Transcriptional regulation of secondary metabolism and autophagy genes in response to DNA replication stress in Setosphaeria turcica

The fungal pathogen Setosphaeria turcica causes northern corn leaf blight(NCLB),which leads to considerable crop losses.Setosphaeria turcica elaborates a specialized infection structures called appressorium for maize infection.Previously,we demonstrated that the S.turcica triggers an S-phase checkpoint and ATR(Ataxia Telangiectasia and Rad3 related)-dependent self-protective response to DNA genotoxic insults during maize infection.However,how the regulatory mechanism works was still largely unknown.Here,we report a genome wide transcriptional profile analysis during appressorium formation in the present of DNA replication stress.We performed RNA-Seq analysis to identify S.tuicica genes responsive to DNA replication stress.In the current work,we found that appressorium-mediated maize infection by S.turcica is significantly blocked by S-phase checkpoint.A large serial of secondary metabolite and melanin biosynthesis genes were blocked in appressorium formation of S.turcica during the replication stress.The secondary metabolite biosynthesis genes including alcohol dehydrogenase GroES-like domain,multicopper oxidase,ABCtransporter families,cytochrome P450 and FAD-containing monooxygenase were related to plant pathogen infection.In addition,we demonstrated that autophagy in S.turcica is up-regulated by ATR as a defense response to stress.We identified StATG3,StATG4,StATG5,StATG7 and StATG16 genes for autophagy were induced by ATR-mediated S-phase checkpoint.We therefore propose that in response to genotoxic stress,S.turcica utilizes ATR-dependent pathway to turn off transcription of genes governing appressorium-mediated infection,and meanwhile inducing transcription of autophagy genes likely as a mechanism of self-protection,aside from the more conservative responses in eukaryotes.

MAP kinase gene STK1 is required for hyphal, conidial, and appressorial development, toxin biosynthesis, pathogenicity, and hypertonic stress response in the plant pathogenic fungus Setosphaeria turcica

The mitogen-activated protein kinase （MAPK）, a key signal transduction component in the MAPK cascade pathway, regulates a variety of physiological activities in eukaryotes. However, little is known of the role MAPK plays in phytopathogenic fungi. In this research, we cloned the MAPK gene STK1 from the northern corn leaf blight pathogen Setosphaeria turcica and found that the gene shared high homology with the high osmolality glycerol （HOG） MAPK gene HOG1 of Saccharomy- ces cerevisiae. In addition, gene knockout technology was employed to investigate the function of STKI. Gene knockout mutants （KOs） were found to have altered hyphae morphology and no conidiogenesis, though they did show similar radial growth rate compared to the wild-type strain （WT）. Furthermore, microscope observations indicated that STK1 KOs did not form normal appressoria at 48 h post-inoculation on a hydrophobic surface. STK1 KOs had reduced virulence, a significantly altered Helminthosporium turcicum （HT）-toxin composition, and diminished pathogenicity on the leaves of susceptible inbred corn OH43. Mycelium morphology appeared to be significantly swollen and the radial growth rates of STK1 KOs declined in comparison with WT under high osmotic stress. These results suggested that STK1 affects the hyphae development, conidiogenesis, and pathogenicity of S. turcica by regulating appressorium development and HT-toxin biosynthesis. Moreover, the gene appears to be involved in the hypertonic stress response in S. turcica.

Effects of MEK-Specific Inhibitor U0126 on the Conidial Germination,Appressorium Production,and Pathogenicity of Setosphaeria turcica

Systemic studies on the effects of mitogen-activated protein kinase （MAPK） signal transduction pathway on the growth and development of Setosphaeria turcica is helpful not only in understanding the molecular mechanism of pathogenhost interaction but also in the effective control of the diseases caused by S. turcica. U0126, the specific MEK inhibitor, is used to treat S. turcica before the observation of the conidial germination, appressorium production, and pathogenicity of the pathogen. There is no significant effect of U0126 on the colony morphology and mycelium growth of the pathogen. After treatment with U0126, the growth of mycelium and conidia are normal, but the conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are significantly inhibited. Under the definite concentration scope, an increase in U0126 concentration increases the inhibition degree of conidial germination and appressorium production, but the inhibition degree decreases with elongation of treatment time. The conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are regulated by the MAPK pathway inhibited by U0126.

