The Cucurbita genus contains several economically important species in the Cucurbitaceae family. Here, we report high-quality genome sequences of C. maxima and C. moschata and provide evidence supporting an allotetrap...The Cucurbita genus contains several economically important species in the Cucurbitaceae family. Here, we report high-quality genome sequences of C. maxima and C. moschata and provide evidence supporting an allotetraploidization event in Cucurbita. We are able to partition the genome into two homoeologous subgenomes based on different genetic distances to melon, cucumber, and watermelon in the Benincaseae tribe. We estimate that the two diploid progenitors successively diverged from Benincaseae around 31 and 26 million years ago （Mya）, respectively, and the allotetraploidization happened at some point between 26 Mya and 3 Mya, the estimated date when C. maxima and C. moschata diverged. The subgenomes have largely maintained the chromosome structures of their diploid progenitors. Such long-term karyotype stability after polyploidization has not been commonly observed in plant polyploids. The two subgenomes have retained similar numbers of genes, and neither subgenome is globally dominant in gene expression. Allele-specific expression analysis in the C. maxima ×C. moschata interspecific F1 hybrid and their two parents indicates the predominance of trans-regulatory effects underlying expression divergence of the parents, and detects transgressive gene expression changes in the hybrid correlated with heterosis in important agronomic traits. Our study provides insights into polyploid genome evolution and valuable resources for genetic improvement of cucurbit crops.展开更多
Kiwifruit is a recently domesticated horticultural fruit crop with substantial economic and nutritional value,especially because of the high content of vitamin C in its fruit.In this study,we de novo assembled two tel...Kiwifruit is a recently domesticated horticultural fruit crop with substantial economic and nutritional value,especially because of the high content of vitamin C in its fruit.In this study,we de novo assembled two telomere-to-telomere kiwifruit genomes from Actinidia chinensis var.‘Donghong’(DH)and Actinidia latifolia‘Kuoye’(KY),with total lengths of 608327852 and 640561626 bp for 29 chromosomes,respectively.With a burst of structural variants involving inversion,translocations,and duplications within 8.39 million years,the metabolite content of DH and KY exhibited differences in saccharides,lignans,and vitamins.A regulatory ERF098 transcription factor family has expanded in KY and Actinidia eriantha,both of which have ultra-high vitamin C content.With each assembly phased into two complete haplotypes,we identified allelic variations between two sets of haplotypes,leading to protein sequence variations in 26494 and 27773 gene loci and allele-specific expression of 4687 and 12238 homozygous gene pairs.Synchronized metabolome and transcriptome changes during DH fruit development revealed the same dynamic patterns in expression levels and metabolite contents;free fatty acids and flavonols accumulated in the early stages,but sugar substances and amino acids accumulated in the late stages.The AcSWEET9b gene that exhibits allelic dominance was further identified to positively correlate with high sucrose content in fruit.Compared with wild varieties and other Actinidia species,AcSWEET9b promoters were selected in red-flesh kiwifruits that have increased fruit sucrose content,providing a possible explanation on why red-flesh kiwifruits are sweeter.Collectively,these two gap-free kiwifruit genomes provide a valuable genetic resource for investigating domestication mechanisms and genome-based breeding of kiwifruit.展开更多
Divergence of gene expression and alter native splicing is a crucial driving force in the evolution of species;to date, however the molecular mechanism remains unclear. Hybrids of closely related species provide a sui...Divergence of gene expression and alter native splicing is a crucial driving force in the evolution of species;to date, however the molecular mechanism remains unclear. Hybrids of closely related species provide a suitable model to analyze allele-specific expressi on (ASE) and allele-specific alter native splicing (ASS). Analysis of ASE and ASS can uncover the differences in cis-regulatory elements between closely related species, while eliminating interferenee of trans-regulatory elements. Here, we provide a detailed characterization of ASE and ASS from 19 and 10 transcriptome datasets across five tissues from reciprocal-cross hybrids of horsex don key (mule/hi nny) and cattlexyak (dzo), respectively. Results showed that 4.8%-8.7% and 10.8%-16.7% of genes exhibited ASE and ASS, respectively. Notably, IncRNAs and pseudogenes were more likely to show ASE than protein-coding genes. In addition, genes showing ASE and ASS in mule/hinny were found to be involved in the regulation of muscle strength, whereas those of dzo were involved in high-altitude adaptati on. In con clusi on, our study dem on strated that explorati on of genes showing ASE and ASS in hybrids of closely related species is feasible for species evolution research.展开更多
目的根据allele-specific PCR(AS-PCR)原理,建立具有良好敏感性与特异性的百日咳鲍特菌23S r RNA位点变异检测方法。方法基于百日咳鲍特菌23S r RNA A2047G位点突变与其对红霉素耐药的相关性,设计包含特异变异位点的引物进行两阶段PCR反...目的根据allele-specific PCR(AS-PCR)原理,建立具有良好敏感性与特异性的百日咳鲍特菌23S r RNA位点变异检测方法。方法基于百日咳鲍特菌23S r RNA A2047G位点突变与其对红霉素耐药的相关性,设计包含特异变异位点的引物进行两阶段PCR反应,根据电泳有无特异大小目的片段的出现,确定百日咳鲍特菌23S r RNA A2047G位点是否有变异。结果以基因序列测定法为金标准的评价结果显示,AS-PCR法检测突变型菌株的灵敏度为96%(144/150),特异度为100%(100/100),kappa=0.95(P<0.01);检测野生型菌株的灵敏度为100%(18/18),特异度为100%(100/100),kappa=1(P<0.01)。结论 AS-PCR方法检测百日咳鲍特菌23S r RNA A2047G位点变异具有较高灵敏度和特异度,适用于普通微生物实验室开展百日咳鲍特菌对红霉素耐药性的快速检测。展开更多
Background Mycoplasma pneumoniae （M. pneumoniae） is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies hav...Background Mycoplasma pneumoniae （M. pneumoniae） is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent, followed by the mutation at position 2064. Reported molecular detection methods for the identification of these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time polymerase chain reaction （PCR） with high-resolution melt analysis, and nested PCR-linked with capillary electrophoresis, etc. The most commonly used method for monitoring resistance-conferring mutations in M. pneumoniae is direct DNA sequencing of PCR or nested PCR products. However, these methods are time-consuming, labor-intensive or need expensive equipments. Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally. Methods In this study, we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis, and all results were compared with the sequencing data. We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results. Results Among 97 M. pneumoniae specimens, 88 were found to possess mutations by this method, and all modified allele-specific PCR analysis results were consistent with the sequencing data. The data of the clinical courses of these 97 cases showed that they suffered from severe pneumonia. Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens. However, in some cases from which mutations were detected, erythromycin monotherapy had poor efficacy, and on these patients severe symptoms improved only when azithromycin was added to the treatment. Conclusions The drug-resistant M. pneumoniae is very common in Beijing, 展开更多
