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

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

The Influence of Aerial Exposure on Sea Anemones Aulactinia veratra Mucin Genes Expression Using the RNA Sequencing

Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air during the low tide and produces large quantities of mucus as an external covering. The relation between low tide and mucus secretion is still unclear, and what is the role of mucin during arial exposure is not yet investigated. This study hypothesised that the mucin genes in A. veratra would have significantly high expression in response to aerial exposure. Therefore, the aim of current study was to examine and analyses the response of A. veratra mucins in response to an experiment involving three hours of aerial exposure. To achieve this, aim the RNA-sequencing and bioinformatics analyses were used to examine the expression profile of A. veratra mucin genes in response to aerial exposure. The generated results have shown that, Mucin4-like and mucin5B-like were up-regulated in response to the three hours of aerial exposure in A. veratra. This finding shows a significant role of mucin5B-like and mucin4-like genes in response to air stress at low tide. The data generated from this study could be used in conjunction with future mucin gene studies of sea anemones and other cnidarians to compare A. veratra mucin gene expression results across time, and to extend our understanding of mucin stress response in this phylum.

Bioinformatics analysis of microarray data to explore the key genes involved in HSF4 mutation-induced cataract

AIM： To reveal the mechanisms of heat-shock transcription factor 4 （HSF4） mutation-induced cataract.METHODS： GSE22362, including 3 HSF4-null lens and 3 wild-type lens, was obtained from Gene Expression Omnibus database. After data preprocessing, the differentially expressed genes （DEGs） were identified using the limma package. Based on Database for Annotation, Visualization and Integrated Discovery （DAVID） tool, functional and pathway enrichment analyses were performed for the DEGs. Followed by protein-protein interaction （PPI） network was constructed using STRING database and Cytoscape software. Furthermore, the validated microRNA （miRNA）-DEG pairs were obtained from miRWalk2.0 database, and then miRNA-DEG regulatory network was visualized by Cytoscape software. RESULTS： A total of 176 DEGs were identified in HSF4-null lens compared with wild-type lens. In the PPI network, FBJ osteosarcoma oncogene （FOS）, early growth response 1 （EGR1） and heme oxygenase （decycling） 1 （HMOX1） had higher degrees and could interact with each other. Besides, mmu-miR-15a-5p and mmu-miR-26a-5p were among the top 10 miRNAs in the miRNA-DEG regulatory network. Additionally, mmu-miR-26a-5p could target EGR1 in the regulatory network. CONCLUSION： FOS, EGR1, HMOX1, mmu-miR-26a-5p and mmu-miR-15a-5p might function in the pathogenesis of HSF4 mutation-induced cataract.

Changes of chlorogenic acid content and its synthesis-associated genes expression in Xuehua pear fruit during development

According to synthetic pathway of plant chlorogenic acid （CGA）, the expression patterns of genes encoding enzymes that are associated with CGA synthesis were studied in normally developed Xuehua pear fruit. The study demonstrated that CGA content in peel and flesh of Xuehua pear decreased as fruit development progressed, with a higher level in peel. The expression levels of PbPAL 1, PbPAL2, PbC3H, PbC4H, Pb4CL 1, Pb4CL2, Pb4CL6, PbHC T1 and PbHC T3 genes decreased in fruit, which was consistent with the pattern of variation in CGA content. That indicated that these genes might be key genes for influencing fruit CGA synthesis in Xuehua pear. However, Pb4CL7 gene expression profile is not consistent with variation of CGA content, hence, it may not be a key gene involved in CGA synthesis.

The relationship between loss expression of DPC4/Smad4 gene and carcinogenesis of pancreatobiliary carcinoma

