Comprehensive analysis of the full-length transcripts and alternative splicing involved in clubroot resistance in Chinese cabbage

Chinese cabbage is an economically important Brassica vegetable worldwide, and clubroot, which is caused by the soilborne protist plant pathogen Plasmodiophora brassicae is regarded as a destructive disease to Brassica crops. Previous studies on the gene transcripts related to Chinese cabbage resistance to clubroot mainly employed RNA-seq technology,although it cannot provide accurate transcript assembly and structural information. In this study, PacBio RS II SMRT sequencing was used to generate full-length transcriptomes of mixed roots at 0, 2, 5, 8, 13, and 22 days after P. brassicae infection in the clubroot-resistant line DH40R. Overall, 39 376 high-quality isoforms and 26 270 open reading frames(ORFs) were identified from the SMRT sequencing data. Additionally, 426 annotated long noncoding RNAs(lncRNAs),56 transcription factor(TF) families, 1 883 genes with poly(A) sites and 1 691 alternative splicing(AS) events were identified. Furthermore, 1 201 of the genes had at least one AS event in DH40R. A comparison with RNA-seq data revealed six differentially expressed AS genes(one for disease resistance and five for defensive response) that are potentially involved in P. brassicae resistance. The results of this study provide valuable resources for basic research on clubroot resistance in Chinese cabbage.

Full-Length Transcriptome Analysis of Cultivated and Wild Tetraploid Peanut

The high-quality genomes and large-scale full-length cDNA sequences of allotetraploid peanuts have been sequenced and released,which has accelerated the functional genomics and molecular breeding research of peanut.In order to understand the difference in the transcriptional levels of wild and cultivated peanuts.In this study,we integrated of second-and third-generation sequencing technologies to sequence full-length transcriptomes in peanut cv.Pingdu9616 and its putative ancestor Arachis monticola.The RNA extracted from six different tissues(i.e.,roots,stems,leaves,flowers,needles and pods)were sampled at 20 days after flowering.A total of 31,764 and 33,981 high-quality transcripts were obtained from Monticola and Pingdu9616,respectively.The number of alternative splicing,the unit point mutation of variable adenylation,the number of open reading frames and the two-site mutation were identified in Pingdu9616 more than in Monticola,but the three-site mutation in Pingdu9616 was lower than in Monticola.1,691 LncRNAs,and 4,000 bp of maximum length of LncRNA was identified in Monticola and Pingdu9616.Furthermore,comparative analysis between transcript data shown that 56 transcription factor families were involved in Monticola,and Pingdu9616 and the number of transcription factors in Pingdu9616 was higher than that in Monticola,the number of expressed genes estimated in flower,root,young pod and leaf organs was higher in Monticola than Pingdu9616.Over all,our study provided a valuable resource of large-scale full-length transcripts for further research of the molecular breeding and functional analysis of genes.

Full-length transcriptome sequence and SSR marker development for genetic diversity research in yellowfin seabream Acanthopagrus latus

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.

Construction and Quality Analysis of Full-length cDNA Library of Phyllostachys heterocycla Germinating Seeds

[Objective] This study aimed to construct the full-length cDNA library for ger- minating seeds of Phyllostachys heterocycla [Method] Germinating seeds of P. hetero- cycla were used as experimental materials to construct the full-length cDNA library by using Oligo-capping method. [Result] The constructed library has a total capacity of 6.5×10^6 recombinant clones, and a low proportion of clones without inserted frag- ments; the size of inserted fragments ranges between 0.3-5.0 kb, with strict classifi- cation and ideal consistency. Furthermore, the proportion of clones harboring long in- serted fragments （1.0-5.0 kb） is as high as 30%, achieving the standard for high- quality full-length cDNA library. [Conclusion] The full-length cDNA library of germinat- ing seeds of P. heterocycla was successfully constructed, which laid important foun- dation for the functional genomics research of bamboo plants.

2,3,5,4’-Tetrahydroxystilbene-2-O-b-D-Glucoside modulates CHEK2 and CCND1 alternative splicing to inhibit MCF-7 cells proliferation

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 inhibited the expression of CHK2 and CyclinD1.Conclusion:THSG modulated the alternative splicing of CHEK2 and CCND1 by inducing G0/G1 cell cycle arrest,consequently suppressing MCF-7 cell proliferation.

Distinct gut microbiomes in Thai patients with colorectal polyps

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-associated bacteria.Furthermore,the co-occurrence of SCFA-producing bacteria was lower in TA patients than HP patients.CONCLUSION This study revealed distinct gut microbiome signatures associated with pathways of colorectal polyp development,providing insights concerning the roles of microbial species,functional pathways,and microbial interactions in colorectal carcinogenesis.