Background It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequenci...Background It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20%-30%. The aim of this study was to evaluate allele-specific real-time PCR （ASPCR） to detect the resistance-related mutations located at positions 103, 184 and 215.Methods We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140clinical samples using this method.Results The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M1841, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.Conclusions Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Yemerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.展开更多
Hybrid rice(Oryza sativa)generally outperforms its inbred parents in yield and stress tolerance,a phenomenon termed heterosis,but the underlying mechanism is not completely understood.Here,we combined transcriptome,pr...Hybrid rice(Oryza sativa)generally outperforms its inbred parents in yield and stress tolerance,a phenomenon termed heterosis,but the underlying mechanism is not completely understood.Here,we combined transcriptome,proteome,physiological,and heterosis analyses to examine the salt response of super hybrid rice Chaoyou1000(CY1000).In addition to surpassing the mean values for its two parents(mid-parent heterosis),CY1000 exhibited a higher reactive oxygen species scavenging ability than both its parents(over-parent heterosis or heterobeltiosis).Nonadditive expression and allele-specific gene expression assays showed that the glutathione S-transferase gene OsGSTU26 and the amino acid transporter gene OsAAT30 may have major roles in heterosis for salt tolerance,acting in an overdominant fashion in CY1000.Furthermore,we identified OsWRKY72 as a common transcription factor that binds and regulates OsGSTU26 and OsAAT30.The salt-sensitive phenotypes were associated with the OsWRKY72^(paternal)genotype or the OsAAT30^(maternal)genotype in core rice germplasm varieties.OsWRKY72^(paternal)specifically repressed the expression of OsGSTU26 under salt stress,leading to salinity sensitivity,while OsWRKY72^(maternal)specifically repressed OsAAT30,resulting in salinity tolerance.These results suggest that the OsWRKY72-OsAAT30/OsGSTU26 module may play an important role in heterosis for salt tolerance in an overdominant fashion in CY1000 hybrid rice,providing valuable clues to elucidate the mechanism of heterosis for salinity tolerance in hybrid rice.展开更多
Embryo abortion stage and rescue system of hybrids were studied in the distant hybridizationbetween plum and apricot. Identification of the hybrids was also made. The resultsshowed: (1) Embryo abortion started from th...Embryo abortion stage and rescue system of hybrids were studied in the distant hybridizationbetween plum and apricot. Identification of the hybrids was also made. The resultsshowed: (1) Embryo abortion started from three weeks after pollination. (2) The germinationand growth of embryos were different at different growth stages, which could germinateand grow with PF value>0.5, but failed with PF value<0.5. In embryo rescue system ofhybrids, the best germination and differentiation medium was MS+6-BA 2mgL-1+IAA 0.3mgL-1,the rate of germination and differentiation reached up to 80%, bud induction andmultiplication medium was MS+6-BA 1.5mgL-1+IAA 0.3mgL-1, rooting medium was 1/2 MS+IAA0.8mgL-1. Some hybrids were transplanted into the field successfully. (3) Leaf shapeinvestigation and identification by S allele-specific PCR and RAPDs showed that thehybrids were true ones.展开更多
High-density genetic markers are required for genotyping and linkage mapping in identifying genes from crops with complex genomes, such as barley. As the most common variation, single nucleotide polymorphisms(SNPs) ar...High-density genetic markers are required for genotyping and linkage mapping in identifying genes from crops with complex genomes, such as barley. As the most common variation, single nucleotide polymorphisms(SNPs) are suitable for accurate genotyping by using the next-generation sequencing(NGS) technology. Reduced representation libraries(RRLs) of five barley accessions and one mutant were sequenced using NGS technology for SNP discovery. Twenty million short reads were generated and the proportion of repetitive sequences was reduced by more than 56%. A total of 6061 SNPs were identified, and 451 were mapped to the draft sequence of the barley genome with pairing reads. Eleven SNPs were validated using length polymorphic allele-specific PCR markers.展开更多
Translational regulation is a critical step in the process of gene expression and governs the synthesis of proteins from mRNAs.Many studies have revealed translational regulation in plants in response to various envir...Translational regulation is a critical step in the process of gene expression and governs the synthesis of proteins from mRNAs.Many studies have revealed translational regulation in plants in response to various environmental stimuli.However,there have been no studies documenting the comprehensive landscape of translational regulation and allele-specific translational efficiency in multiple plant tissues,especially those of rice,a main staple crop that feeds nearly half of the world’s population.Here we used RNA sequencing and ribosome profiling data to analyze the transcriptome and translatome of an elite hybrid rice,Shanyou 63(SY63),and its parental varieties Zhenshan 97 and Minghui 63.The results revealed that gene expression patterns varied more among tissues than among varieties at the transcriptional and translational levels.We identified 3392 upstream open reading frames(uORFs),and the uORF-containing genes were enriched in transcription factors.Only 668 of 13492 long non-coding RNAs could be translated into peptides.Finally,we discovered numerous genes with allele-specific translational efficiency in SY63 and demonstrated that some cis-regulatory elements may contribute to allelic divergence in translational efficiency.Overall,these findings may improve our understanding of translational regulation in rice and provide information for molecular breeding research.展开更多