Objective: To clarify the relationship between loss of DPCA gene expression and pathogenesis of pancreato- biliary carcinoma. Methods: 75 slides of normal duct (20), hyperplasia (15), dysplasia (15), invasive carcinoma (25) from patients with pancreatic diseases including pancreatic carcinoma (25 patients), chronic pancreatitis (6), pancreas injury (2) and 71 slides of common bile duct (CBD) carcinoma (38), gallbladder carcinoma (18), hilar bile duct (HBD) carcinoma (15) from patients with primary biliary tract carcinoma were analyzed for the expression of DPC4 protein by im- munohistochemical staining. Results: All specimens from 20 cases of normal duct and 15 cases of hyperplasia showed marked expres- sion of DPC4 protein. The frequency of loss expres- sion of the DPC4 gene was 33 % in dysplasia, and 48% in invasive carcinoma. There was a significant statistical difference between byperplasia and dyspla- sia (P<0.01) and in dysplasia vs invasive carcinoma (P<0.05). The frequency of loss expression of the DPC4 gene was 47.3% in CBD carcinoma, 11% in gallbladder carcinoma, and 13% in HBD carcinoma. The frequency of loss expression of the DPCA gene was significantly different in CBD carcinoma vs gall- bladder carcinoma and HBD carcinoma (P<0.01). Conclusions: Inactivation of the DPC4 gene occurs late in the neoplastic progression of pancreatic carci- noma. The frequency of DPC4 gene alternation was different in various locations of biliary tract carcino- ma. In CBD carcinoma, this frequency is similar to that in pancreatic carcinoma, indicating their similar molecular alternations.

Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice

Endogenous reference genes (ERGs) provide vital information regarding genetically modified organisms (GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified (GM) ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase (SPS), phospholipase D (PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs, a new ERG gene, phosphoenolpyruvate carboxylase (PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R2 of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection, which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.

Sequence analysis of VP4 genes of wild type and culture adapted human rotavirus G1P[8]strains

Objective:To conduct a comparative analysis of the VP4 gene sequences of Indian wild type (06361,0613158,061060 and 0715880) and cell culture adapted(06361-CA,0613158-CA.061060- CA and 0715880-CA) G1P[8]rotavirus strains.Methods:Full-length VP4 genes of each of the four wild type G1P[8]rotavirus strains and their cell culture adapted counterparts displaying consistent cytopathic effect were subjected to RT-PCR amplification and nucleotide sequencing. Results:All four cell culture adapted G1P[8]rotavirus strains showed nucleotide and amino acid substitutions in the VP4 gene as compared to their wild type strains.The number of substitutions however,varied from 1-64 and 1-13 respectively.The substitutions were distributed in both VP5* and VP8* subunits of VP4 gene respectively of permeabilizalion and hemagglutinaling activity. The presence of unique amino acid substitutions was identified in two of the four wild type(V377G. S387N in 061060 and 1644L in 0715880) and all four cell culture adapted(A46V in 0613158-CA. T60R in 06361-CA,L237V.G389V and Q480H in 061060-CA and S615G and T625P in 0715880-CA) strains for the first time in the VP4 gene of P[8]specificity.Amino acid substitutions generated increase in the hydrophilicity in the cell culture adapted rotavirus strains as compared to their corresponding wild type strains.Conclusions:Amino acid substitutions detected in the VP4 genes of G1P[8]rotavirus strains from this study together with those from other studies highlight occurrence of only strain and/or host specific substitutions during cell culture adaptation. Further evaluation of such substitutions for their role in attenuation,immunogenicity and conformation is needed for the development of newer rolavirus vaccines.

Fine-mapping and characterisation of genes on barley(Hordeum vulgare)chromosome 2H for salinity stress tolerance during germination

Salinity causes a detrimental impact on plant growth,particularly when the stress occurs during germination and early development stages.Barley is one of the most salt-tolerant crops;previously we mapped two quantitative trait loci(QTL)for salinity tolerance during germination on the short arm of chromosome 2 H using a CM72/Gairdner doubled haploid(DH)population.Here,we narrowed down the major QTL to a region of 0.341 or 0.439 Mb containing 9 or 24 candidate genes belonging to 6 or 20 functional gene families according to barley reference genomes v1 and v3 respectively,using two DH populations of CM72/Gairdner and Skiff/CM72,F_(2)and F;generations of CM72/Gairdner/;Spartacus CL,Two Receptorlike kinase 4(RLPK4)v1 or Receptor-like kinase(RLK)v3 could be the candidates for enhanced germination under salinity stress because of their upregulated expression in salt-tolerant variety CM72.Besides,several insertion/deletion polymorphisms were identified within the 3 rd exon of the genes between CM72 and Gairdner.The sequence variations resulted in shifted functional protein domains,which may be associated with differences in salinity tolerance.Two molecular markers were designed for selecting the locus with receptor-like protein kinase 4,and one was inside HORVU2 Hr1 G111760.1 or HORVU.MOREX.r3.2 HG0202810.1.The diagnostic markers will allow for pyramiding of 2 H locus in barley varieties and facilitate genetic improvement for saline soils.Further,validation of the genes to elucidate the mechanisms involved in enhancing salinity tolerance at germination and designing RLPK4 specific markers is proposed.For this publication,all the analysis was based on barley reference genome of2017(v1),and it was used throughout for consistence.However,the positions of the markers and genes identified were updated according to new genome(v3)for reference.

