Sesame （Sesamue indicum L.） is one of the most important oilseed crops with high oil yield. Here, we described a simple and efficient method for constructing a normalized cDNA library from a high oil content cultiva...Sesame （Sesamue indicum L.） is one of the most important oilseed crops with high oil yield. Here, we described a simple and efficient method for constructing a normalized cDNA library from a high oil content cultivar of sesame Zhongzhi 14, during its oil accumulation stages. It combined switching mechanism at 5？end of RNA transcript （SMART） technique and duplex-specific nuclease （DSN） normalization methods. Double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into Escherichia coli DH10B by electroporation. The capacity of the library was 1.0？06 clones in this library. Gel electrophoresis results indicated the fragments ranged from 700 to 2 000 bp, with the average size of 1 800 bp. Random picking clones showed that the recombination rate was 100%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of oil synthesis.展开更多
Hepatitis B immunoglobulin （HBIG） is important in the management of hepatitis B virus （HBV） infection. Aiming to develop recombinant monoclona# antibodies as an alternative to HBIG, we report the successful identi...Hepatitis B immunoglobulin （HBIG） is important in the management of hepatitis B virus （HBV） infection. Aiming to develop recombinant monoclona# antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen （HBsAg）-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the FIp recombinase-mediated integration （FIp-ln） system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting （FACS）, eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression （FSE）, fluorescent strength of binding （FSB） and relative binding ability （RBA） were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.展开更多
Aconitum carmichaelii is a high-value medicinal herb widely used across China,Japan,and other Asian countries.Aconitine-type diterpene alkaloids(DAs)are the characteristic compounds in Aconitum.Although six transcript...Aconitum carmichaelii is a high-value medicinal herb widely used across China,Japan,and other Asian countries.Aconitine-type diterpene alkaloids(DAs)are the characteristic compounds in Aconitum.Although six transcriptomes,based on short-read next generation sequencing technology,have been reported from the Aconitum species,the terpene synthase(TPS)corresponding to DAs biosynthesis remains unidentified.We apply a combination of Pacbio isoform sequencing and RNA sequencing to provide a comprehensive view of the A.carmichaelii transcriptome.Nineteen TPSs and five alternative splicing isoforms belonging to TPS-b,TPS-c,and TPS-e/f subfamilies were identified.In vitro enzyme reaction analysis functional identified two sesqui-TPSs and twelve di TPSs.Seven of the TPS-c subfamily genes reacted with GGPP to produce the intermediate ent-copalyl diphosphate.Five Ac KSLs separately reacted with ent-CPP to produce ent-kaurene,ent-atiserene,and ent-13-epi-sandaracopimaradie:a new diterpene found in Aconitum.Ac TPSs gene expression in conjunction DAs content analysis in different tissues validated that ent-CPP is the sole precursor to all DAs biosynthesis,with Ac KSL1,Ac KSL2 s and Ac KSL3-1 responsible for C20 atisine and napelline type DAs biosynthesis,respectively.These data clarified the molecular basis for the C20-DAs biosynthetic pathway in A.carmichaelii and pave the way for further exploration of C19-DAs biosynthesis in the Aconitum species.展开更多
A full-length cDNA of proteinase inhibitor gene with completed open reading frame of 116 amino acids was cloned from Ralstonia solanacearum （Rs） resistant potato leaves using the rapid amplification of cDNA ends （R...A full-length cDNA of proteinase inhibitor gene with completed open reading frame of 116 amino acids was cloned from Ralstonia solanacearum （Rs） resistant potato leaves using the rapid amplification of cDNA ends （RACE） method and designated as StPI. BLAST search against NCBI showed that the StPI gene shared 89% identity with potato proteinase inhibitor I precursor in nucleotide and 74% in amino acid. Analysis of semi-quantitative RT-PCR indicated that this gene was induced by Rs as well as up-regulated by jasmonic acid （JA）. The StPI gene expression reached the highest level during 6-12 h post Rs-inoculation or JA-treatment, and then leveled off. Moreover, this gene was strongly induced by JA and its mRNA accumulation increased more quickly than that of Rs-inoculation. The StPI gene may play a role in potato resistance against Rs. The induction of StPI by Rs invasion may have a similar signal transduction pathway with JA treatment.展开更多
