome arm 5BL that included 61 annotated genes.Transcriptome analysis of two NIL pairs identified 64 upregulated differentially expressed genes(DEGs)in the resistant NILs(NILs-R).Annotations indicated that many of these genes have roles in plant disease resistance pathways.Through a combined approach of fine-mapping and transcriptome sequencing,we identified a serine/threonine-protein kinase SRPK as a candidate gene underlying YrAYH.A unique 25 bp insertion was identified in the NILs-R compared to the NILs-S and previously published wheat genomes.An InDel marker was developed and co-segregated with YrAYH.Agronomic trait evaluation of the NILs suggested that YrAYH not only reduces the impact of stripe rust but was also associated with a gene that increases plant height and spike length.

Genetic dissection and origin of pleiotropic loci underlying multilevel fiber quality traits in upland cotton(Gossypium hirsutum L.)

Cotton fiber quality is a persistent concern that determines planting benefits and the quality of finished textile products.However,the limitations of measurement instruments have hindered the accurate evaluation of some important fiber characteristics such as fiber maturity,fineness,and neps,which in turn has impeded the genetic improvement and industrial utilization of cotton fiber.Here,12 single fiber quality traits were measured using Advanced Fiber Information System(AFIS)equipment among 383 accessions of upland cotton(Gossypium hirsutum L.).In addition,eight conventional fiber quality traits were assessed by the High Volume Instrument(HVI)System.Genome-wide association study(GWAS),linkage disequilibrium(LD)block genotyping and functional identification were conducted sequentially to uncover the associated elite loci and candidate genes of fiber quality traits.As a result,the previously reported pleiotropic locus FL_D11 regulating fiber length-related traits was identified in this study.More importantly,three novel pleiotropic loci(FM_A03,FF_A05,and FN_A07)regulating fiber maturity,fineness and neps,respectively,were detected based on AFIS traits.Numerous highly promising candidate genes were screened out by integrating RNA-seq and qRT-PCR analyses,including the reported GhKRP6 for fiber length,the newly identified GhMAP8 for maturity and GhDFR for fineness.The origin and evolutionary analysis of pleiotropic loci indicated that the selection pressure on FL_D11,FM_A03 and FF_A05 increased as the breeding period approached the present and the origins of FM_A03 and FF_A05 were traced back to cotton landraces.These findings reveal the genetic basis underlying fiber quality and provide insight into the genetic improvement and textile utilization of fiber in G.hirsutum.

Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene(VdThit)confers resistance to Verticillium wilt in cotton

Verticillium wilt(VW),induced by the soil-borne fungus Verticillium dahliae(Vd),poses a substantial threat to a diverse array of plant species.Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures.In this study,we successfully generated two stable transgenic lines of cotton(Gossypium hirsutum L.),VdThitRNAi-1 and VdThit-RNAi-2,using host-induced gene silencing(HIGS)technology to introduce double-stranded RNA(dsRNA)targeting the thiamine transporter protein gene(VdThit).Southern blot analysis confirmed the presence of a single-copy insertion in each line.Microscopic examination showed marked reductions in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type(WT).The corresponding disease index and fungal biomass of VdThit-RNAi-1/2 also exhibited significant reductions.Real-time quantitative PCR(qRT-PCR)analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton.Small RNA sequencing(sRNA-Seq)analysis revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines.Additionally,the silencing of VdThit by the siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd.Under field conditions,VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT.In summary,our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton,thereby potentially enabling the development of cotton breeding as a promising strategy for managing VW.

Genome wide investigation of Hsf gene family in Phoebe bournei:identification,evolution,and expression after abiotic stresses

Heat shock transcription factors(Hsfs)have important roles during plant growth and development and responses to abiotic stresses.The identification and func-tion of Hsf genes have been thoroughly studied in various herbaceous plant species,but not woody species,especially Phoebe bournei,an endangered,unique species in China.In this study,17 members of the Hsf gene family were identi-fied from P.bournei using bioinformatic methods.Phyloge-netic analysis indicated that PbHsf genes were grouped into three subfamilies:A,B,and C.Conserved motifs,three-dimensional structure,and physicochemical properties of the PbHsf proteins were also analyzed.The structure of the PbHsf genes varied in the number of exons and introns.Pre-diction of cis-acting elements in the promoter region indi-cated that PbHsf genes are likely involved in responses to plant hormones and stresses.A collinearity analysis dem-onstrated that expansions of the PbHsf gene family mainly take place via segmental duplication.The expression levels of PbHsf genes varied across different plant tissues.On the basis of the expression profiles of five representative PbHsf genes during heat,cold,salt,and drought stress,PbHsf pro-teins seem to have multiple functions depending on the type of abiotic stress.This systematic,genome-wide investigation of PbHsf genes in P.bournei and their expression patterns provides valuable insights and information for further func-tional dissection of Hsf proteins in this endangered,unique species.