Potential effect of hepatitis C Virus non-structural protein 4B on liver carcinogenesis

Objective：To investigate the effect of hepatitis C virus non-structural protein 4B（HCV NS4B） on c-Myc, P53, ras gene expression＂ and apoptosis in hepatic cells and study the possible role that NS4B played in the carcinogenesis of heparoma. Methods： The recombinant plasmid（PCXN2-NS4B, PCXN2-P53） and the empty, vector were transfected or co-transfected into Chang liver cells with liposome. Screening was performed with G418. Plasmid mRNA was detected by RT-PCR. The pro rein expressions of c-Myc and ras genes were analyzed by immunocytochemistry. The expressions of wild-type P53 （wtp53） gene were detected by in situ hybridization. TUNEL（flow cytometry） was used for assessing the rate of apoptosis. Results：No expression of c-Myc gene was found in PCXN2 group. The expression of c-Myc gene in NS4B group was 21.3% ＋ 1.2%. The ex pression of ras gene in PCXN2 group was lower than that in NS4B group. Compared with PCXN2 group, the expression of P53 mRNA was not promoted or inhibited in NS4B group. But the expression of P53 mRNA in NS4B-P53 group was lower than that in P53 group. In PCXN2, NS4B, P53 and NS4B-P53 group, the rates of apoptosis were 17.02% ± 1.24%, 11.94% ± 2.24%, 25.84% ± 3.49% and 18.34% ± 1.55% respectively. Conclusion ：HCV NS4B induces the expression of c-Myc and ras gene. HCV NS4B may play a role in the inhibition of cell death through P53-dependent manner. Results from this study suggested that HCV NS4B might contribute to the viral carcinogenesis.

Expression Profiles of <i>psbA, ALS, EPSPS</i>, and Other Chloroplastic Genes in Response to PSII-, ALS-, and EPSPS-Inhibitor Treatments in <i>Kochia scoparia</i>

Kochia (Kochia scoparia L. Schrad.), also known as tumbleweed, is an economically important annual C4 broadleaf weed found throughout the US Great Plains. Several herbicides with different modes of action are used in the management of kochia. The effect of commonly used herbicides on the expression of their target site(s) and photosynthetic/chloroplastic genes is poorly understood in weed species, including kochia. The objective of this research was to characterize the expression profiles of herbicide target-site genes, KspsbA, KsALS, and KsEPSPS upon treatment with PSII- (e.g. atrazine), ALS- (e.g. chlorsulfuron), and EPSPS- (e.g. glyphosate)-inhibitors, respectively, in kochia. Furthermore, the expression of genes involved in photosynthesis (e.g. KsRubisco, KsCAB, and KsPPDK) was also determined in response to these herbicide treatments. KspsbA was strongly upregulated (>200-fold) 24 h after atrazine treatment. Transcript levels of the KsALS or KsEPSPS genes were 7 and 3-fold higher 24 h after chlorsulfuron or glyphosate treatment, respectively. KsRubisco, a Calvin cycle gene important for CO2 fixation, was upregulated 7 and 2.6-fold 8 and 24 h after glyphosate and chlorsulfuron treatments, whereas it downregulated 8 and 24 h after atrazine treatment. The transcript levels of KsPPDK remained unchanged after glyphosate treatment but increased 1.8-fold and decreased 2-fold at 24 h after chlorsulfuron and atrazine treatments, respectively. KsCAB remained unchanged after chlorsulfuron treatment, but was downregulated after glyphosate and atrazine treatments. The results show that herbicide treatments not only affect the respective target-site gene expression, but also influence the genes involved in the critical photosynthetic pathway.