The carbonic anhydrases(CAs)are a group of enzymes that play an important role in the absorption and transportation of CO_(2) in Saccharina japonica.They are encoded by a superfamily of genes with seven subtypes that ...The carbonic anhydrases(CAs)are a group of enzymes that play an important role in the absorption and transportation of CO_(2) in Saccharina japonica.They are encoded by a superfamily of genes with seven subtypes that are unrelated in sequence but share conserved function in catalyzing the reversible conversion of CO_(2) and HCO_(3)^(-).Here we have characterized the CA members in the transcriptome of S.japonica using Single-molecule real-time(SMRT)sequencing technology.Approximately 9830.4 megabases from 5,028,003 quality subreads were generated,and they were assembled into 326,512 full-length non-chimeric(FLNC)reads,with an average flnc read length of 2181 bp.After removing redundant sequences,79,010 unique transcripts were obtained of which 38,039 transcripts were successfully annotated.From the full-length transcriptome,we have identified 7 full-length cDNA sequences for CA genes(4α-CAs,1β-CAs and 2γ-CAs)and assessed for their potential functions based on phylogenetic analysis.Characterizations of CAs will provide the ground for future studies to determine the involvement of CAs in inorganic carbon absorption and transportation in S.japonica.展开更多
Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extract...Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5’RACE and bioinformatics. Results Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b).Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.展开更多
This study was aimed to isolate ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit （RbcS） from tea plant [Camellia sinensis （L.） O. Kuntze]. In the study of transcriptional profiling of gene expression ...This study was aimed to isolate ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit （RbcS） from tea plant [Camellia sinensis （L.） O. Kuntze]. In the study of transcriptional profiling of gene expression from tea flower bud development stage by cDNA-AFLP （cDNA amplified fragment length polymorphism）, we have isolated some transcript-derived fragments （TDFs） occurring in both the young and mature flower bud. One of them showed a high degree of similarity to RbcS. Based on the fragment, the full length of RbcS with 769-bp （EF011075） cDNA was obtained via rapid amplification of cDNA ends （RACE）. It contained an open reading frame of 176 amino acids consisting of a chloroplast transit peptide with 52 amino acids and a mature protein of 124 amino acids. The amino acids sequence presented a high identity to those of other plant RbcS genes. It also contains three conserved domains and a protein kinase C phosphorylation site, one tyrosine kinase phosphorylation site and two N-myristoylation sites. Analysis by RT-PCR showed that the expression of RbcS in tea from high to low was leaf, young stem, young flower bud and mature flower bud, respectively. The isolation of the tea Rubisco small subunit gene establishes a good foundation for further study on the photosynthesis of tea plant.展开更多
BACKGROUND Colorectal polyps that develop via the conventional adenoma-carcinoma sequence[e.g.,tubular adenoma(TA)]often progress to malignancy and are closely associated with changes in the composition of the gut mic...BACKGROUND Colorectal polyps that develop via the conventional adenoma-carcinoma sequence[e.g.,tubular adenoma(TA)]often progress to malignancy and are closely associated with changes in the composition of the gut microbiome.There is limited research concerning the microbial functions and gut microbiomes associated with colorectal polyps that arise through the serrated polyp pathway,such as hyperplastic polyps(HP).Exploration of microbiome alterations asso-ciated with HP and TA would improve the understanding of mechanisms by which specific microbes and their metabolic pathways contribute to colorectal carcinogenesis.AIM To investigate gut microbiome signatures,microbial associations,and microbial functions in HP and TA patients.METHODS Full-length 16S rRNA sequencing was used to characterize the gut microbiome in stool samples from control participants without polyps[control group(CT),n=40],patients with HP(n=52),and patients with TA(n=60).Significant differences in gut microbiome composition and functional mechanisms were identified between the CT group and patients with HP or TA.Analytical techniques in this study included differential abundance analysis,co-occurrence network analysis,and differential pathway analysis.RESULTS Colorectal cancer(CRC)-associated bacteria,including Streptococcus gallolyticus(S.gallolyticus),Bacteroides fragilis,and Clostridium symbiosum,were identified as characteristic microbial species in TA patients.Mediterraneibacter gnavus,associated with dysbiosis and gastrointestinal diseases,was significantly differentially abundant in the HP and TA groups.Functional pathway analysis revealed that HP patients exhibited enrichment in the sulfur oxidation pathway exclusively,whereas TA patients showed dominance in pathways related to secondary metabolite biosynthesis(e.g.,mevalonate);S.gallolyticus was a major contributor.Co-occurrence network and dynamic network analyses revealed co-occurrence of dysbiosis-associated bacteria in HP patients,whereas TA patients exhibited co-occurrence of CRC展开更多
