Action of circulating and infiltrating B cells in the immune microenvironment of colorectal cancer by single-cell sequencing analysis

BACKGROUND The complexity of the immune microenvironment has an impact on the treatment of colorectal cancer(CRC),one of the most prevalent malignancies worldwide.In this study,multi-omics and single-cell sequencing techniques were used to investigate the mechanism of action of circulating and infiltrating B cells in CRC.By revealing the heterogeneity and functional differences of B cells in cancer immunity,we aim to deepen our understanding of immune regulation and provide a scientific basis for the development of more effective cancer treatment strategies.AIM To explore the role of circulating and infiltrating B cell subsets in the immune microenvironment of CRC,explore the potential driving mechanism of B cell development,analyze the interaction between B cells and other immune cells in the immune microenvironment and the functions of communication molecules,and search for possible regulatory pathways to promote the anti-tumor effects of B cells.METHODS A total of 69 paracancer(normal),tumor and peripheral blood samples were collected from 23 patients with CRC from The Cancer Genome Atlas database(https://portal.gdc.cancer.gov/).After the immune cells were sorted by multicolor flow cytometry,the single cell transcriptome and B cell receptor group library were sequenced using the 10X Genomics platform,and the data were analyzed using bioinformatics tools such as Seurat.The differences in the number and function of B cell infiltration between tumor and normal tissue,the interaction between B cell subsets and T cells and myeloid cell subsets,and the transcription factor regulatory network of B cell subsets were explored and analyzed.RESULTS Compared with normal tissue,the infiltrating number of CD20+B cell subsets in tumor tissue increased significantly.Among them,germinal center B cells(GCB)played the most prominent role,with positive clone expansion and heavy chain mutation level increasing,and the trend of differentiation into memory B cells increased.However,the number of plasma cells in the tumor microenvironment decreased significantly,and the plasma cells secreting IgA antibodies decreased most obviously.In addition,compared with the immune microenvironment of normal tissues,GCB cells in tumor tissues became more closely connected with other immune cells such as T cells,and communication molecules that positively regulate immune function were significantly enriched.CONCLUSION The role of GCB in CRC tumor microenvironment is greatly enhanced,and its affinity to tumor antigen is enhanced by its significantly increased heavy chain mutation level.Meanwhile,GCB has enhanced its association with immune cells in the microenvironment,which plays a positive anti-tumor effect.

Safety assessment of a novel marine multi-stress-tolerant yeast Meyerozyma guilliermondii GXDK6 according to phenotype and whole genome-sequencing analysis

The application of microorganisms as probiotics is limited due to lack of safety evaluation.Here,a novel multi-stress-tolerant yeast Meyerozyma guilliermondii GXDK6 with aroma-producing properties was identified from marine mangrove microorganisms.Its safety and probiotic properties were assessed in accordance with phenotype and whole-genome sequencing analysis.Results showed that the genes and phenotypic expression of related virulence,antibiotic resistance and retroelement were rarely found.Hyphal morphogenesis genes(SIT4,HOG1,SPA2,ERK1,ICL1,CST20,HSP104,TPS1,and RHO1)and phospholipase secretion gene(VPS4)were annotated.True hyphae and phospholipase were absent.Only one retroelement(Tad1-65_BG)was found.Major biogenic amines(BAs)encoding genes were absent,except for spermidine synthase(JA9_002594),spermine synthase(JA9_004690),and tyrosine decarboxylase(inx).The production of single BAs and total BAs was far below the food-defined thresholds.GXDK6 had no resistance to common antifungal drugs.Virulence enzymes,such as gelatinase,DNase,hemolytic,lecithinase,and thrombin were absent.Acute toxicity test with mice demonstrated that GXDK6 is safe.GXDK6 has a good reproduction ability in the simulation gastrointestinal tract.GXDK6 also has a strong antioxidant ability,β-glucosidase,and inulinase activity.To sum up,GXDK6 is considered as a safe probiotic for human consumption and food fermentation.