Unique genes carried by abundant species enhance CH_(4) emissions during the growing season at the Tibetan Plateau

CH_(4) emission rates followed an increased pattern during the growing season at Tibetan Plateau.•Unique genes carried by abundant species were positively correlated with CH_(4) emission rates.•Climate factors influenced CH_(4) emission rates by regulating microbial community and their genes.Microorganisms play pivotal roles in soil methane(CH_(4))emissions and their functional genes are origins of a key mechanism for soil CH4-cycling.However,understanding of the roles of specific genes(e.g.,unique or shared genes carried by species)underlying CH_(4)-cycling remains elusive.Here,we measured CH_(4) emission rates and investigated variations in microbial community and the abundance of genes carried by species during the growing season in alpine meadow on the Tibetan Plateau.We discovered that CH_(4) emission rates increased from 394.4,745.9,and 1092.7µg CH4 m−2 h−1,in April,June,and August,respectively,and had a positive correlation with unique genes carried by abundant species during the growing season.Moreover,we found that unique genes carried by abundant species involved in methanogenesis processes have a higher abundance than methanotrophic processes.Further analysis indicated that climate factors(i.e.,mean monthly temperature(MMT)and mean monthly precipitation(MMP))influenced microbial community and their functional genes,and therefore affected the CH_(4) emission rates.Overall,the present study provides a novel insight into the variation of soil CH4 emissions from a functional gene perspective,highlighting the important roles of unique genes carried by abundant species in CH4 emissions in the Tibetan Plateau under seasonal variation.

SCN4A基因R669H变异的家族性低钾性周期性麻痹1家系分析

目的 探讨一个低钾性周期性麻痹(HOKPP)家系的临床及遗传学特点。方法 回顾性分析1个HOKPP家系的临床资料。结果 先证者表现为周期性瘫痪,发作时四肢无力,血钾明显降低(1~2 mmol/L),先证者的父亲及表哥有相似症状。基因检测发现,先证者、父亲、小姑和表哥SCN4A基因存在1个杂合错义变异c.2006G>A(p.R669H),其大姑及叔叔未发现该变异。结论 该家系呈现不规则显性遗传,男性杂合子严重程度、发病频率和起病年龄有所不同,而女性杂合子无临床表型。本研究首次明确在亚洲种族人群中R669H变异导致男性完全外显,而女性可能为携带者。

H3K27me3 and H3K4me3 Chromatin Environment at Super-Induced Dehydration Stress Memory Genes of Arabidopsis thaliana

Pre-exposure to a stress may alter the plant＇s cellular, biochemical, and/or transcriptional responses during future encounters as a ＂memory＇ from the previous stress. Genes increasing transcription in response to a first dehydra- tion stress, but producing much higher transcript levels in a subsequent stress, represent the super-induced ＇transcription memory＇ genes in Arabidopsis thaliana. The chromatin environment （histone H3 tri-methylations of Lys 4 and Lys 27, H3K4me3, and H3K27me3） studied at five dehydration stress memory genes revealed existence of distinct memory- response subclasses that responded differently to CLF deficiency and displayed different transcriptional activities dur- ing the watered recovery periods. Among the most important findings is the novel aspect of the H3K27me3 function observed at specific dehydration stress memory genes. In contrast to its well-known role as a chromatin repressive mechanism at developmentally regulated genes, H3K27me3 did not prevent transcription from the dehydration stress- responding genes. The high H3K27me3 levels present during transcriptionally inactive states did not interfere with the transition to active transcription and with H3K4me3 accumulation. H3K4me3 and H3K27me3 marks function indepen- dently and are not mutually exclusive at the dehydration stress-responding memory genes.

SCN4A基因突变导致的低钾性周期性麻痹2例报道并文献复习

本文报道2例SCN4A基因突变导致的家族性低钾性周期性麻痹病例的基因及临床特点,全外显子组测序提示2例先证者的SCN4A基因存在杂合性突变,其中1例携带的突变(R672L)尚未见文献及数据库报道。本文进一步对文献中已报道的相关病例进行回顾性分析。