Background:In our previous study,we observed a synergistic effect of 2,3,5,4’-Tetrahydroxystilbene-2-O-b-D-glucoside combined with adriamycin to induce apoptosis in MCF-7 breast cancer cells.However,the underlying me...Background:In our previous study,we observed a synergistic effect of 2,3,5,4’-Tetrahydroxystilbene-2-O-b-D-glucoside combined with adriamycin to induce apoptosis in MCF-7 breast cancer cells.However,the underlying mechanisms of epigenetic modifications,such as alternative splicing,have not been explored.In this study,we aimed to investigate the mechanism by which THSG inhibits MCF-7 cell proliferation using full-length transcriptome sequencing.Methods:First,cell viability was examined using the methyl thiazolyl tetrazolium method and full-length transcriptome sequencing was performed to identify genes and pathways.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to identify the principal pathways and targets of THSG.Flow cytometry analysis of cell cycle distribution was performed.Meanwhile,the analysis of alternative splicing and domains of the key proteins was conducted.Quantitative polymerase chain reaction and western blotting were performed for verification.Results:THSG showed significant cytotoxic activity in MCF-7 cells.Full-length transcriptome sequencing revealed differential alternative splicing with 173 upregulated and 263 downregulated genes.Further analysis identified distinct differential expression of genes(CHEK2-211 and CCND1-201)involved in the cell cycle in the THSG-treated group.Subsequently,alternative splicing types of CHEK2(mutually exclusive exon)and CCND1(intron retention).We found that THSG downregulated mRNA expression,as confirmed by quantitative polymerase chain reaction analysis.Interestingly,protein structural analysis revealed that THSG treatment led to the generation of CHK2-211,which was the result of a mutation in the amino acid residues(GLU-150,ASN-151)of the CHEK2 domain(VAL-150,GLY-151).and CyclinD1-201 were obtained when an amino acid(ASP-267)in the domain was lost in CyclinD1.Moreover,molecular docking analysis demonstrated that the domains of key proteins could bind THSG more effectively,with no difference in affinity.Western blotting confirmed that THSG展开更多
Circular RNAs(circ RNAs),covalently closed continuous RNA loops,are generated from cognate linear RNAs through back splicing events,and alternative splicing events may generate different circ RNA isoforms at the same ...Circular RNAs(circ RNAs),covalently closed continuous RNA loops,are generated from cognate linear RNAs through back splicing events,and alternative splicing events may generate different circ RNA isoforms at the same locus.However,the challenges of reconstruction and quantification of alternatively spliced full-length circ RNAs remain unresolved.On the basis of the internal structural characteristics of circ RNAs,we developed Circ AST,a tool to assemble alternatively spliced circ RNA transcripts and estimate their expression by using multiple splice graphs.Simulation studies showed that Circ AST correctly assembled the full sequences of circ RNAs with a sensitivity of 85.63%–94.32%and a precision of 81.96%–87.55%.By assigning reads to specific isoforms,Circ AST quantified the expression of circ RNA isoforms with correlation coefficients of 0.85–0.99 between theoretical and estimated values.We evaluated Circ AST on an in-house mouse testis RNA-seq dataset with RNase R treatment for enriching circ RNAs and identified 380 circ RNAs with full-length sequences different from those of their corresponding cognate linear RNAs.RT-PCR and Sanger sequencing analyses validated 32 out of 37 randomly selected isoforms,thus further indicating the good performance of Circ AST,especially for isoforms with low abundance.We also applied Circ AST to published experimental data and observed substantial diversity in circular transcripts across samples,thus suggesting that circ RNA expression is highly regulated.Circ AST can be accessed freely at https://github--com.3pco.8686c.com/xiaofengsong/CircAST.展开更多
Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle ...Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identified in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1 076 bp, and the cDNA fullness ratio was 86.2%. A total of 1 058 unique sequences with 342 contigs （32.3%） and 716 singleton （67.7%） expressed sequence tags （EST） were obtained by clustering and assembling. Meanwhile, 826 （78.1%） ESTs were categorized as known genes, and 232 （21.9%） ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index of Sus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank （accession numbers： EU650784-EU650788, GE843306, GH228978-GH229100）. The abundantly expressed genes in porcine muscle tissue were related to muscle fiber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.展开更多
Yellowfin seabream Acanthopagrus latus is an important economic fish in Chinese coastal areas.Given its narrow distribution and overfishing,the genetic diversity of yellowfin seabream has been restricted for artificia...Yellowfin seabream Acanthopagrus latus is an important economic fish in Chinese coastal areas.Given its narrow distribution and overfishing,the genetic diversity of yellowfin seabream has been restricted for artificial breeding and reproduction.We performed full-length transcriptome sequencing and assembly of the genome of yellowfin seabream.A total of 68086 unigenes were obtained,with an N50 of 3391 bp on average length of 2933 bp.A total number of 50593 expressed sequence tags linked to simple sequence repeats(EST-SSR)were identified,among them dinucleotide repeats(40.6%)and AC/GT motifs(38.5%)were the most frequent.Of the 190 EST-SSRs for which PCR primer pairs were designed,150 primer pairs successfully amplified target loci and 15 SSRs showed high polymorphism.The alleles per locus ranged 6-50 on average of 25.3.The expected and observed heterozygosity varied from 0.632 to 0.969 and from 0.519 to 0.953,respectively.The polymorphic index content(PIC)values of each locus ranged 0.587-0.966 on average of 0.851.Among six yellowfin seabream population samples preliminarily tested for genetic diversity and differentiation,the Fangchenggang(FCG)population in Guangxi Province had the highest mean observed heterozygosity(H_(o))value(0.786),whereas the Zhangzhou(ZZ)population in Fujian Province had the lowest(0.678).The pairwise fixation index(Fst)values indicated significant population differentiation among six yellowfin seabream populations.This study provided evidence for the usefulness of the transcriptomic resource information and EST-SSR markers for natural resource conservation,population genetics,and breeding studies of yellowfin seabream in South China.展开更多
基金supported by the National Basic Research Program of China (2011cb109305)the Genetically Modified Organisms Breeding Major Projects, China (2009zx08004-002B)+1 种基金the Open Project Program of Key Laboratory for Oil Crops Biology, the Ministry of Agriculture, China (200703)the Foundation of Oil Crops Research Institute, Chinese Academy of Agricultural Sciences
文摘Sesame （Sesamue indicum L.） is one of the most important oilseed crops with high oil yield. Here, we described a simple and efficient method for constructing a normalized cDNA library from a high oil content cultivar of sesame Zhongzhi 14, during its oil accumulation stages. It combined switching mechanism at 5？end of RNA transcript （SMART） technique and duplex-specific nuclease （DSN） normalization methods. Double-stranded cDNAs were synthesized from mRNAs, processed by normalization and Sfi I restriction endonuclease, and finally the cDNAs were ligated to pDNR-LIB vector. The ligation mixture was transformed into Escherichia coli DH10B by electroporation. The capacity of the library was 1.0？06 clones in this library. Gel electrophoresis results indicated the fragments ranged from 700 to 2 000 bp, with the average size of 1 800 bp. Random picking clones showed that the recombination rate was 100%. The results showed that the cDNA library constructed successfully was a full-length library with high quality, and could be used to screen the genes related to development of oil synthesis.
文摘Hepatitis B immunoglobulin （HBIG） is important in the management of hepatitis B virus （HBV） infection. Aiming to develop recombinant monoclona# antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen （HBsAg）-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the FIp recombinase-mediated integration （FIp-ln） system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting （FACS）, eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression （FSE）, fluorescent strength of binding （FSB） and relative binding ability （RBA） were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.