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

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

Complete Genome Sequencing and Genetic Variation Analysis of Two H9N2 Subtype Avian Influenza Virus Strains

[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.

Association between childhood obesity and gut microbiota:16S rRNA gene sequencing-based cohort study

BACKGROUND This study aimed to identify characteristic gut genera in obese and normal-weight children(8-12 years old)using 16S rDNA sequencing.The research aimed to provide insights for mechanistic studies and prevention strategies for childhood obesity.Thirty normal-weight and thirty age-and sex-matched obese children were included.Questionnaires and body measurements were collected,and fecal samples underwent 16S rDNA sequencing.Significant differences in body mass index(BMI)and body-fat percentage were observed between the groups.Analysis of gut microbiota diversity revealed lowerα-diversity in obese children.Differences in gut microbiota composition were found between the two groups.Prevotella and Firmicutes were more abundant in the obese group,while Bacteroides and Sanguibacteroides were more prevalent in the control group.AIM To identify the characteristic gut genera in obese and normal-weight children(8-12-year-old)using 16S rDNA sequencing,and provide a basis for subsequent mechanistic studies and prevention strategies for childhood obesity.METHODS Thirty each normal-weight,1:1 matched for age and sex,and obese children,with an obese status from 2020 to 2022,were included in the control and obese groups,respectively.Basic information was collected through questionnaires and body measurements were obtained from both obese and normal-weight children.Fecal samples were collected from both groups and subjected to 16S rDNA sequencing using an Illumina MiSeq sequencing platform for gut microbiota diversity analysis.RESULTS Significant differences in BMI and body-fat percentage were observed between the two groups.The Ace and Chao1 indices were significantly lower in the obese group than those in the control group,whereas differences were not significant in the Shannon and Simpson indices.Kruskal-Wallis tests indicated significant differences in unweighted and weighted UniFrac distances between the gut microbiota of normal-weight and obese children(P<0.01),suggesting substantial disparities in both the species and quantity of gut microbiota between the two groups.Prevotella,Firmicutes,Bacteroides,and Sanguibacteroides were more abundant in the obese and control groups,respectively.Heatmap results demonstrated significant differences in the gut microbiota composition between obese and normal-weight children.CONCLUSION Obese children exhibited lowerα-diversity in their gut microbiota than did the normal-weight children.Significant differences were observed in the composition of gut microbiota between obese and normal-weight children.

Transcriptomic and bioinformatics analysis of the mechanism by which erythropoietin promotes recovery from traumatic brain injury in mice

Recent studies have found that erythropoietin promotes the recovery of neurological function after traumatic brain injury.However,the precise mechanism of action remains unclea r.In this study,we induced moderate traumatic brain injury in mice by intrape ritoneal injection of erythro poietin for 3 consecutive days.RNA sequencing detected a total of 4065 differentially expressed RNAs,including 1059 mRNAs,92 microRNAs,799 long non-coding RNAs,and 2115circular RNAs.Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses revealed that the coding and non-coding RNAs that were differentially expressed after traumatic brain injury and treatment with erythropoietin play roles in the axon guidance pathway,Wnt pathway,and MAPK pathway.Constructing competing endogenous RNA networks showed that regulatory relationship between the differentially expressed non-coding RNAs and mRNAs.Because the axon guidance pathway was repeatedly enriched,the expression of Wnt5a and Ephb6,key factors in the axonal guidance pathway,was assessed.Ephb6 expression decreased and Wnt5a expression increased after traumatic brain injury,and these effects were reversed by treatment with erythro poietin.These findings suggest that erythro poietin can promote recove ry of nerve function after traumatic brain injury through the axon guidance pathway.