Construction of a Normalized Full-Length cDNA Library of Sesame Developing Seed by DSN and SMART^(TM)

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.

Cloning and Sequencing of a Full-Length cDNA Encoding the RuBPCase Small Subunit (RbcS) in Tea (Camellia sinensis)

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.

Generation and Analysis of Expressed Sequence Tags(ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

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.

De novo assembly of Zea nicaraguensis root transcriptome identified 5261 full-length transcripts

Zea nicaraguensis, a wild relative of cultivated maize （Zea mays subsp, mays）, is considered to be a valuable germplasm to improve the waterlogging tolerance of cultivated maize. Use of reverse genetic-based gene cloning and function verifi- cation to discover waterlogging tolerance genes in Z. nicaraguensis is currently impractical, because little gene sequence information for Z. nicaraguensis is available in public databases. In this study, Z. nicaraguensis seedlings were subjected to simulated waterlogging stress and total RNAs were isolated from roots stressed and non-stressed controls. In total, 80 mol L-1 Illumina 100-bp paired-end reads were generated. De novo assembly of the reads generated 81 002 final non-re- dundant contigs, from which 5 261 full-length transcripts were identified. Among these full-length transcripts, 3 169 had at least one Gene Ontology （GO） annotation, 2 354 received cluster of orthologous groups （COG） terms, and 1 992 were assigned a Kyoto encyclopedia of genes and genomes （KEGG） Orthology number. These sequence data represent a valuable resource for identification of Z. nicaraguensis genes involved in waterlogging response.

Full-length transcripts facilitates Portunus trituberculatus genome structure annotation

Portunus trituberculatus is an ide al model for elucidating crustacean genetic networks.Here we combined single molecule real-time(SMRT)sequencing and Illumina RNA-seq to characterize the coding genes,non-coding RNAs and pseudogenes and further to improve the genome annotation information of P.tritub erculatus.In this study,we assembled 9694 non-redundancy full-length transcripts,and 658737307-bp repetitive sequences were identified in the P.trituberculatus full-length transcriptome.We also predicted the P.tritub erculatus genome structure based on full-length transcripts,including 18602 genes,28686 non-coding RNAs,1407 pseudogenes,740 motif,and 26434 domain.Meanwhile,14460,10211,5412,7314,and 14448 genes had significant matches with sequences in the NR,KOG,GO,KEGG,and TrEMBL database,respectively.Overall,our work firstly provided the long-read transcriptome and we believed that these data are very necessary to improve the annotation information of P.trituberculatus genome structure,and useful information for the future studies on evolution and physiological regulation of P.trituberculatus.

Cloning and Sequence Analysis of the Full-Length Genome of Japanese encephalitis virus Strain SXBJ07 Isolated from Swine

A virus strain, showing cytopathic effect in BHK-21 cell, was isolated from swine brains in Shaanxi Province, China, in 2007. The isolate was confirmed as Japanese encephalitis virus （JEV） by immunofluorescence assay （IFA） and reverse transcription polymerase chain reaction （RT-PCR）, and named SXBJ07. The complete nueleotide and deduced amino acid sequences of the JEV strain SXBJ07 were determined. Its single open reading frame has a total of 3 432 amino acid residues. An extensive E gene based phylogenetic analysis was performed, the result showed that SXBJ07 strain belongs to genotype I. Comparison of the SXBJ07 genomic sequence with those of the 24 fully sequenced JEV strains in published databases showed nucleotide homology ranging from 99.0 to 83.7%; amino acid homology ranged from 99.8 to 94.8%. Compared SXBJ07 with SA14-14-2 strain, the current live vaccine strain in China, the homology of amino acid in envelope gene was 97.0%; and there were amino acid substitutions in 13 sites of the active domains of E protein （E1-E411）.

Amplification of the Full-Length PAMP Gene and Difference of the mRNA Expression Among Three Lean Pig Breeds

To understand the function of porcine adipocyte-special membrane protein （PAMP） gene and the difference of fat deposition ability among various lean pig breeds, a full-length porcine adipocyte-special membrane protein （PAMP） gene was successfully amplified using reverse transcription polymerase chain reaction （RT-PCR） and 5＇-rapid amplification of cDNA end （5＇-RACE）. The open reading frame was 1 587 bp encoding 529 amino acids. The nucleotide sequence of the fulllength PAMP gene was deposited in the GenBank under the accession number EF433431. The PAMP gene mRNA expression was analyzed on three lean pig breeds by quantitative reverse transcription polymerase chain reaction （QRTPCR）. The PAMP gene mRNA levels in YHM （Yorkshire × Hampshire × Meishan） pig and DLY （Duroc × Landrance × Yorkshire） pig were about 0.82 and 0.38 times of that in SW （Shanxi-White） pig, respectively.