Despite the scientific and medicinal importance of diploid sika deer(Cervus nippon),its genome resources are limited and haplotype-resolved chromosome-scale assembly is urgently needed.To explore mechanisms underlying...Despite the scientific and medicinal importance of diploid sika deer(Cervus nippon),its genome resources are limited and haplotype-resolved chromosome-scale assembly is urgently needed.To explore mechanisms underlying the expression patterns of the allele-specific genes in antlers and the chromosome evolution in Cervidae,we report,for the first time,a high-quality haplotype-resolved chromosome-scale genome of sika deer by integrating multiple sequencing strategies,which was anchored to 32 homologous groups with a pair of sex chromosomes(XY).Several expanded genes(RET,PPP2R1A,PPP2R1B,YWHAB,YWHAZ,and RPS6)and positively selected genes(eIF4E,Wnt8A,Wnt9B,BMP4,and TP53)were identified,which could contribute to rapid antler growth without carcinogenesis.A comprehensive and systematic genome-wide analysis of allele expression patterns revealed that most alleles were functionally equivalent in regulating rapid antler growth and inhibiting oncogenesis.Comparative genomic analysis revealed that chromosome fission might occur during the divergence of sika deer and red deer(Cervus elaphus),and the olfactory sensation of sika deer might be more powerful than that of red deer.Obvious inversion regions containing olfactory receptor genes were also identified,which arose since the divergence.In conclusion,the high-quality allele-aware reference genome provides valuable resources for further illustration of the unique biological characteristics of antler,chromosome evolution,and multi-omics research of cervid animals.展开更多
Lung oncogenesis relies on intracellular cysteine to overcome oxidative stress.Several tumor types,including non-small cell lung cancer(NSCLC),upregulate the system x-c cystine/glutamate antiporter(xCT)through overexp...Lung oncogenesis relies on intracellular cysteine to overcome oxidative stress.Several tumor types,including non-small cell lung cancer(NSCLC),upregulate the system x-c cystine/glutamate antiporter(xCT)through overexpression of the cystine transporter SLC7A11,thus sustaining intracellular cysteine levels to support glutathione synthesis.Nuclear factor erythroid 2-related factor 2(NRF2)serves as a master regulator of oxidative stress resistance by regulating SLC7A11,whereas Kelch-like ECH-associated protein(KEAP1)acts as a cytoplasmic repressor of the oxidative responsive transcription factor NRF2.Mutations in KEAP1/NRF2 and p53 induce SLC7A11 activation in NSCLC.Extracellular cystine is crucial in supplying the intracellular cysteine levels necessary to combat oxidative stress.Disruptions in cystine availability lead to iron-dependent lipid peroxidation,thus resulting in a type of cell death called ferroptosis.Pharmacologic inhibitors of xCT(either SLC7A11 or GPX4)induce ferroptosis of NSCLC cells and other tumor types.When cystine uptake is impaired,the intracellular cysteine pool can be sustained by the transsulfuration pathway,which is catalyzed by cystathionine-B-synthase(CBS)and cystathionine g-lyase(CSE).The involvement of exogenous cysteine/cystine and the transsulfuration pathway in the cysteine pool and downstream metabolites results in compromised CD8^(+)T cell function and evasion of immunotherapy,diminishing immune response and potentially reducing the effectiveness of immunotherapeutic interventions.Pyroptosis is a previously unrecognized form of regulated cell death.In NSCLCs driven by EGFR,ALK,or KRAS,selective inhibitors induce pyroptotic cell death as well as apoptosis.After targeted therapy,the mitochondrial intrinsic apoptotic pathway is activated,thus leading to the cleavage and activation of caspase-3.Consequently,gasdermin E is activated,thus leading to permeabilization of the cytoplasmic membrane and cell-lytic pyroptosis(indicated by characteristic cell membrane ballooning).Breakthroughs i展开更多
Hybrids are always a focus of botanical research and have a high practical value in agricultural production.To better understand allele regulation and differences in DNA methylation in hybrids,we developed a phasing p...Hybrids are always a focus of botanical research and have a high practical value in agricultural production.To better understand allele regulation and differences in DNA methylation in hybrids,we developed a phasing pipeline for hybrid rice based on two parental genomes(PP2PG),which is applicable for Iso-Seq,RNA-Seq,and Bisulfite sequencing(BS-Seq).Using PP2PG,we analyzed differences in gene transcription,alternative splicing,and DNA methylation in an allele-specific manner between parents and progeny or different progeny alleles.The phasing of Iso-Seq data provided a great advantage in separating the whole gene structure and producing a significantly higher separation ratio than RNA-Seq.The interaction of hybrid alleles was studied by constructing an allele co-expression network that revealed the dominant allele effect in the network.The expression variation between parents and the parental alleles in progeny showed tissue-or environment-specific patterns,which implied a preference for trans-acting regulation under different conditions.In addition,by comparing allele-specific DNA methylation,we found that CG methylation was more likely to be inherited than CHG and CHHmethylation,and its enrichment in genic regions was connected to gene structure.In addition to an effective phasing pipeline,we also identified differentiation in OsWAK38 gene structure that may have led to the expansion of allele functions in hybrids.In summary,we developed a phasing pipeline and provided valuable insights into alternative splicing,interaction networks,trans-acting regulation,and the inheritance of DNA methylation in hybrid rice.展开更多
Gene expression variation is a key component underlying phenotypic variation and heterosis. Transcriptome profiling was performed on 23 different tissues or developmental stages of two maize inbreds, B73 and Mo17, as ...Gene expression variation is a key component underlying phenotypic variation and heterosis. Transcriptome profiling was performed on 23 different tissues or developmental stages of two maize inbreds, B73 and Mo17, as well as their hybrid. The obtained large-scale datasets provided opportunities to monitor the developmental dynamics of differential expression, additivity for gene expression, and regulatory variation. The transcriptome can be divided into .30 000 genes that are expressed in at least one tissue of one in bred and an additional ~10 000 “silent” genes that are not expressed in any tissue of any genotype, 90% of which are non-syntenic relative to other grasses. Many (.74%) of the expressed genes exhibit differential expression in at least one tissue. However, the majority of genes with differential expression do not exhibit consistent differential expression in different tissues. These genes often exhibit tissue-specific differential expression with equivalent expression in other tissues, and in many cases they switch the directionality of differential expression in different tissues. This suggests widespread variation for tissue-specific regulation of gene expression between the two maize inbreds B73 and Mo17. Nearly 5000 genes are expressed in only one parent in at least one tissue (single parent expression) and 97% of these genes are expressed at mid-parent levels or higher in the hybrid, providing extensive opportunities for hybrid complementation in heterosis. In general, additive expression patterns are much more common than non-additive patterns, and this trend is more pronounced for genes with strong differential expression or single pare nt expressi on. There is relatively little evidence for non-additive expression patterns that are maintained in multiple tissues. The analysis of allele-specific expression allowed classification of cis. and trans-regulatory variation. Genes with c/s-regulatory variation often exhibit additive expression and tend to have more consistent regulatory variatio展开更多