Development and parentage analysis of SNP markers for Chestnut-vented Nuthatch(Sitta nagaensis)based on ddRAD-seq data

Extra-pair paternity(EPP)is commonly found in socially monogamous birds,especially in small passerine birds,and there are interspecific and intraspecific variations in the extent of EPP.The Chestnut-vented Nuthatch(Sitta nagaensis)is a socially monogamous passerine bird,and verifying whether this species has EPP relies on parentage testing-S.nagaensis is not known to have EPP.In this study,we developed SNP markers of this species that are informative for parentage analysis from double digest restriction site-associated DNA sequencing(ddRAD-seq)data.A panel consisting of 50 SNP markers,with a mean heterozygosity of 0.343,was used to resolve 95% of nestlings to fathers.The combined exclusion probabilities for the first parent and second parent were 0.991 and 0.9999,respectively.This panel of SNP markers is a powerful tool for parentage assignments in S.nagaensis.In addition,we found that three offspring(7.9%)from three nests(23.1%)were the result of extra-pair fertilization out of 38 offspring in 13 nests.Our study provided information on parentage analysis that has not been reported before in S.nagaensis.It also supplemented the understudied EPP behavior of birds in Asia,contributing to a general understanding of the EPP behaviors of birds.

Fine-mapping and primary analysis of candidate genes associated with seed coat color in mung bean(Vigna radiata L.)

Seed coat color affects the appearance and commodity quality of mung beans(Vigna radiata L.).The substances that affect mung bean seed coat color are mainly flavonoids,which have important medicinal value.Mapping the seed coat color gene in mung beans would facilitate the development of new varieties and improve their value.In this study,an F2 mapping population consisting of 546 plants was constructed using Jilv9(black seed coat)and BIS9805(green seed coat).Using bulk segregated analysis(BSA)sequencing and kompetitive allele-specific PCR(KASP)markers,the candidate region related to seed coat color was finally narrowed to 0.66 Mb on chromosome(Chr.)4 and included eight candidate genes.Combined transcriptome and metabolome analyses showed that three of the eight candidate genes(LOC106758748,LOC106758747,and LOC106759075)were differentially expressed,which may have caused the differences in flavonoid metabolite content between Jilv9 and BIS9805.These findings can provide a research basis for cloning the genes related to seed coat color and accelerate molecular markerassisted selection breeding in mung beans.

Revolutionizing stem cell research:unbiased insights through single-cell sequencing

Stem cells have shown great application potential in wound repair,tissue regeneration,and disease treatment.Therefore,a full understanding of stem cells and their related regulatory mechanisms in disease treatment is conducive to improving the therapeutic effect of stem cells.However,thus far,there are still many unsolved mysteries in thefield of stem cells due to technical limitations,which hinder the in-depth exploration of stem cells and their wide clinical application.Single-cell sequencing(SCS)has provided very powerful and unbiased insights into cell gene expression profiles at the single-cell level,bringing exciting results to the stem cellfield.At present,SCS has been widely applied in thefield of stem cells,covering various aspects,including lineage tracing the development of stem cells,identifying new stem cell types,exploring cellular heterogeneity,and identifying internal functional subpopulations.In this paper,we focus on the latest research progress and discuss the application of SCS technology in stem cells.

Comparative analysis of dideoxy sequencing,the KRAS StripAssay and pyrosequencing for detection of KRAS mutation