香蕉枯萎病菌内源报告基因Foc4carS的鉴定及其应用

香蕉枯萎病是由尖孢镰刀菌古巴转化型(Fusarium oxysporum f. sp. cubense, Foc)引起的香蕉毁灭性土传病害,其中4号生理小种(Foc4)能感染几乎所有的香蕉品系,危害最严重。carS基因通过调控下游car结构基因参与调控镰刀菌类胡萝卜素的生物合成,本研究克隆鉴定了Foc4carS基因(FOIG_05085),Foc4carS蛋白具有典型的RING-finger蛋白结构域。利用分割标记法(Split-marker PCR)获得Foc4carS基因的融合片段,同时构建含有Foc4carS基因sgRNA591序列的pUC-fFuCas9-HTBNLS-hph-Foc4carS基因编辑载体,通过PEG介导的原生质体转化获得该基因的敲除突变体、回补突变体以及基因编辑敲除体,并对敲除和回补突变体的生物学特性和致病力进行分析。结果显示:ΔFoc4carS突变体的菌落直径、产孢量和致病力等生物学表型与野生菌株Foc4无显著差异,而ΔFoc4carS突变体菌落颜色呈深橙色,Foc4carS基因的缺失影响了次生代谢产物类胡萝卜素的生物合成;基因编辑的ΔFoc4carS(HDR)突变体不论是再生筛选板还是继代后的PDA平板,其菌落均出现典型的深橙色,表明Foc4carS可作为内源报告基因,在香蕉枯萎菌Foc4中进行基因质粒型CRISPR/Cas9编辑可行。

香蕉枯萎和细菌性软腐病菌的多重PCR检测

香蕉枯萎病菌Fusarium oxysporum f.sp.cubense和细菌性软腐病菌Dickeya zeae的复合侵染为害给香蕉产业发展带来严重挑战,有必要建立相关病害的多重聚合酶链式反应(multiplex polymerase chain reaction, multiplex PCR)检测技术。本文基于尖孢镰刀菌古巴专化型1号生理小种(F.oxysporum f.sp.cubense race 1,FOC1)基因组contig 438区间(35 631-37 693 bp)(GenBank:AMGP01000438.1)和4号生理小种(F.oxysporum f.sp.cubense race 4,FOC4)基因组contig 195区间(4 028-6 126 bp)(GenBank:AMGQ01000195.1)存在160 bp插入序列差异设计特异扩增引物FOC-F/-R,同时以香蕉细菌性软腐病菌D.zeae的促旋酶B亚单位基因(the subunit B of gyrase gene)(GenBank:JQ284039)序列设计特异扩增引物gyrB-F/-R。多重PCR检测结果显示:本技术可在一次PCR扩增反应内同时检测香蕉枯萎病菌1号、4号生理小种和细菌性软腐病菌;多重PCR的灵敏度结果表明:检测香蕉枯萎病菌的DNA浓度最低限为0.1 ng/μL,细菌性软腐病菌的灵敏度为103cfu/mL;检测结果稳定可靠。因此,本研究建立的多重PCR检测方法可有效应用于检测香蕉发病组织中的香蕉枯萎病菌和细菌性软腐病菌,也可用于香蕉种苗和田间土壤带病菌的监测,为香蕉种植保驾护航。

常染色体显性遗传性微管聚集性肌病存在SCN4A基因突变(附1家系报告)

目的探讨显性遗传性微管聚集性肌病的临床、骨骼肌病理和基因改变特点。方法对显性遗传性微管聚集性肌病1家系进行分析研究,先证者为17岁男性,1岁开始出现发作性面肌、咀嚼肌和四肢肌无力,以及寒冷或活动诱发的肌肉僵硬,发作间期持续性肢体乏力。家族中连续4代10例出现类似临床表现。对先证者及其母亲进行肌肉活检。对母子及家族中无症状者行SCN4A基因序列检测。结果在先证者和其母亲的Ⅱ型肌纤维肌纤维内均发现异常沉积物,分别占所有肌纤维的10%和3%。免疫组织化学染色见病变肌纤维内tau、dysferlin和泛素阳性表达。电镜检查显示沉积物为大量聚集的微管结构。基因测序发现母子两人的SCN4A基因第13号外显子存在T704M突变。结论病理检查证实显性遗传性微管聚集性肌病,该病和SCN4A基因突变有关,出现副肌强直性周期性瘫痪的临床表型。