基金supported by the Major Program of National Natural Science Foundation of China(81891010,81891013)the National Natural Science Foundation of China(81822046)+1 种基金National Key R&D Program of China(2018YFA0900600,2020YFA0908000)Key project at central government level:the ability to establish sustainable use of valuable Chinese Medicine Resources(2060302,China)。
文摘Aconitum carmichaelii is a high-value medicinal herb widely used across China,Japan,and other Asian countries.Aconitine-type diterpene alkaloids(DAs)are the characteristic compounds in Aconitum.Although six transcriptomes,based on short-read next generation sequencing technology,have been reported from the Aconitum species,the terpene synthase(TPS)corresponding to DAs biosynthesis remains unidentified.We apply a combination of Pacbio isoform sequencing and RNA sequencing to provide a comprehensive view of the A.carmichaelii transcriptome.Nineteen TPSs and five alternative splicing isoforms belonging to TPS-b,TPS-c,and TPS-e/f subfamilies were identified.In vitro enzyme reaction analysis functional identified two sesqui-TPSs and twelve di TPSs.Seven of the TPS-c subfamily genes reacted with GGPP to produce the intermediate ent-copalyl diphosphate.Five Ac KSLs separately reacted with ent-CPP to produce ent-kaurene,ent-atiserene,and ent-13-epi-sandaracopimaradie:a new diterpene found in Aconitum.Ac TPSs gene expression in conjunction DAs content analysis in different tissues validated that ent-CPP is the sole precursor to all DAs biosynthesis,with Ac KSL1,Ac KSL2 s and Ac KSL3-1 responsible for C20 atisine and napelline type DAs biosynthesis,respectively.These data clarified the molecular basis for the C20-DAs biosynthetic pathway in A.carmichaelii and pave the way for further exploration of C19-DAs biosynthesis in the Aconitum species.
文摘A full-length cDNA of proteinase inhibitor gene with completed open reading frame of 116 amino acids was cloned from Ralstonia solanacearum （Rs） resistant potato leaves using the rapid amplification of cDNA ends （RACE） method and designated as StPI. BLAST search against NCBI showed that the StPI gene shared 89% identity with potato proteinase inhibitor I precursor in nucleotide and 74% in amino acid. Analysis of semi-quantitative RT-PCR indicated that this gene was induced by Rs as well as up-regulated by jasmonic acid （JA）. The StPI gene expression reached the highest level during 6-12 h post Rs-inoculation or JA-treatment, and then leveled off. Moreover, this gene was strongly induced by JA and its mRNA accumulation increased more quickly than that of Rs-inoculation. The StPI gene may play a role in potato resistance against Rs. The induction of StPI by Rs invasion may have a similar signal transduction pathway with JA treatment.
基金This research was supported by Shanghai Universities Peak Discipline Project of Aquaculture and“Double First-Class Initiative”program for its First-Class Discipline of aquaculture.
文摘The carbonic anhydrases(CAs)are a group of enzymes that play an important role in the absorption and transportation of CO_(2) in Saccharina japonica.They are encoded by a superfamily of genes with seven subtypes that are unrelated in sequence but share conserved function in catalyzing the reversible conversion of CO_(2) and HCO_(3)^(-).Here we have characterized the CA members in the transcriptome of S.japonica using Single-molecule real-time(SMRT)sequencing technology.Approximately 9830.4 megabases from 5,028,003 quality subreads were generated,and they were assembled into 326,512 full-length non-chimeric(FLNC)reads,with an average flnc read length of 2181 bp.After removing redundant sequences,79,010 unique transcripts were obtained of which 38,039 transcripts were successfully annotated.From the full-length transcriptome,we have identified 7 full-length cDNA sequences for CA genes(4α-CAs,1β-CAs and 2γ-CAs)and assessed for their potential functions based on phylogenetic analysis.Characterizations of CAs will provide the ground for future studies to determine the involvement of CAs in inorganic carbon absorption and transportation in S.japonica.
文摘Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5’RACE and bioinformatics. Results Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b).Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.
基金supported by the National Natural Science Foundation of China (30871568)National Key Technology R&D Program of China(2008BAC0B03).
文摘This study was aimed to isolate ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit （RbcS） from tea plant [Camellia sinensis （L.） O. Kuntze]. In the study of transcriptional profiling of gene expression from tea flower bud development stage by cDNA-AFLP （cDNA amplified fragment length polymorphism）, we have isolated some transcript-derived fragments （TDFs） occurring in both the young and mature flower bud. One of them showed a high degree of similarity to RbcS. Based on the fragment, the full length of RbcS with 769-bp （EF011075） cDNA was obtained via rapid amplification of cDNA ends （RACE）. It contained an open reading frame of 176 amino acids consisting of a chloroplast transit peptide with 52 amino acids and a mature protein of 124 amino acids. The amino acids sequence presented a high identity to those of other plant RbcS genes. It also contains three conserved domains and a protein kinase C phosphorylation site, one tyrosine kinase phosphorylation site and two N-myristoylation sites. Analysis by RT-PCR showed that the expression of RbcS in tea from high to low was leaf, young stem, young flower bud and mature flower bud, respectively. The isolation of the tea Rubisco small subunit gene establishes a good foundation for further study on the photosynthesis of tea plant.