The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in ...The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.展开更多
Objective：To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo^＋ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods： We used the mt...Objective：To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo^＋ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods： We used the mtDNA 10400 locus to design unrnodifled and 3 ＇ phosphorothioate-modified allele-specific primers for PCR, which was performed using polymerases with and without 3 ＇ exonuclease activities. The effects of these primers on primer-extension were evaluated by agarose gel electrophoresis. Results： The unmodified primers were extended by both exo and exo^＋ polymerase irrespective of whether the primers were matched or mismatched with the templates. However, the 3＇ phosphorothioate-modified primers with a terminal mismatch triggered an ＂off-switch＂ of exo~ polymerase when compared to exopolymerase. Conclusion： The＂ on/off＇switch constituted by the combination of 3 ＇ phosphorothioatemodified primers with exo^＋ polymerase is a cost-effective, high-throughput and reliable method for SNP typing, which will be of enormous application in association studies by single nucleotide polymorphism screening.展开更多
Recent years have witnessed enormous progress in our understanding of the genetic predisposition to colorectal cancer (CRC). Estimates suggest that all or most genetic susceptibility mechanisms proposed so far, rangi... Recent years have witnessed enormous progress in our understanding of the genetic predisposition to colorectal cancer (CRC). Estimates suggest that all or most genetic susceptibility mechanisms proposed so far, ranging from high-penetrance genes to low-risk alleles, account for about 60% of the population-attributable fraction of CRC predisposition. In this context, there is increasing interest in the gene encoding the transforming growth factor β receptor 1 (TGFBR1 ); first when over a decade ago a common polymorphism in exon 1 (rs11466445, TGFBR1 *6A/9A) was suggested to be a risk allele for CRC, then when linkage studies identified the chromosomal region where the gene is located as susceptibility locus for familial CRC, and more recently when the allele-specific expression (ASE) of the gene was proposed as a risk factor for CRC. Published data on the association of TGFBR1 with CRC, regarding polymorphisms and ASE and including sporadic and familial forms of the disease, are often contradictory. This review gives a general overview of the most relevant studies in order to clarify the role of TGFBR1 in the field of CRC genetic susceptibility.展开更多
Allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions(DMRs),and aberrant genomic imprinted DNA methylation is associated with some human diseases,includ...Allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions(DMRs),and aberrant genomic imprinted DNA methylation is associated with some human diseases,including Prader-Willi syndrome and cancer.Thus,the development of an effective strategy for the precise editing of allele-specific methylated genes is essential for the functional clarification of imprinting elements and the correction of imprinting disorders in human diseases.To discover a feasible allele-specific genome editing tool based on the CRISPR/Cas system,which is an efficient genetargeting technique in various organisms,we examined the targeting efficiency of Staphylococcus aureus Cas9(SaCas9)and Streptococcus pyogenes Cas9(SpCas9)in response to DNA methylation interference.We found that the targeting efficiency of SaCas9,but not SpCas9,was enhanced by targeted DNA demethylation using the d Cas9-Tet1 catalytic domain(CD)but suppressed by targeted DNA methylation using Dnmt3l-Dnmt3a-d Cas9.An in vitro cleavage assay further demonstrated that SaCas9 nuclease activity was inhibited by 5-methylcytosine(5mC)in a synthesized Cp G-containing context.Further analysis with Ch IP-Q-PCR demonstrated that the non-methylated sequence targeting of Sa Cas9 depends on the binding preference of SaCas9 to non-methylated sequences.Taking advantage of this feature of SaCas9,we have successfully obtained non-methylated allele-biased targeted embryos/mice for two imprinting genes,H19 and Snrpn,with relatively high efficiencies of 28.6%and 47.4%,respectively.These results indicate that the targeting efficiency of SaCas9 was strongly reduced by DNA methylation.By using SaCas9,we successfully achieved allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci.展开更多
文摘The Cucurbita genus contains several economically important species in the Cucurbitaceae family. Here, we report high-quality genome sequences of C. maxima and C. moschata and provide evidence supporting an allotetraploidization event in Cucurbita. We are able to partition the genome into two homoeologous subgenomes based on different genetic distances to melon, cucumber, and watermelon in the Benincaseae tribe. We estimate that the two diploid progenitors successively diverged from Benincaseae around 31 and 26 million years ago （Mya）, respectively, and the allotetraploidization happened at some point between 26 Mya and 3 Mya, the estimated date when C. maxima and C. moschata diverged. The subgenomes have largely maintained the chromosome structures of their diploid progenitors. Such long-term karyotype stability after polyploidization has not been commonly observed in plant polyploids. The two subgenomes have retained similar numbers of genes, and neither subgenome is globally dominant in gene expression. Allele-specific expression analysis in the C. maxima ×C. moschata interspecific F1 hybrid and their two parents indicates the predominance of trans-regulatory effects underlying expression divergence of the parents, and detects transgressive gene expression changes in the hybrid correlated with heterosis in important agronomic traits. Our study provides insights into polyploid genome evolution and valuable resources for genetic improvement of cucurbit crops.
