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.

A large-scale functional approach to uncover human genes and pathways in Drosophila

We demonstrate the feasibility of performing a systematic screen for human gene functions in Drosophila by assaying for their ability to induce overexpression phenotypes. Over 1 500 transgenic fly lines corresponding to 236 human genes have been established. In all, 51 lines are capable of eliciting a phenotype suggesting that the human genes are functional. These heterologous genes are functionally relevant as we have found a similar mutant phenotype caused either by a dominant negative mutant form of the human ribosomal protein L8 gene or by RNAi downregulation of the Drosophila RPL8. Significantly, the Drosophila RPL8 mutant can be rescued by wild-type human RPL8. We also provide genetic evidence that Drosophila RPL8 is a new member of the insulin signaling pathway. In summary, the functions of many human genes appear to be highly conserved, and the ability to identify them in Drosophila represents a powerful genetic tool for large-scale analysis of human transcripts in vivo.

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.

Tumor suppress genes screening analysis on 4q in sporadic colorectal carcinoma

AIM: To search candidate tumor suppressor genes (TSGs) on chromosome 4q through detecting high loss of heterozygosity (LOH) regions in sporadic colorectal carcinoma in Chinese patients. METHODS: Thirteen fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by polymerase chain reaction (PCR). PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. Comparison between LOH frequency and clinicopathological factors were performed by χ2 test. RESULTS: Data were collected on all informative loci. The average LOH frequency on 4q was 28.56%. The D4S2915 locus showed highest LOH frequency (36.17%). Two obvious deletion regions were detected: one between D4S3000 and D4S2915 locus (4q12-21.1), another flanked by D4S407 and D4S2939 locus (4q25-31.1). None case showed complete deletion of 4q, most cases displayed interstitial deletion pattern solely. Furthermore, compared with clinicopathological features, a significant relationship was observed between LOH frequencies on D4S3018locus. In tumors larger than 5 cm in diameter, LOH frequency was significantly higher than tumors that were less than 5 cm (56% vs 13.79%, P = 0.01). On D4S1534 locus, LOH was significantly associated with liver metastasis (80% vs 17.25%, P = 0.012). No relationship was detected on other locus compared with clinicopathologial features. CONCLUSION: By high resolution deletion mapping, two high frequency regions of LOH (4q12-21.1 and 4q25-31.1) were detected, which may contribute to locate TSGs on chromosome 4q involved in carcinogenesis and progression of sporadic colorectal carcinoma.

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.

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.

Are there tumor suppressor genes on chromosome 4p in sporadic colorectal carcinoma?

AIM: To study the candidate tumor suppressor genes (TSG) on chromosome 4p by detecting the high frequency of loss of heterozygosity (LOH) in sporadic colorectal carcinoma in Chinese patients.METHODS: Seven fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. The same procedure was performed by the other six microsatellite markers spanning D4S3013 locus to make further detailed deletion mapping. Comparison between LOH frequency and clinicopathological factors was performed by χ2 test.RESULTS: Data were collected from all informative loci. The average LOH frequency on 4p was 24.25%, and 42.3% and 35.62% on D4S405 and D4S3013 locus, respectively. Adjacent markers of D4S3013 displayed a low LOH frequency (< 30%) by detailed deletion mapping. Significant opposite difference was observed between LOH frequency and tumor diameter on D4S412 and D4S1546 locus (0% vs 16.67%, P = 0.041; 54.55% vs 11.11%, P = 0.034, respectively). On D4S403 locus, LOH was significantly associated with tumor gross pattern (11.11%, 0, 33.33%, P = 0.030). No relationship was detected on other loci compared with clinicopathologial features.CONCLUSION: By deletion mapping, two obvious high frequency LOH regions spanning D4S3013 (4p15.2) and D4S405 (4p14) locus are detected. Candidate TSG, which is involved in carcinogenesis and progression of sporadic colorectal carcinoma on chromosome 4p, may be located between D4S3017 and D4S2933 (about 1.7 cm).

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.

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.

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.

血清4型禽腺病毒的分离鉴定及致病性分析

旨在从送检的病鸡中分离鉴定出禽腺病毒(fowl adenovirus,FAdV),为临床诊断和疫病防控提供参考依据。本研究将阳性样品接种SPF鸡胚并传代,利用PCR检测、基因序列分析、动物回归试验等方法鉴定病毒。结果:经过鸡胚传代及病毒特异性检测获得了纯净的FAdV,命名为HB2306;分析Hexon基因序列可知,HB2306株属于血清4型分支,与国内外分离的FAdV-4毒株核苷酸序列同源性为98.2%~99.9%,HB2306株与国内外的4型高致病性毒株氨基酸序列相似性在97.9%~99.8%;受试动物临床病理变化显示,HB2306株以104.5EID50/只的剂量经胸部肌肉注射后,引起宿主心包积液,肝脏变黄肿胀、淤血出血,肾出血、肿胀等典型的病理变化,与流行的高致病性毒株所致临床症状相似,且死亡率为100%。综上,本研究成功分离并鉴定出一株FAdV-4高致病力毒株,丰富了毒种库,为该病毒感染的流行情况调查和综合防控奠定了基础。

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。

冠脉支架内再狭窄患者TLR4基因rs4986790、rs4986791位点多态性及其与临床关系的研究

目的 探讨冠脉支架内再狭窄(ISR)患者Toll样受体4(TLR4)外显子基因突变情况及其与临床的关系。方法 选取2019年1月至2022年12月在右江民族医学院附属医院治疗的ISR患者137例,检测体重指数(BMI)、血压、血脂、血糖、血尿酸、C反应蛋白(CRP)、β2微球蛋白(β2MG)、白细胞介素6(IL-6)和TLR4基因rs4986790、rs4986791位点碱基。并与131例同期行冠脉支架植入术支架内无再狭窄(NISR)患者比较。结果 ISR组患者BMI、收缩压、总胆固醇、甘油三酯、空腹血糖、血尿酸、CRP、β2MG和IL-6水平高于NISR组(P <0.05);ISR组TLR4基因rs4986790位点碱基突变率高于NISR组(P <0.05),TLR4基因rs4986791位点碱基突变率高于NISR组(P <0.05);TLR4基因突变表型组BMI、收缩压、总胆固醇、甘油三酯、空腹血糖、血尿酸、CRP、β2MG和IL-6水平高于TLR4野生表型组(P <0.05)。结论 冠脉支架内再狭窄患者TLR4基因外显子突变率高,TLR4基因外显子突变的患者其代谢和炎症因子异常情况有叠加作用。