LIPI-3和LIPI-4共存对致病性单核细胞增生李斯特菌毒力的影响

【目的】探究单核细胞增生李斯特菌(Listeria monocytogenes,LM)毒力岛3(LIPI-3)和LIPI-4共存对LM毒力的影响,揭示它们对细菌毒力的潜在调节作用,为进一步研究LM的致病机制提供依据。【方法】以LM928(存在LIPI-4)和LM873(同时存在LIPI-3和LIPI-4)2个LM菌株为研究对象,通过侵染HCMEC/D3细胞监测2种菌株对HCMEC/D3细胞的黏附和侵袭情况;通过鸡胚感染试验评估2株菌对鸡胚半数致死量(LD 50)的影响;采用溶血试验来测定菌株的溶血活性;通过流式细胞仪分析HCMEC/D3细胞凋亡情况,以评估菌株诱导细胞凋亡的能力;利用实时荧光定量PCR技术比较2株菌在BHI培养条件下及感染HCMEC/D3细胞后毒力基因的转录水平差异。【结果】LM928和LM873株的黏附率无显著差异(P>0.05),LM928株的侵袭率极显著高于LM873株(P<0.01)。LM873株的LD 50是LM928株的约1000倍;LM928和LM873株的溶血价分别为24和23。LM928株感染24和48 h后诱导的细胞凋亡率显著或极显著高于LM873株(P<0.05;P<0.01),而LM873株的凋亡率与对照组间无显著差异(P>0.05)。LM928和LM873株在BHI培养基中培养时,与LM928株相比,LM873株毒力基因mpl、inlB、inlC、inlP、actA、plcA、plcB和sigB的转录水平均显著或极显著上调(P<0.05;P<0.01),毒力基因hly、inlA和iap的转录水平均极显著或显著下调(P<0.01;P<0.05)。LM928和LM873株侵染HCMEC/D3细胞后,与LM928株相比,LM873株毒力基因inlP、actA、plcA、plcB和prfA的转录水平均显著或极显著上调(P<0.05;P<0.01);毒力基因hly、mpl、inlA、inlB、inlC和iap的转录水平均极显著下调(P<0.01)。【结论】LIPI-3和LIPI-4共存降低了LM的毒力,该结果对进一步研究LIPI-3和LIPI-4在细菌毒力调控中的分子机制和复杂的相互作用具有重要意义。

Relationship between alterations of p16^( INK4a) and p14^(ARF) genes of CDKN2A locus and gastric carcinogenesis

Objective To investigate the relationship between alterations of p16 INK4a and p14 ARF genes and gastric carcinogenesis Methods The tumors and neighboring gastric tissues from 48 patients with gastric cancer were studied The homozygous deletion, mutation, methylation of the CpG islands, and mRNA expression of p16 INK4a and p14 ARF genes were assessed by PCR, PCR SSCP, PCR based methylation assay, and RT PCR Results ① The homozygous deletion rate of p16 INK4a and p14 ARF was 35 4% (17/48), and no homozygous deletion was examined in any gastric tissue neighboring the tumor ② There was no point mutation of p16 INK4a and p14 ARF in 31 gastric cancers without homozygous deletion or in the matched gastric tissues adjacent to the tumor ③ Methylation of the CpG islands of p16 INK4a and p14 ARF was detected in 47 9% (23/48) of gastric cancers, while methylation was observed only in 2 of 48 gastric tissues neighboring the cancer with a significant difference ( P <0 01) ④ The loss rate of p16 INK4a mRNA was 47 9% (23/48) in gastric cancer, and the patients of the combined methylation of exons 1α and 2 had a higher loss rate (100%, 6/6) of p16 INK4a mRNA than those of the methylation of the other exons (11 8%, 2/17, P <0 01); the loss rate of p14 ARF mRNA was 45 8%(22/48) in gastric cancer, and patients with the combined methylation of exons 1β and 2 had a higher loss rate (100%, 3/3) of p14 ARF mRNA than those of the methylation of the other exons (15%, 3/20, P <0 05) ⑤ The combined loss of p16 INK4a and p14 ARF mRNAs was examined in 1 (5 6%) of 18 patients of well and moderately differentiated carcinomas, and 11 (36 7%) of 30 patients of poorly and not differentiated carcinomas with a significant difference ( P <0 05) Conclusion p16 INK4a and p14 ARF genes are frequently inactivated by homozygous deletion and methylation of the 5'CpG islands in gastric cancer, which may play an important role in the carcinogenesis of gastric cancer