基金supported by the Provincial Technology Innovation Program of Shandongan award from the Natural Science Foundation of Shandong Province(ZR2021ZD30)+2 种基金the Director’s Award from the Peking University Institute of Advanced Agricultural Sciences,the National Top Young Talents Program of Chinathe Boya Postdoctoral Program of Peking University,the National Key R&D Program of China(2019YFD1000200)the Youth Innovation Promotion Association CAS(2018376).
文摘Kiwifruit is a recently domesticated horticultural fruit crop with substantial economic and nutritional value,especially because of the high content of vitamin C in its fruit.In this study,we de novo assembled two telomere-to-telomere kiwifruit genomes from Actinidia chinensis var.‘Donghong’(DH)and Actinidia latifolia‘Kuoye’(KY),with total lengths of 608327852 and 640561626 bp for 29 chromosomes,respectively.With a burst of structural variants involving inversion,translocations,and duplications within 8.39 million years,the metabolite content of DH and KY exhibited differences in saccharides,lignans,and vitamins.A regulatory ERF098 transcription factor family has expanded in KY and Actinidia eriantha,both of which have ultra-high vitamin C content.With each assembly phased into two complete haplotypes,we identified allelic variations between two sets of haplotypes,leading to protein sequence variations in 26494 and 27773 gene loci and allele-specific expression of 4687 and 12238 homozygous gene pairs.Synchronized metabolome and transcriptome changes during DH fruit development revealed the same dynamic patterns in expression levels and metabolite contents;free fatty acids and flavonols accumulated in the early stages,but sugar substances and amino acids accumulated in the late stages.The AcSWEET9b gene that exhibits allelic dominance was further identified to positively correlate with high sucrose content in fruit.Compared with wild varieties and other Actinidia species,AcSWEET9b promoters were selected in red-flesh kiwifruits that have increased fruit sucrose content,providing a possible explanation on why red-flesh kiwifruits are sweeter.Collectively,these two gap-free kiwifruit genomes provide a valuable genetic resource for investigating domestication mechanisms and genome-based breeding of kiwifruit.
基金supported by grants from the National Natural Science Foundation of China(31572381)National Thousand Youth Talents Plan of the International Science and Technology Cooperation Project of China(2013DFA31420)Science and Technology Innovation Capability Promotion Program of the Science and Technology Department of Qinghai Province(2015-ZJ-712)
文摘Divergence of gene expression and alter native splicing is a crucial driving force in the evolution of species;to date, however the molecular mechanism remains unclear. Hybrids of closely related species provide a suitable model to analyze allele-specific expressi on (ASE) and allele-specific alter native splicing (ASS). Analysis of ASE and ASS can uncover the differences in cis-regulatory elements between closely related species, while eliminating interferenee of trans-regulatory elements. Here, we provide a detailed characterization of ASE and ASS from 19 and 10 transcriptome datasets across five tissues from reciprocal-cross hybrids of horsex don key (mule/hi nny) and cattlexyak (dzo), respectively. Results showed that 4.8%-8.7% and 10.8%-16.7% of genes exhibited ASE and ASS, respectively. Notably, IncRNAs and pseudogenes were more likely to show ASE than protein-coding genes. In addition, genes showing ASE and ASS in mule/hinny were found to be involved in the regulation of muscle strength, whereas those of dzo were involved in high-altitude adaptati on. In con clusi on, our study dem on strated that explorati on of genes showing ASE and ASS in hybrids of closely related species is feasible for species evolution research.
文摘目的根据allele-specific PCR(AS-PCR)原理,建立具有良好敏感性与特异性的百日咳鲍特菌23S r RNA位点变异检测方法。方法基于百日咳鲍特菌23S r RNA A2047G位点突变与其对红霉素耐药的相关性,设计包含特异变异位点的引物进行两阶段PCR反应,根据电泳有无特异大小目的片段的出现,确定百日咳鲍特菌23S r RNA A2047G位点是否有变异。结果以基因序列测定法为金标准的评价结果显示,AS-PCR法检测突变型菌株的灵敏度为96%(144/150),特异度为100%(100/100),kappa=0.95(P<0.01);检测野生型菌株的灵敏度为100%(18/18),特异度为100%(100/100),kappa=1(P<0.01)。结论 AS-PCR方法检测百日咳鲍特菌23S r RNA A2047G位点变异具有较高灵敏度和特异度,适用于普通微生物实验室开展百日咳鲍特菌对红霉素耐药性的快速检测。
基金Beijing Natural Science Foundation，Beijing City Talent Training Project Fund
文摘Background Mycoplasma pneumoniae （M. pneumoniae） is one of the common pathogens causing atypical pneumonia. In recent years, resistance to macrolides has become more common, especially in China. Previous studies have confirmed that the mutation at position 2063 in domain V of the 23S rRNA is the most prevalent, followed by the mutation at position 2064. Reported molecular detection methods for the identification of these mutations include direct sequencing, restriction fragment length polymorphism analysis, real-time polymerase chain reaction （PCR） with high-resolution melt analysis, and nested PCR-linked with capillary electrophoresis, etc. The most commonly used method for monitoring resistance-conferring mutations in M. pneumoniae is direct DNA sequencing of PCR or nested PCR products. However, these methods are time-consuming, labor-intensive or need expensive equipments. Therefore the development of rapid and sensitive methods is very important for monitoring the resistance globally. Methods In this study, we reported a fast and cost-effective method for detecting 2063 and/or 2064 macrolide resistant mutations from specimens using a modified allele-specific PCR analysis, and all results were compared with the sequencing data. We also analyzed the clinical courses of these samples to confirm the modified allele-specific PCR results. Results Among 97 M. pneumoniae specimens, 88 were found to possess mutations by this method, and all modified allele-specific PCR analysis results were consistent with the sequencing data. The data of the clinical courses of these 97 cases showed that they suffered from severe pneumonia. Erythromycin showed better efficacy on cases from which no macrolide resistance mutation was found on their specimens. However, in some cases from which mutations were detected, erythromycin monotherapy had poor efficacy, and on these patients severe symptoms improved only when azithromycin was added to the treatment. Conclusions The drug-resistant M. pneumoniae is very common in Beijing,
基金This work was supported-by a grant from the National Natural Science Foundation of China （No. 30830088 and No. 30800938）.