Stk2,a Mitogen-Activated Protein Kinase from Setosphaeria turcica,Specifically Complements the Functions of the Fus3 and Kss1 of Saccharomyces cerevisiae in Filamentation,Invasive Growth,and Mating Behavior

Setosphaeria turcica,an essential phytopathogenic fungus,is the primary cause of serious yield losses in corn; however,its pathogenic mechanism is poorly understood.We cloned STK2,a newly discovered mitogen-activated protein kinase gene with a deduced amino acid sequence that is 96% identical to MAK2 from Phaeosphaeria nodorum,56% identical to KSS1 and 57% identical to FUS3 from Saccharomyces cerevisiae.To deduce Stk2 function in S.turcica and to identify the genetic relationship between STK2 and KSS1/FUS3 from S.cerevisiae,a restructured vector containing the open reading frame of STK2 was transformed into a fus3/kss1 double deletion mutant of S.cerevisiae.The results show that the STK2 complementary strain clearly formed pseudohyphae and ascospores,and the strain grew on the surface of the medium after rinsing with sterile water and the characteristics of the complementary strain was the same as the wild-type strain.Moreover,STK2 complemented the function of KSS1 in filamentation and invasive growth,as well as the mating behavior of FUS3 in S.cerevisiae,however,its exact functions in S.turcica will be studied in the future research.

Ocular Symptoms of Tumors at Sella Turcica Region

Background: The sella tumors were found in 15% of intracranial tumors. The sella turcica region locates in the base of skull, so the carcinoma patients in early stage often showed no intracranial hypertension and other positive signs of neural system. However, the patients presented visual field defect and hypopsia, and often went to see the doctor of ophthalmology, which resulted from that the tumor often compressed optic nerve and chiasm. We analysed the symptomatic changes of the visual organs in order to provide a diagnositic basis for sellar tumor in oph-thalmological department.Clinical materials: Of the 407 cases of sellar tumors, 296 were pituitary adenoma, 52 were craniopharyngioma, 43 were meningioma, and the rest 16 were other kinds of sellar tumors. We analysed their ocular changes of the sellar tumors and the characteristics of the tumors.Results: The sellar tumor in its early stage is mainly symptomized as visual field changes. Visual field defect was found in 92. 9% of pituitary adenoma,

Purification and Structural Analysis of a Selective Toxin Fraction Produced by the Plant Pathogen Setosphaeria turcica

Thirteen fractions from the pathogenic plant fungus Setosphaeria turcica race 1 were separated and collected using high performance liquid chromatography （HPLC）. Their toxic activities were assayed through leaf puncturing on corn differentials （OH43, OH43Ht1, OH43Ht2, and OH43HtN）, and the results revealed that eight fractions were toxic and fraction 6 was specifically toxic to OH43Ht1, which could be taken as a gene-selective toxin fraction. Fraction 6 was finely purified via HPLC and condensed by freeze desiccation. Its chemical structure was analyzed with EI-MS, IR, HMBC, ^1H-NMR, and two-dimensional NMR. The results suggested that fraction 6 contained an unsaturated double bond, carbonyl and methylene groups with molecular weight of 142.