PbrARF4 contributes to calyx shedding of fruitlets in ‘Dangshan Suli’ pear by partly regulating the expression of abscission genes

Fruitlet calyx shedding in pear plants is apparently regulated via numerous pathways that involve both environmental triggers and phytohormones cues such as auxin. In this study, we found at 10 days after full bloom (DAFB) higher levels of indoleacetic acid (IAA) and tryptophan (Trp) in calyx persistence fruitlet (CPF) than calyx shedding fruitlet (CSF) ofDanshan Suli’ pear (Pyrus bretschneideri Rhed.). Consisting with this, the activity of indolealdehyde oxidase (IAAIdO), which promotes IAA synthesis, was remarkably increased, and that of peroxidase(POD), which degrades IAA, dropped markedly in CPF but not in CSF. Further, qRT-PCR results revealed that most of 31 PbrARFs (encoding auxin response factors) in Pyrus bretschneideri were highly expressed in CPF, whereas PbrARF4, PbrARF24 and PbrARF26 were significantly downregulated in CPF vis-a-vis CSF. Phylogenetic analysis revealed that 6 PbrARFs clustered in the group III, where PbrARF4 showed the closest affinity with AtARF1 that promotes organ abscission, indicating a putative role of PbrARF4 in mediating the process of calyx shedding in pear. In fact, the ectopic overexpression of PbrARF4 in Solanum lycopersicum resulted in an earlier-formed and deeper abscission layer (AL) in the transgenic plants, whose calyxes were more prone to wilt at the mature red stage (MR) compared with the control plants (wild-type). More importantly, expression levels of the abscission genes SILS and Sl Cel2 in transgenic plants overexpressing PbrARF4 were significantly upregulated in comparation with the WT, whereas those of Sl BI and Sl TAPG2 were considerably inhibited. Further, PbrJOINTLESS and PbrIDA,the two genes related to calyx shedding in pear, were up-regulated more in CSF than CPF. The findings contribute to a better understanding of PbrARFs involved in fruitlet calyx shedding of pear, which could prove beneficial to improving the quality of pear fruit.

Genome-wide identification,characterization and functional prediction of the SRS gene family in sesame(Sesamum indicum L.)

Sesame(Sesamum indicum L.)is an ancient oilseed crop of the Pedaliaceae family with high oil content and potential health benefits.SHI RELATED SEQUENCE(SRS)proteins are the transcription factors(TFs)specific to plants that contain RING-like zinc finger domain and are associated with the regulation of several physiological and biochemical processes.They also play vital roles in plant growth and development such as root formation,leaf development,floral development,hormone biosynthesis,signal transduction,and biotic and abiotic stress responses.Nevertheless,the SRS gene family was not reported in sesame yet.In this study,identification,molecular characterization,phylogenetic relationship,cis-acting regulatory elements,protein-protein interaction,syntenic relationship,duplication events and expression pattern of SRS genes were analyzed in S.indicum.We identified total six SiSRS genes on seven different linkage groups in the S.indicum genome by comparing with the other species,including the model plant Arabidopsis thaliana.The SiSRS genes showed variation in their structure like2–5 exons and 1–4 introns.Like other species,SiSRS proteins also contained‘RING-like zinc finger'and‘LRP1'domains.Then,the SiSRS genes were clustered into subclasses via phylogenetic analysis with proteins of S.indicum,A.thaliana,and some other plant species.The cis-acting regulatory elements analysis revealed that the promoter region of SiSRS4(SIN_1011561)showed the highest 13 and 16 elements for light-and phytohormone-responses whereas,SiSRS1(SIN_1015187)showed the highest 15 elements for stress-response.The ABREs,or ABA-responsive elements,were found in a maximum of 8 copies in the SiSRS3(SIN 1009100).Moreover,the available RNA-seq based expression of SiSRS genes revealed variation in expression patterns between stress-treated and non-treated samples,especially in drought and salinity conditions in.S.indicum.Two SiSRS genes like SiSRS1(SIN_1015187)and SiSRS5(SIN_1021065),also exhibited variable expression patterns between control vs PEG-treated sesame root samples and three SiSRS genes,including SiSRS1(SIN_1015187),SiSRS2(SIN_1003328)and SiSRS5(SIN_1021065)were responsive to salinity treatments.The present outcomes will encourage more research into the gene expression and functionality analysis of SiSRS genes in S.indicum and other related species.