文摘Background It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20%-30%. The aim of this study was to evaluate allele-specific real-time PCR （ASPCR） to detect the resistance-related mutations located at positions 103, 184 and 215.Methods We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140clinical samples using this method.Results The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M1841, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.Conclusions Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Yemerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.
基金supported by grants from the National Natural Science Foundation of China(Grant No.32272050 and U21A20208)the National Center of Technology Innovation for Saline-Alkali Tolerant Rice(2022PT1005)+2 种基金the Hunan Natural Science Foundation(Grant No.2022JJ30021)the Science and Technology Innovation Program of Hunan Province(2023NK1010)the Changsha Science and Technology Project(Grant No.kq2202221)。
文摘Hybrid rice(Oryza sativa)generally outperforms its inbred parents in yield and stress tolerance,a phenomenon termed heterosis,but the underlying mechanism is not completely understood.Here,we combined transcriptome,proteome,physiological,and heterosis analyses to examine the salt response of super hybrid rice Chaoyou1000(CY1000).In addition to surpassing the mean values for its two parents(mid-parent heterosis),CY1000 exhibited a higher reactive oxygen species scavenging ability than both its parents(over-parent heterosis or heterobeltiosis).Nonadditive expression and allele-specific gene expression assays showed that the glutathione S-transferase gene OsGSTU26 and the amino acid transporter gene OsAAT30 may have major roles in heterosis for salt tolerance,acting in an overdominant fashion in CY1000.Furthermore,we identified OsWRKY72 as a common transcription factor that binds and regulates OsGSTU26 and OsAAT30.The salt-sensitive phenotypes were associated with the OsWRKY72^(paternal)genotype or the OsAAT30^(maternal)genotype in core rice germplasm varieties.OsWRKY72^(paternal)specifically repressed the expression of OsGSTU26 under salt stress,leading to salinity sensitivity,while OsWRKY72^(maternal)specifically repressed OsAAT30,resulting in salinity tolerance.These results suggest that the OsWRKY72-OsAAT30/OsGSTU26 module may play an important role in heterosis for salt tolerance in an overdominant fashion in CY1000 hybrid rice,providing valuable clues to elucidate the mechanism of heterosis for salinity tolerance in hybrid rice.
基金supported by the Technological Production Transformation Foundation by the Ministry of Science and Technology of China(2002370010495)the foundation of Shandong Fruit Tree Three-Zero Project
文摘Embryo abortion stage and rescue system of hybrids were studied in the distant hybridizationbetween plum and apricot. Identification of the hybrids was also made. The resultsshowed: (1) Embryo abortion started from three weeks after pollination. (2) The germinationand growth of embryos were different at different growth stages, which could germinateand grow with PF value>0.5, but failed with PF value<0.5. In embryo rescue system ofhybrids, the best germination and differentiation medium was MS+6-BA 2mgL-1+IAA 0.3mgL-1,the rate of germination and differentiation reached up to 80%, bud induction andmultiplication medium was MS+6-BA 1.5mgL-1+IAA 0.3mgL-1, rooting medium was 1/2 MS+IAA0.8mgL-1. Some hybrids were transplanted into the field successfully. (3) Leaf shapeinvestigation and identification by S allele-specific PCR and RAPDs showed that thehybrids were true ones.
基金supported by the National Natural Science Foundation of China (31000711, 31370032)China Agriculture Research System (CARS-05)the Agricultural Science and Technology Innovation Program
文摘High-density genetic markers are required for genotyping and linkage mapping in identifying genes from crops with complex genomes, such as barley. As the most common variation, single nucleotide polymorphisms(SNPs) are suitable for accurate genotyping by using the next-generation sequencing(NGS) technology. Reduced representation libraries(RRLs) of five barley accessions and one mutant were sequenced using NGS technology for SNP discovery. Twenty million short reads were generated and the proportion of repetitive sequences was reduced by more than 56%. A total of 6061 SNPs were identified, and 451 were mapped to the draft sequence of the barley genome with pairing reads. Eleven SNPs were validated using length polymorphic allele-specific PCR markers.
基金supported by the National Natural Science Foundation of China(31871269 and 32270712)the Hubei Provincial Natural Science Foundation of China(2019CFA014)a starting research grant for High-level Talents from Guangxi University.
文摘Translational regulation is a critical step in the process of gene expression and governs the synthesis of proteins from mRNAs.Many studies have revealed translational regulation in plants in response to various environmental stimuli.However,there have been no studies documenting the comprehensive landscape of translational regulation and allele-specific translational efficiency in multiple plant tissues,especially those of rice,a main staple crop that feeds nearly half of the world’s population.Here we used RNA sequencing and ribosome profiling data to analyze the transcriptome and translatome of an elite hybrid rice,Shanyou 63(SY63),and its parental varieties Zhenshan 97 and Minghui 63.The results revealed that gene expression patterns varied more among tissues than among varieties at the transcriptional and translational levels.We identified 3392 upstream open reading frames(uORFs),and the uORF-containing genes were enriched in transcription factors.Only 668 of 13492 long non-coding RNAs could be translated into peptides.Finally,we discovered numerous genes with allele-specific translational efficiency in SY63 and demonstrated that some cis-regulatory elements may contribute to allelic divergence in translational efficiency.Overall,these findings may improve our understanding of translational regulation in rice and provide information for molecular breeding research.