Proteomics Identification of Differentially Expressed Leaf Proteins in Response to Setosphaeria turcica Infection in Resistant Maize

Northern corn leaf blight （NCLB）, caused by the heterothallic ascomycete fungus Setosphaeria turcica, is a destructive foliar disease of maize and represents a serious threat to maize production worldwide. A comparative proteomic study was conducted to explore the molecular mechanisms underlying the defense responses of the maize resistant line A619 Ht2 to S. turcica race 13. Leaf proteins were extracted from mock and S. turcica-infected leaves after inoculated for 72 h and analyzed for differentially expressed proteins using two-dimensional electrophoresis and mass spectrometry identification. 137 proteins showed reproducible differences in abundance by more than 2-fold at least, including 50 up-regulated proteins and 87 down-regulated proteins. 48 protein spots were successfully identified by MS analysis, which included 10 unique, 6 up-regulated, 20 down-regulated and 12 disappeared protein spots. These identified proteins were classified into 9 functional groups and involved in multiple functions, particularly in energy metabolism （46%）, protein destination and storage （12%）, and disease defense （18%）. Some defense-related proteins were upregulated such as 13-glucosidase, SOD, polyamines oxidase, HSC 70 and PPIases; while the expressions of photosynthesis- and metabolism-related proteins were down-regulated, by inoculation with S. turcica. The results indicated that a complex regulatory network was functioned in interaction between the resistant line A619 Ht2 and S. turcica. The resistance processes of A619 Ht2 mainly resided on directly releasing defense proteins, modulation of primary metabolism, affecting photosyntesis and carbohydrate metabolism.

Bioinformatic analysis and functional characterization of CFEM proteins in Setosphaeria turcica

Common in Fungal Extracellular Membrane(CFEM)domains are uniquely found in fungal extracellular membrane proteins which are important for pathogens.This study identified 13 StCFEM proteins in the genome of Setosphaeria turcica,the hemibiotrophic fungus that causes northern corn leaf blight.Sequence alignment and WebLogo analysis of their CFEM domains indicated that the amino acids were highly conserved and that,with the exception of StCFEM1,2,3,and 6,they contained eight cysteines.Phylogenic analysis suggested that these 13 proteins(StCFEM1–13)could be divided into 2 clades based on the presence of the trans-membrane domain.Six StCFEM proteins with a signal peptide and without a trans-membrane domain were considered as candidate effector proteins.The CFEM domain in the candidate effector proteins could form a helical-basket structure homologous to Csa2 in Candida albicans.Transcriptome analysis suggested that the 13 genes were expressed during fungal infection and a yeast secretion assay revealed that these candidate effectors were secreted proteins.It was also found that StCFEM3,4,and 5 couldn't affect BAX/INF1-induced programmed cell death(PCD)in Nicotiana benthamiana and while StCFEM12 could suppress INF1-induced PCD,it showed no effect on BAXinduced PCD.This study found that there were 13 members of the S.turcica CFEM protein family and that StCFEM12 was a candidate effector.This study laid the foundation for illustrating the roles of CFEM proteins during the pathogenic processes of phytopathogens.

Characterization of laccase gene StLAC6 and its involvement in the pathogenicity and peroxisome function in Setosphaeria turcica

Laccases, as a kind of multicopper oxidase, play an important role in pigment synthesis and growth in fungi and are involved in their interactions with host plants. In Setosphaeria turcica, 9 laccase-like multicopper oxidases have been identified, and StLAC2 is involved in the synthesis of the melanin that accumulates in the cell wall. The function of another major laccase gene, StLAC6, was studied here. The knockout of StLAC6 had no effect on the growth, morphology or invasion ability of S. turcica, but the morphology and function of peroxisomes of knockout mutants were abnormal. The knockout of the StLAC6 gene resulted in increased contents of phenolic compounds and melanin and the sensitivity to fungicides increased compared with wild type strains. In the mutants of StLAC6, there is a significant change of the expression levels of other laccase genes. This study provides a new insight into laccase functions and the relationship of the laccase gene family in plant pathogenic fungi.