Full-length model with multi-point settings for finishing rolling

Thickness,width,temperature,and profile are considered as control targets in process control of hot strip finishing rolling. The pre-calculated settings of the model information include rolling force,cooling water flow between stands,bending force,and roll shifting position. Without changing the load distribution,the interaction among thread speed,rolling force,rolling power,and cooling water flow between stands is comprehensively considered based on the quantifiable relationship among speed,force,and temperature. This paper proposes a full-length multi-point-setting model that uses the settings of strip head to combine the rolling speed diagram with the target finishing mill delivery temperature( FDT) to achieve the calculation of the control parameters along full length of strip. Traditional models cannot effectively predict rolling force of strip body or the maximum and minimum temperatures of FDT. It is also difficult for traditional models to suppress fluctuations in shape accuracy of full-length strip or improve the shape accuracy of the product. Calculation results show that the proposed full-length multi-point-setting model can provide the control parameters for temperature and rolling-force over full length of strip,predict the risk of rolling exceeding the equipment capability,and improve the shape accuracy and rolling stability of hot-rolled products.

Phyllosphere eukaryotic microalgal communities in rainforests:Drivers and diversity

Phyllosphere algae are common in tropical rainforests,forming visible biofilms or spots on plant leaf surfaces.However,knowledge of phyllosphere algal diversity and the environmental factors that drive that diversity is limited.The aim of this study is to identify the environmental factors that drive phyllosphere algal community composition and diversity in rainforests.For this purpose,we used single molecule real-time sequencing of full-length 18S rDNA to characterize the composition of phyllosphere microalgal communities growing on four host tree species(Ficus tikoua,Caryota mitis,Arenga pinnata,and Musa acuminata) common to three types of forest over four months at the Xishuangbanna Tropical Botanical Garden,Yunnan Province,China.Environmental 18S rDNA sequences revealed that the green algae orders Watanabeales and Trentepohliales were dominant in almost all algal communities and that phyllosphere algal species richness and biomass were lower in planted forest than in primeval and reserve rainforest.In addition,algal community composition differed significantly between planted forest and primeval rainforest.We also found that algal communities were affected by soluble reactive phosphorous,total nitrogen,and ammonium contents.Our findings indicate that algal community structure is significantly related to forest type and host tree species.Furthermore,this study is the first to identify environmental factors that affect phyllosphere algal communities,significantly contributing to future taxonomic research,especially for the green algae orders Watanabeales and Trentepohliales.This research also serves as an important reference for molecular diversity analysis of algae in other specific habitats,such as epiphytic algae and soil algae.

基于SMRT技术的水杉全长转录组分析及基因功能注释

以水杉原生种为材料,提取其幼嫩植株的根、茎和叶的总RNA,经mRNA纯化、反转录、全长转录组文库构建等过程,再采用SMRT技术测定其全长转录本序列,运用生物信息学对获得的原始转录组数据进行分析。结果显示:提取的总RNA符合建库要求;全长转录组测序共获得了包含5914711个亚读段(subreads)的14.0 Gb的数据,质量控制处理后包括236130个全长非嵌合读段(reads)和97626个一致性reads;转录本经去冗余处理后共获得61057个全长一致性读段(unigenes),其中有54099个被成功注释到7个数据库中,注释比达88.60%;对水杉unigenes作CDS(coding sequence)长度分布及转录因子分析,其CDS长度范围为144~6477 nt,平均长度约为679 nt;共检测到2386个转录因子,这些转录因子可以归类为29个家族。

Development of an Improved DNA-launched Porcine Reproductive and Respiratory Syndrome Virus Reverse Genetics System

[Objective] This study was to improve the virus replication efficiency of full length infectious cDNA clones by making use of the ribozyme＇s self incision property.[Method] By employing three-step PCR,HDV ribozyme（HdvRz）cDNA was isolated,and cloned into the downstream flanking the genome of the porcine reproductive and respiratory syndrome virus,and into which the bovine growth hormone polyadenylation sequence（BGH）was inserted via enzyme digestion and ligation,yielding pAPRRS-HB.The newly constructed pAPRRS-HB was used to transfect MARC-145 cells,in which the N protein and non-structural protein（nsp2）were determined by indirect immunofluorescence assay after 72 h of expression;meanwhile the virus titer of cell supernatant was tested using TCID50 assay.[Result] pAPRRS-HB containing complete infectious PRRSV cDNA has been successfully developed,and it performed about 10-fold higher virus rescue rate than pAPRRS without the engineered ribozyme element.[Conclusion] The results laid a foundation for revealing the structure and function of PRRSV gene.