基金the National Key R&D Program of China(Grant No.2018YFC1706601)the Natural Science Foundation of Heilongjiang Province of China(Grant No.C2017012)。
文摘Despite the scientific and medicinal importance of diploid sika deer(Cervus nippon),its genome resources are limited and haplotype-resolved chromosome-scale assembly is urgently needed.To explore mechanisms underlying the expression patterns of the allele-specific genes in antlers and the chromosome evolution in Cervidae,we report,for the first time,a high-quality haplotype-resolved chromosome-scale genome of sika deer by integrating multiple sequencing strategies,which was anchored to 32 homologous groups with a pair of sex chromosomes(XY).Several expanded genes(RET,PPP2R1A,PPP2R1B,YWHAB,YWHAZ,and RPS6)and positively selected genes(eIF4E,Wnt8A,Wnt9B,BMP4,and TP53)were identified,which could contribute to rapid antler growth without carcinogenesis.A comprehensive and systematic genome-wide analysis of allele expression patterns revealed that most alleles were functionally equivalent in regulating rapid antler growth and inhibiting oncogenesis.Comparative genomic analysis revealed that chromosome fission might occur during the divergence of sika deer and red deer(Cervus elaphus),and the olfactory sensation of sika deer might be more powerful than that of red deer.Obvious inversion regions containing olfactory receptor genes were also identified,which arose since the divergence.In conclusion,the high-quality allele-aware reference genome provides valuable resources for further illustration of the unique biological characteristics of antler,chromosome evolution,and multi-omics research of cervid animals.
基金supported by a Spanish Association Against Cancer(AECC)grant,(grant No.PROYE18012ROSE)support from Julián Santamaría Vali?o to the IOR Foundation。
文摘Lung oncogenesis relies on intracellular cysteine to overcome oxidative stress.Several tumor types,including non-small cell lung cancer(NSCLC),upregulate the system x-c cystine/glutamate antiporter(xCT)through overexpression of the cystine transporter SLC7A11,thus sustaining intracellular cysteine levels to support glutathione synthesis.Nuclear factor erythroid 2-related factor 2(NRF2)serves as a master regulator of oxidative stress resistance by regulating SLC7A11,whereas Kelch-like ECH-associated protein(KEAP1)acts as a cytoplasmic repressor of the oxidative responsive transcription factor NRF2.Mutations in KEAP1/NRF2 and p53 induce SLC7A11 activation in NSCLC.Extracellular cystine is crucial in supplying the intracellular cysteine levels necessary to combat oxidative stress.Disruptions in cystine availability lead to iron-dependent lipid peroxidation,thus resulting in a type of cell death called ferroptosis.Pharmacologic inhibitors of xCT(either SLC7A11 or GPX4)induce ferroptosis of NSCLC cells and other tumor types.When cystine uptake is impaired,the intracellular cysteine pool can be sustained by the transsulfuration pathway,which is catalyzed by cystathionine-B-synthase(CBS)and cystathionine g-lyase(CSE).The involvement of exogenous cysteine/cystine and the transsulfuration pathway in the cysteine pool and downstream metabolites results in compromised CD8^(+)T cell function and evasion of immunotherapy,diminishing immune response and potentially reducing the effectiveness of immunotherapeutic interventions.Pyroptosis is a previously unrecognized form of regulated cell death.In NSCLCs driven by EGFR,ALK,or KRAS,selective inhibitors induce pyroptotic cell death as well as apoptosis.After targeted therapy,the mitochondrial intrinsic apoptotic pathway is activated,thus leading to the cleavage and activation of caspase-3.Consequently,gasdermin E is activated,thus leading to permeabilization of the cytoplasmic membrane and cell-lytic pyroptosis(indicated by characteristic cell membrane ballooning).Breakthroughs i
基金supported by the National Natural Science Foundation of China(31871269)the Hubei Provincial Natural Science Foundation of China(2019CFA014).
文摘Hybrids are always a focus of botanical research and have a high practical value in agricultural production.To better understand allele regulation and differences in DNA methylation in hybrids,we developed a phasing pipeline for hybrid rice based on two parental genomes(PP2PG),which is applicable for Iso-Seq,RNA-Seq,and Bisulfite sequencing(BS-Seq).Using PP2PG,we analyzed differences in gene transcription,alternative splicing,and DNA methylation in an allele-specific manner between parents and progeny or different progeny alleles.The phasing of Iso-Seq data provided a great advantage in separating the whole gene structure and producing a significantly higher separation ratio than RNA-Seq.The interaction of hybrid alleles was studied by constructing an allele co-expression network that revealed the dominant allele effect in the network.The expression variation between parents and the parental alleles in progeny showed tissue-or environment-specific patterns,which implied a preference for trans-acting regulation under different conditions.In addition,by comparing allele-specific DNA methylation,we found that CG methylation was more likely to be inherited than CHG and CHHmethylation,and its enrichment in genic regions was connected to gene structure.In addition to an effective phasing pipeline,we also identified differentiation in OsWAK38 gene structure that may have led to the expansion of allele functions in hybrids.In summary,we developed a phasing pipeline and provided valuable insights into alternative splicing,interaction networks,trans-acting regulation,and the inheritance of DNA methylation in hybrid rice.
基金a grant from the National Science Foundation (#1546899) to S.P.B and N.M.S.