Antimicrobial activity against periodontopathogenic bacteria,antioxidant and cytotoxic effects of various extracts from endemic Thermopsis turcica

Objective:To investigate the in vitro antimicrobial potential of Thermopsis turcica Kit Tan,Vural&Kckdk against periodoutopathogenie bacteria,its antioxidant activity and cytotoxic effect on various cancer cell lines.Methods:In vitro antimicrobial activities of elhanol.methanol,ethyl acetate(ElAc,n-hexane and water extracts of Thermopsis turcica herb against periodontopathogenic bacteria,Aggregatibacter actinnmycelemconilans ATCC 29523 and Porphyromonas gingivalis ATCC 33277 were tested by agar well diffusion,minimum inhibitory concentration(MIC)and minimal bactericidal concentration(MBC),Antioxidant properties of the extracts were evaluatod by 1,1-diphenyl-2-picryl-hydrazyl radical scavenging activity and p-carotene bleaching methods.Amounts of phenolic contents of the extracts were also analysed by using the Folin-Ciocalteu reagent.Additionally,cytotoxic activity of the extracts on androgcn-insensilivc prostate cancer,androgen—sensitive prostate cancer,chronic myelogenous leukemia and acute promyelocytic leukemia bunian cancer cell lines were determined by 3-4,5-dimelhylthiazol-2-yh-2,5-diphenyltclrazolium bromide assay.Human gingival fibroblast cells were used as a control.Results:Our data showed that ELAc extract had the highest antimicrobial effect on Aggregatibacter actinomycetemitans(MIC:1.562 mg/ml_MHC:3.124 mg/ml.)and Porph yromonas gingiralis(MIC:0.781 mg/mL,MBC:1.562 mg/mL).In antioxidant assays.ElAc extract exhibited also the highest radical scavenging activity[IC_(50)=(30.0±0.3)μg/ml.]and the highest inhibition[(74.35±0.30)%]|against lineloic acide oxidation.The amount of phenolic content of it was also the highest[(l62.5±l.2)μg/mg gallic acid].In cytotoxic assay,only etbanol[IC_(50)=(80.00±1.21)μg/ml.]and EtAc extract[IC_(50)=(70.0±0.9)μg/ml]were toxic on acute promyeloeytic leukemia cells at 20—100μg/mL.P<0.05>.However,no toxic effect was observed on human gingival fibroblast cells.Cunclusions:According to our findings,owing to its antioxidant and cytotoxic potential,EtAc exlrael might include anticancer agents for acute promyelocytic leukemia.

Melanin,DNA replication,and autophagy affect appressorium development in Setosphaeria turcica by regulating glycerol accumulation and metabolism

Setosphaeria turcica(syn.Exserohilum turcicum)is the pathogenic fungus of maize(Zea mays)that causes northern leaf blight,which is a major maize disease worldwide.Melanized appressoria are highly specialized infection structures formed by germinated conidia of S.turcica that infect maize leaves.The appressorium penetrates the plant cuticle by generating turgor,and glycerol is known to be the main source of the turgor.Here,the infection position penetrated by the appressorium on maize leaves was investigated,most of the germinated conidia entered the leaf interior by directly penetrating the epidermal cells,and the appressorium structure was necessary for the infection,whether it occurred through epidermal cells or stomata.Then,to investigate the effects of key factors in the development of the appressorium,we studied the effects of three inhibitors,including a melanin inhibitor(tricyclazole,TCZ),a DNA replication inhibitor(hydroxyurea,HU),and an autophagy inhibitor(3-methyladenine,3-MA),on appressorium turgor and glycerol content.As results,appressorium turgor pressure and glycerol concentration in the appressorium reached their highest levels at the mature stage of the appressorium under the control and inhibitor treatments.The three inhibitors had the greatest effects on appressorium turgor pressure at this stage.Glycogen and liposomes are the main substances producing glycerol.It was also found inhibitors affected the distribution of glycogen and liposomes,which were detected in the conidia,the germ tube,and the appressorium during appressorium development.This study provides profound insight into the relationship between appressorium turgor pressure and glycerol content,which was affected by the synthesis of melanin,DNA replication,and autophagy in the developing appressorium during a S.turcica infection.