基金Supported by Chulabhorn Royal Academy(Fundamental Fund:Fiscal year 2022 by National Science Research and Innovation Fund),No.FRB650039/0240 Project Code 165422.
文摘BACKGROUND Colorectal polyps that develop via the conventional adenoma-carcinoma sequence[e.g.,tubular adenoma(TA)]often progress to malignancy and are closely associated with changes in the composition of the gut microbiome.There is limited research concerning the microbial functions and gut microbiomes associated with colorectal polyps that arise through the serrated polyp pathway,such as hyperplastic polyps(HP).Exploration of microbiome alterations asso-ciated with HP and TA would improve the understanding of mechanisms by which specific microbes and their metabolic pathways contribute to colorectal carcinogenesis.AIM To investigate gut microbiome signatures,microbial associations,and microbial functions in HP and TA patients.METHODS Full-length 16S rRNA sequencing was used to characterize the gut microbiome in stool samples from control participants without polyps[control group(CT),n=40],patients with HP(n=52),and patients with TA(n=60).Significant differences in gut microbiome composition and functional mechanisms were identified between the CT group and patients with HP or TA.Analytical techniques in this study included differential abundance analysis,co-occurrence network analysis,and differential pathway analysis.RESULTS Colorectal cancer(CRC)-associated bacteria,including Streptococcus gallolyticus(S.gallolyticus),Bacteroides fragilis,and Clostridium symbiosum,were identified as characteristic microbial species in TA patients.Mediterraneibacter gnavus,associated with dysbiosis and gastrointestinal diseases,was significantly differentially abundant in the HP and TA groups.Functional pathway analysis revealed that HP patients exhibited enrichment in the sulfur oxidation pathway exclusively,whereas TA patients showed dominance in pathways related to secondary metabolite biosynthesis(e.g.,mevalonate);S.gallolyticus was a major contributor.Co-occurrence network and dynamic network analyses revealed co-occurrence of dysbiosis-associated bacteria in HP patients,whereas TA patients exhibited co-occurrence of CRC
基金This research was funded by the Science and Technology Project of Jiaxing City(2019AD32251,2020AY30010)Scientific Research Foundation of Traditional Chinese Medicine of Zhejiang Province(2021ZB291)+2 种基金Medical Scientific Research Foundation of Zhejiang Province(2020KY9482019)the 2023 Jiaxing Key Discipline of Medicine-Clinical Diagnostics(Supporting Subject 2023-ZC-002)Project of Education Commission of Hubei Province(D20202802,B2022192).
文摘Background:In our previous study,we observed a synergistic effect of 2,3,5,4’-Tetrahydroxystilbene-2-O-b-D-glucoside combined with adriamycin to induce apoptosis in MCF-7 breast cancer cells.However,the underlying mechanisms of epigenetic modifications,such as alternative splicing,have not been explored.In this study,we aimed to investigate the mechanism by which THSG inhibits MCF-7 cell proliferation using full-length transcriptome sequencing.Methods:First,cell viability was examined using the methyl thiazolyl tetrazolium method and full-length transcriptome sequencing was performed to identify genes and pathways.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to identify the principal pathways and targets of THSG.Flow cytometry analysis of cell cycle distribution was performed.Meanwhile,the analysis of alternative splicing and domains of the key proteins was conducted.Quantitative polymerase chain reaction and western blotting were performed for verification.Results:THSG showed significant cytotoxic activity in MCF-7 cells.Full-length transcriptome sequencing revealed differential alternative splicing with 173 upregulated and 263 downregulated genes.Further analysis identified distinct differential expression of genes(CHEK2-211 and CCND1-201)involved in the cell cycle in the THSG-treated group.Subsequently,alternative splicing types of CHEK2(mutually exclusive exon)and CCND1(intron retention).We found that THSG downregulated mRNA expression,as confirmed by quantitative polymerase chain reaction analysis.Interestingly,protein structural analysis revealed that THSG treatment led to the generation of CHK2-211,which was the result of a mutation in the amino acid residues(GLU-150,ASN-151)of the CHEK2 domain(VAL-150,GLY-151).and CyclinD1-201 were obtained when an amino acid(ASP-267)in the domain was lost in CyclinD1.Moreover,molecular docking analysis demonstrated that the domains of key proteins could bind THSG more effectively,with no difference in affinity.Western blotting confirmed that THSG
基金the National Natural Science Foundation of China(Grant No.61571223)the National Key R&D Program of China(Grant No.2016YFA0503300)+4 种基金supported by the National Natural Science Foundation of China(Grant Nos.61171191,31471403,and 81771641)the 333 Project of Jiangsu Province(Grant No.BRA2016386)the Program for Distinguished Talents of Six Domains in Jiangsu Province(Grant No.YY-019)the Fundamental Research Funds for the Central Universities(Grant No.NP2018109)the Fok Ying Tung Education Foundation(Grant No.161037),China.