文摘Gene expression variation is a key component underlying phenotypic variation and heterosis. Transcriptome profiling was performed on 23 different tissues or developmental stages of two maize inbreds, B73 and Mo17, as well as their hybrid. The obtained large-scale datasets provided opportunities to monitor the developmental dynamics of differential expression, additivity for gene expression, and regulatory variation. The transcriptome can be divided into .30 000 genes that are expressed in at least one tissue of one in bred and an additional ~10 000 “silent” genes that are not expressed in any tissue of any genotype, 90% of which are non-syntenic relative to other grasses. Many (.74%) of the expressed genes exhibit differential expression in at least one tissue. However, the majority of genes with differential expression do not exhibit consistent differential expression in different tissues. These genes often exhibit tissue-specific differential expression with equivalent expression in other tissues, and in many cases they switch the directionality of differential expression in different tissues. This suggests widespread variation for tissue-specific regulation of gene expression between the two maize inbreds B73 and Mo17. Nearly 5000 genes are expressed in only one parent in at least one tissue (single parent expression) and 97% of these genes are expressed at mid-parent levels or higher in the hybrid, providing extensive opportunities for hybrid complementation in heterosis. In general, additive expression patterns are much more common than non-additive patterns, and this trend is more pronounced for genes with strong differential expression or single pare nt expressi on. There is relatively little evidence for non-additive expression patterns that are maintained in multiple tissues. The analysis of allele-specific expression allowed classification of cis. and trans-regulatory variation. Genes with c/s-regulatory variation often exhibit additive expression and tend to have more consistent regulatory variatio
基金Financial support received from Department of Biotechnology,Government of India vide grant No.BT/PR-8953/BCE/08/533/2007project sanctioned against grant No.BT/04/NE/2009financial support from Department of Science&Technology,Government of India in the form of a research fellowship under the INSPIRE program
文摘The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.
基金supported by a grant from the "135" Foundation of Jiangsu Province(RC2002052).
文摘Objective：To apply reformed AS-PCR, which combined phosphorothioate-modified primers with exo^＋ polymerase, in single nucleotide polymorphism discrimination of mitochondrial DNA 10400 locus. Methods： We used the mtDNA 10400 locus to design unrnodifled and 3 ＇ phosphorothioate-modified allele-specific primers for PCR, which was performed using polymerases with and without 3 ＇ exonuclease activities. The effects of these primers on primer-extension were evaluated by agarose gel electrophoresis. Results： The unmodified primers were extended by both exo and exo^＋ polymerase irrespective of whether the primers were matched or mismatched with the templates. However, the 3＇ phosphorothioate-modified primers with a terminal mismatch triggered an ＂off-switch＂ of exo~ polymerase when compared to exopolymerase. Conclusion： The＂ on/off＇switch constituted by the combination of 3 ＇ phosphorothioatemodified primers with exo^＋ polymerase is a cost-effective, high-throughput and reliable method for SNP typing, which will be of enormous application in association studies by single nucleotide polymorphism screening.
基金Supported by The Spanish Ministry of Science and Innovation(Grant BFU2009-10281 and Ramón y Cajal contract)the Scientific Foundation of Asociación Espa ola Contra el Cáncer
文摘 Recent years have witnessed enormous progress in our understanding of the genetic predisposition to colorectal cancer (CRC). Estimates suggest that all or most genetic susceptibility mechanisms proposed so far, ranging from high-penetrance genes to low-risk alleles, account for about 60% of the population-attributable fraction of CRC predisposition. In this context, there is increasing interest in the gene encoding the transforming growth factor β receptor 1 (TGFBR1 ); first when over a decade ago a common polymorphism in exon 1 (rs11466445, TGFBR1 *6A/9A) was suggested to be a risk allele for CRC, then when linkage studies identified the chromosomal region where the gene is located as susceptibility locus for familial CRC, and more recently when the allele-specific expression (ASE) of the gene was proposed as a risk factor for CRC. Published data on the association of TGFBR1 with CRC, regarding polymorphisms and ASE and including sporadic and familial forms of the disease, are often contradictory. This review gives a general overview of the most relevant studies in order to clarify the role of TGFBR1 in the field of CRC genetic susceptibility.
基金supported by the National Key R&D Program of China(2016YFC0905901,2018YFC1004700)the National Natural Science Foundation of China(31471400)
文摘Allele-specific DNA methylation is the most important imprinting marker localized to differentially methylated regions(DMRs),and aberrant genomic imprinted DNA methylation is associated with some human diseases,including Prader-Willi syndrome and cancer.Thus,the development of an effective strategy for the precise editing of allele-specific methylated genes is essential for the functional clarification of imprinting elements and the correction of imprinting disorders in human diseases.To discover a feasible allele-specific genome editing tool based on the CRISPR/Cas system,which is an efficient genetargeting technique in various organisms,we examined the targeting efficiency of Staphylococcus aureus Cas9(SaCas9)and Streptococcus pyogenes Cas9(SpCas9)in response to DNA methylation interference.We found that the targeting efficiency of SaCas9,but not SpCas9,was enhanced by targeted DNA demethylation using the d Cas9-Tet1 catalytic domain(CD)but suppressed by targeted DNA methylation using Dnmt3l-Dnmt3a-d Cas9.An in vitro cleavage assay further demonstrated that SaCas9 nuclease activity was inhibited by 5-methylcytosine(5mC)in a synthesized Cp G-containing context.Further analysis with Ch IP-Q-PCR demonstrated that the non-methylated sequence targeting of Sa Cas9 depends on the binding preference of SaCas9 to non-methylated sequences.Taking advantage of this feature of SaCas9,we have successfully obtained non-methylated allele-biased targeted embryos/mice for two imprinting genes,H19 and Snrpn,with relatively high efficiencies of 28.6%and 47.4%,respectively.These results indicate that the targeting efficiency of SaCas9 was strongly reduced by DNA methylation.By using SaCas9,we successfully achieved allele-specific genome editing of imprinting genes by preferentially targeting non-methylated loci.