Changes of SOD Activity in Corn Leaves Infected by Setosphaeria turcica with Different Pathogenicity

[ Objective] The paper was to explore the relationship between SOD activity and resistance of corn against Setosphaeria turcica. [ Method] Strain YC and 01-23T of S. turcica with different pathogenicity were inoculated into corn leaves by fungal disc inoculation method, and the dynamic changes of SOD activity in infected corn leaves was measured. [Result] After inoculation of higly virulent strain YC and weakly virulent strain 01-23T of S. turcica, SOD activity in corn leaves within 1 -5 d showed obvious stages, which gradually decreased within 1 -3 d, then gradually increased within 3 -4 d, and decreased again within 4 -5 d. The SOD activity of corn leaves in inoculation treatment was higher than that in the treatment without inoculation within 1 - 4 d, which significantly decreased at fifth day. The pathogen with different pathogenicity would lead to different changes in SOD activity of corn leaves. SOD activity of corn leaves infected by weakly virulent strain 01-23T for the first day was extremely higher than that infected by strong virulent strain YC, but had no significant difference with highly virulent strain YC at 2 -3 d. [ Conclusion] SOD activity played an important role in initial resistant ability of corn against pathogen infection, and had no significant effect on resistance against pathogen extension after incidence.

Isolation and Expression Analysis of Meristem Gene CLV2 and Carpel Generation Gene (CRC) from Multi-Carpel Structure of Thermopsis turcica

Thermopsis turcica is an endemic species of wild legume in Türkiye. The plant produces flower pistils with unusual morphological properties. The aim of this study was to determine the factors controlling the interesting feature of this plant. Particularly the controlling features of the flower meristem and the pistils that mutations in two genes and helps occurring the number of flower organs and can cause abnormalities, This study has attempted to explore the controlling feature of Thermopsis turcica, the young bud Arabidopsis CLV2, and CRC gene homologs were isolated and analyzed in different tissues. First, with total RNA isolated from the first thread degenerated primers synthesized cDNA expressions, second with a partial length cDNA cloning process made and the replicated set of analyses. The full-length cDNA of genes related to RACE analysis was carried out with the sequencing process. The real-time PCR method was applied for gene functional analyses with buds, sepals, petals and stamen and relative expression analysis of genes in the tissue of pistils. Full-length cDNA sequence for the gene CLV2 2557 bp, the encoded protein 716 amino acids, CRC 901 for full-length cDNA sequence of the gene—bp, the encoded protein 174 identified as amino acids. Alignment and phylogenetic analyses of CLV2 and CRC genes were found to be similar to other types of legumes in homologous sets. Real-time (RT-qPCR) PCR results relative to study courses with genes.

Empty Sella Turcica Syndrome: About a Case Revealed by Adrenal Insufficiency

Background: Empty sella turcica corresponds to an arachnocele which may be complete or partial, primary or secondary. The primary form is rarer. Its cause is unknown, but etiological factors have been identified. The prevalence of this condition is variable, depending on techniques of diagnosis. It may be detected fortuitously on imaging or in the presence of neurological, ocular or endocrine manifestations. Aim: We aim to report a case of empty sella turcica syndrome revealed by adrenal insufficiency in a young African black woman. Case presentation: It was an overweight young woman with signs of intracranial hypertension associated with central adrenal insufficiency. The diagnosis of empty sella was confirmed by magnetic resonance imaging. Secondarily, she developed a hypo metabolism syndrome and biology confirmed central hypothyroidism. After correction of the adrenal cortical deficit, the treatment of the thyroid deficit was administered. Evolution was favourable clinically and biologically. Conclusion: The empty sella turcica is rare and its revelation by a central adrenal insufficiency is even more. The case we report is a perfect illustration.