AIM:To compare the differences between dideoxy sequencing/KRAS StripAssay/pyrosequencing for detection of KRAS mutation in Chinese colorectal cancer (CRC) patients.METHODS:Formalin-f ixed, paraff in-embedded (FFPE) samples with tumor cells ≥ 50% were collected from 100 Chinese CRC patients at Beijing Cancer Hospital. After the extraction of genome DNA from FFPE samples, fragments contained codons 12 and 13 of KRAS exon 2 were amplified by polymerase chain reaction and analyzed by dideoxy sequencing, the KRAS Strip Assay and pyrosequencing. In addition, the sensitivities of the 3 methods were compared on serial dilutions (contents of mutant DNA: 100%,50%,20%, 5%,10%, 5%,1%,0%) of A549 cell line DNA (carrying the codon 12 Gly>Ser mutation) into wild-type DNA (human normal intestinal mucosa). The results of dideoxy sequencing,the KRAS StripAssay and pyrosequencing were analyzed by Chromas Software, Collector forKRAS Strip Assay and the pyrosequencing PyroMarkTM Q24 system, respectively.RESULTS: Among 100 patients, KRAS mutations were identif ied in 34%, 37% and 37% of patients by dideoxy sequencing, the KRAS StripAssay and pyrosequencing, respectively. The sensitivity was highest with the KRAS Strip Assay (1%), followed by pyrosequencing (5%), and dideoxy sequencing was lowest (15%). Six different mutation types were found in this study with 3 main mutations Gly12 Asp (GGT>GAT), Gly12 Val (GGT>GTT) and Gly13 Asp (GGC>GAC). Thirty-three patients were identifi ed to have KRAS mutations by the 3 methods, and a total of 8 patients had conflicting results between 3 methods: 4 mutations not detected by dideoxy sequencing and the KRAS StripAssay were identified by pyrosequencing; 3 mutations not detected by dideoxy sequencing and pyrosequencing were identif ied by the KRAS StripAssay; and 1 mutation not detected by pyrosequencing was conf irmed by dideoxy sequencing and the KRAS StripAssay. Among these discordant results, the results identif ied by dideoxy sequencing were consistent either with the KRAS StripAssay or with pyrosequencing, which indicated that the accuracy of dideoxy sequencing was high. CONCLUSION: Taking a worldwide view of reports and our results,dideoxy sequencing remains the most popular method because of its low cost and high accuracy.

Sequencing analysis of matrix metalloproteinase 7-induced genetic changes in Schwann cells

Previous research revealed the positive activity of matrix metalloproteinase 7(MMP7) on migration and myelin regeneration of Schwa nn cells(SCs). However, understanding of the molecular changes and biological activities induced by increased amounts of MMP7 in SCs remains limited. To better understand the underlying molecular events, primary SCs were isolated from the sciatic nerve stump of newborn rats and cultured with 10 nM human MMP7 for 24 hours. The results of genetic testing were analyzed at a relatively relaxed threshold value(fold change ≥ 1.5 and P-value < 0.05). Upon MMP7 exposure, 149 genes were found to be upregulated in SCs, whereas 133 genes were downregulated. Gene Ontology analysis suggested that many differentially expressed molecules were related to cellular processes, single-organism processes, and metabolic processes. Kyoto Enrichment of Genes and Genomes pathway analysis further indicated the critical involvement of cell signaling and metabolism in MMP7-induced molecular regulation of SCs. Results of Ingenuity Pathway Analysis(IPA) also revealed that MMP7 regulates biological processes, molecular functions, cellular components, diseases and functions, biosynthesis, material metabolism, cell movement, and axon guidance. The outcomes of further analysis will deepen our comprehension of MMP7-induced biological changes in SCs. This study was approved by the Laboratory Animal Ethics Committee of Nantong University, China(approval No. 20190225-004) on February 27, 2019.

Analysis of Saccharina japonica transcriptome using the high-throughput DNA sequencing technique and its vanadium-dependent haloperoxidase gene

Saccharina is one of the most important cold-water living marine brown algal genera. In this study we ana-lyzed the transcriptome of S. japonica, which belongs to the 1 000 Plants （OneKP） Project, by using a next-generation high-throughput DNA sequencing technique. About 5.16 GB of raw data were generated, and 65 536 scaffolds with an average length of 454 bp were assembled with SOAP de novo assembly method. In total, 19 040 unigenes were identified by BLAST;25 734 scaffolds were clustered into 37 Gene ontology functional groups;6 760 scaffolds were classified into 25 COG categories, as well as 2 665 scaffolds that were assigned to 306 KEGG pathways. Majority of the unigenes exhibited more similarities to algae including brown algae and diatom than other cyanobacteria, marine diatom, and plant. Saccharina japonica has the outstanding capability to accumulate halogen such as Br and I via halogenation processes from seawater. We acquired 42 different vanadium-dependent haloperoxidases （vHPO） in S. japonica transcriptome data, including 5 segments of vanadium-dependent iodoperoxidase （vIPO） and 37 segments of vanadium-de-pendent bromoperoxidase （vBPO）. Complicated analyses of identified fulllength S. japonica vBPO1 and S. japonica vBPO2 revealed the importance of vBPO among species of brown algae and the strong relationship between marine algal vBPOs and vIPOs. This study will enhance our understanding of the biological charac-teristics and economic values of S. japonica species.