文摘Circular RNAs(circ RNAs),covalently closed continuous RNA loops,are generated from cognate linear RNAs through back splicing events,and alternative splicing events may generate different circ RNA isoforms at the same locus.However,the challenges of reconstruction and quantification of alternatively spliced full-length circ RNAs remain unresolved.On the basis of the internal structural characteristics of circ RNAs,we developed Circ AST,a tool to assemble alternatively spliced circ RNA transcripts and estimate their expression by using multiple splice graphs.Simulation studies showed that Circ AST correctly assembled the full sequences of circ RNAs with a sensitivity of 85.63%–94.32%and a precision of 81.96%–87.55%.By assigning reads to specific isoforms,Circ AST quantified the expression of circ RNA isoforms with correlation coefficients of 0.85–0.99 between theoretical and estimated values.We evaluated Circ AST on an in-house mouse testis RNA-seq dataset with RNase R treatment for enriching circ RNAs and identified 380 circ RNAs with full-length sequences different from those of their corresponding cognate linear RNAs.RT-PCR and Sanger sequencing analyses validated 32 out of 37 randomly selected isoforms,thus further indicating the good performance of Circ AST,especially for isoforms with low abundance.We also applied Circ AST to published experimental data and observed substantial diversity in circular transcripts across samples,thus suggesting that circ RNA expression is highly regulated.Circ AST can be accessed freely at https://github--com.3pco.8686c.com/xiaofengsong/CircAST.
基金supported by the National Basic Research Program of China(2007CB116201)
文摘Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identified in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1 076 bp, and the cDNA fullness ratio was 86.2%. A total of 1 058 unique sequences with 342 contigs （32.3%） and 716 singleton （67.7%） expressed sequence tags （EST） were obtained by clustering and assembling. Meanwhile, 826 （78.1%） ESTs were categorized as known genes, and 232 （21.9%） ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index of Sus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank （accession numbers： EU650784-EU650788, GE843306, GH228978-GH229100）. The abundantly expressed genes in porcine muscle tissue were related to muscle fiber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.
基金Supported by the National Key R&D Program of China (No. 2019YFD0901202)the Key-Area Research and Development Program of Guangdong Province (No. 2021B0202020002)+1 种基金the China Postdoctoral Science Foundation (No. 2021M693677)the Yellow Fin Bream Seed System Building Project (2021)
文摘Yellowfin seabream Acanthopagrus latus is an important economic fish in Chinese coastal areas.Given its narrow distribution and overfishing,the genetic diversity of yellowfin seabream has been restricted for artificial breeding and reproduction.We performed full-length transcriptome sequencing and assembly of the genome of yellowfin seabream.A total of 68086 unigenes were obtained,with an N50 of 3391 bp on average length of 2933 bp.A total number of 50593 expressed sequence tags linked to simple sequence repeats(EST-SSR)were identified,among them dinucleotide repeats(40.6%)and AC/GT motifs(38.5%)were the most frequent.Of the 190 EST-SSRs for which PCR primer pairs were designed,150 primer pairs successfully amplified target loci and 15 SSRs showed high polymorphism.The alleles per locus ranged 6-50 on average of 25.3.The expected and observed heterozygosity varied from 0.632 to 0.969 and from 0.519 to 0.953,respectively.The polymorphic index content(PIC)values of each locus ranged 0.587-0.966 on average of 0.851.Among six yellowfin seabream population samples preliminarily tested for genetic diversity and differentiation,the Fangchenggang(FCG)population in Guangxi Province had the highest mean observed heterozygosity(H_(o))value(0.786),whereas the Zhangzhou(ZZ)population in Fujian Province had the lowest(0.678).The pairwise fixation index(Fst)values indicated significant population differentiation among six yellowfin seabream populations.This study provided evidence for the usefulness of the transcriptomic resource information and EST-SSR markers for natural resource conservation,population genetics,and breeding studies of yellowfin seabream in South China.