Functional identification of lncRNAs in sweet cherry(Prunus avium)pollen tubes via transcriptome analysis using single-molecule long-read sequencing

Sweet cherry(Prunus avium)is a popular fruit with high nutritional value and excellent flavor.Although pollen plays an important role in the double fertilization and subsequent fruit production of this species,little is known about its pollen tube transcriptome.In this study,we identified 16,409 transcripts using single-molecule sequencing.After filtering 292 transposable elements,we conducted further analyses including mRNA classification,gene function prediction,alternative splicing(AS)analysis,and long noncoding RNA(lncRNA)identification to gain insight into the pollen transcriptome.The filtered transcripts could be matched with 3,438 coding region sequences from the sweet cherry genome.GO and KEGG analyses revealed complex biological processes during pollen tube elongation.A total of 2043 AS events were predicted,7 of which were identified in different organs,such as the leaf,pistil and pollen tube.Using BLASTnt and the Coding-Potential Assessment Tool(CPAT),we distinguished a total of 284 lncRNAs,among which 154 qualified as natural antisense transcripts(NATs).As the NATs could be the reverse complements of coding mRNA sequences,they might bind to coding sequences.Antisense transfection assays showed that the NATs could regulate the expression levels of their complementary sequences and even affect the growth conditions of pollen tubes.In summary,this research characterizes the transcripts of P.avium pollen and lays the foundation for elucidating the physiological and biochemical mechanisms underlying sexual reproduction in the male gametes of this species.

Transcriptome Sequencing and Analysis of Agaricus blazei Mycelia Under Cadmium Stress

To explore the mechanism of Agaricus blazei Murrill enrichment of the heavy metal cadmium,we employed Illumina high-throughput sequencing to analyze the transcriptomes of A.blazei mycelia treated with and without exogenous cadmium addition,and then the differentially expressed genes(DEGs)of the strains with high and low cadmium enrichment between the control and cadmium treatment were screened out.The results showed that the DEGs were mainly involved in steroid biosynthesis,antibiotic biosynthesis,protein processing in endoplasmic reticulum,glutathione metabolism and other pathways.Carbon metabolism and glutathione metabolism may play an important role in the response of A.blazei mycelium to cadmium stress.

Comparative analysis on transcriptome sequencings of six Sargassum species in China

Species of Sargassum are distributed worldwide, and are of great ecological and economic importance in marine ecosystems and bioresources. In this study, transcriptome sequencings of six Sargassum species were performed for the first time using an Illumina platform. For each sample, a total of 2.1-2.5 Gb of nucle-otides are collected and assembled into 69 871-116 790 scaffolds, with an average length of 410-550 bp and N50 length of 756-1 462 bp. A total of 20 512-28 684 unigenes of each sample were annotated and compared well with known gene sequences from nr database. Clusters of Orthologous Groups （COG）, gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） analyses were also performed for further un-derstanding of gene functions and regulation pathways. Gene expression levels were calculated based on RPKM values and compared among these species, especially for those genes related to carbohydrate metab-olism. Cluster analyses indicated that the differences of global gene expression between S. fusiforme, which was nominated as Hizikia fusiformis before, and other five species were not significant. Further phylogenet-ic analysis of 108 orthologous genes confirmed that S. fusiforme had closer relationship with S. hemiphyllum rather than S. horneri. These transcriptome data provided valuable information for better understanding of genome and gene characteristics of Sargassum algae and benefiting comparative and phylogenetic studies of Phaeophyceae species in future studies.

rDNA Sequencing and Preliminary Analysis of Microorganisms from Diseased Edible Fungus Using Non-culture Technology

The metagenomic DNA of disease tissue samples from four kinds of major edible fungus was extracted by CTAB method combined with DNA gel recovery kit. The genomie DNA was amplified by polymerase chain reaction using the universal primers of 16S rDNA and 18S rDNA, and then mone, elonal sequenced after ligated and transformed, rDNA sequences of 20 positive clones were selected randomly from each pair of primers for sequence alignment. The results showed that there were two bacterial diseases and two fungul diseases in the samples, respectively.

Complete Genome Sequencing and Analysis of Rehmannia Mosaic Virus Isolate from Shanxi Province

Using double-stranded RNA(dsRNA)technology and sequence-independent amplification(SIA),the molecular identification on infected Rehmannia glutinosa in the field with mosaic symptoms was performed and the whole-genome of the Rehmannia mosaic virus(ReMV)Shanxi isolate(ReMV-SX)was sequenced.Sequencing analysis showed that the virus that infected Rehmannia glutinosa was Rehmannia mosaic virus(ReMV).The full-length of the obtained ReMV-SX sequence(GenBank accession no.JX575184)was 6395 nt,containing four open reading frames(ORFs).The sequence homology analysis of the complete nucleotide sequence showed that ReMV-SX was 93.8%-97.0%homologous to ReMV in Tobamovirus subgroup Ⅰ,while only 49.8%-58.9%homologous to the isolates in subgroups Ⅱ and Ⅲ of the same genus.Phylogenetic analysis showed that ReMV-SX and ReMV-Henan formed a separate branch and had the closest genetic relationship.The results laid the foundation for ongoing researches in the taxonomic status and evolution of ReMV and for further investigating the pathogenic mechanism of ReMV infecting Rehmannia glutinosa.

Genome Sequencing and Phylogenetic Analysis of Three Avian Influenza H9N2 Subtypes in Guangxi

Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylogenetic analysis showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that the Guangxi isolates originated from the same source. However, the eight genes of the three isolates from Guangxi were not in the same sublineages in their respective phylogenetic trees, which suggests that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA which encode amino acids T, G, I were absent between nucleotide 205 and 214 in the open reading frame of the NA gene in the Guangxi isolates. AIV strains that infect human have, in their HA proteins, leucine at position 226. The analysis of deduced amino acid sequence of HA proteins showed that position 226 of these isolates contained glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.

Cloning and Sequence Analysis of Actin Gene from Rehmannia glutinosa

[ Objective ] The aim of this study is to clone and analyze the actin gene from Rehmannia glutinosa. [ Method ] Degenerate primers were designed according to the conserved regions of actin sequences of Rehmannia glutinosa and its similar species, RT-PCR was next conducted to amplify the actin gene from Rehmannia glutinosa. [ Result] The amplified fragment is 724 bp and correspondingly 240 amino acids. The BLAST results indicate that the homology between the amplified fragment and other higher plants for aetin gene sequences and amino acid are more than 80% and 90%, respectively, suggesting that the amplified fragment is the actin gene of Rehmannia glutinosa. [ Conclusion] Phylogenetic analysis shows that the actin gene of Rehmannia glutinosa has an intimate genetic relationship with actin7 gene of Nicotiana tabacum.

Sequence Analysis of Polymorphic Fragments in the Third Intron of Porcine H-FABP Gene

[ Objective] The aim of this study was to provide a theoretical basis for exploring the major genes affecting intramuscular fat （IMF） deposition. [Method] Taking 383 pigs from five breeds including Mashen Pig, Large White Pig, Landrace, Duroc and Shanxi White Pig as the experimental animals, polymorphisms of partial fragments in the third intron of porcine H-FABP gene were detected by PCR-SSCP method, and then the polymorphic fragments were sequenced. [ Result] Two alleles, designated as A and B, were found at the locus 346 in the third intron of porcine H-FABP gene, and the mutation was caused by a A→G substitution. [ Conclusion] A polymorphic locus was discovered in the third intron of porcine H-FABP gene in this experiment, laying a foundation for the further study on the relationship between H-FABP gene and IMF content.