玉米ACO基因家族生物信息学及表达模式分析

【目的】对玉米1-氨基环丙烷-1-羧酸氧化酶ACO基因家族进行全基因组鉴定,分析其在玉米不同器官和不同发育时期以及响应外源激素和病菌侵染中的表达模式,为明确玉米ACO基因家族功能打下基础。【方法】利用生物信息学方法,在玉米B73自交系基因组中鉴定ACO,对其基因结构、蛋白质理化性质、家族成员间的亲缘关系以及保守基序进行分析,利用实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)技术分析ZmACO基因家族的表达模式。【结果】除ZmACO11外,ZmACO家族成员均具有Fe2+离子结合位点和底物抗坏血酸结合位点。系统发育分析显示,ZmACO_(2)与ScACO在同一分支,亲缘关系较近,Bootstrap值达98。基因表达分析表明,ZmACO_(2)、5、9、15、20、35在各发育时期均活跃表达,且在叶片中呈优势表达,因此选择上述6个基因进行下一步检测。喷施乙烯利后,上述6个基因的表达均有所波动,其中ZmACO_(2)的表达量受影响较大,变化幅度在8倍左右。在乙烯利处理的0—24 h内这6个基因的表达量存在波动,但在处理后24 h,6个基因的表达量均接近0。水杨酸处理后,ZmACO5的表达量受影响较大,变化倍数在2倍左右。其他基因的表达量在处理后24 h均接近0。ZmACO9、35在3—12 h的表达量存在波动,ZmACO_(2)、15、20表达量呈下调趋势。在响应生物胁迫方面,接种玉米大斑病菌(Setosphaeria turcica)后,ZmACO5、9的表达量变化幅度最大,在接种后第10天,这两个基因的表达量分别升至对照组的50和60倍。接种玉米小斑病菌(Cochlibolus heterostrophus)后,ZmACO5的表达量变化幅度较大,变化倍数在40—90倍。接种立枯丝核菌(Rhizoctonia solani)后,ZmACO5、35表达量变化幅度最大,在病菌接种的第3天达到200倍。【结论】ZmACO_(2)、5、20、35在玉米生长发育过程中表达变化最活跃;施加外源乙烯利和水杨酸可以对ZmACO的表达水平造成显著影响。病菌侵染玉米后ZmACO的表达水平产生显著变化,与生物胁迫应答关系密切。

玉米大斑病菌cDNA文库的构建及转录因子StMR1互作蛋白的筛选

【目的】筛选玉米大斑病菌(Setosphaeria turcica)转录因子的互作蛋白,解析黑色素调控转录因子StMR1调控玉米大斑病菌致病性的分子机制。为解析玉米大斑病菌侵染过程中转录因子的调控网络,阐明病菌的致病机理提供参考。【方法】收集玉米大斑病菌菌丝和孢子不同萌发阶段作为试验材料,采用Gateway方法构建玉米大斑病菌cDNA文库,使用同源重组的方法构建转录因子StMR1的诱饵载体,采用酵母双杂交技术筛选其互作蛋白并进行一对一验证。【结果】构建的玉米大斑病菌文库插入的平均片段长度大于1000 bp,初级文库及次级文库的库容量为1.2×107和1.04×107CFU,重组率为100%,可以用于酵母双杂交筛选。成功构建可以用于筛库的诱饵载体pGBKT7-StMR1,经初筛与复筛得到3个互作蛋白,一对一验证短链脱氢酶、糖基转移酶、富含亮氨酸重复序列蛋白均与转录因子StMR1存在互作。【结论】成功构建了丰富度高且质量好的玉米大斑病菌cDNA文库并筛选到了与转录因子StMR1互作的蛋白。

经蝶窦鞍区病变手术后迟发性低钠血症

术后迟发性低钠血症是经蝶窦手术的常见内分泌相关并发症,是最常见的再入院原因,通常发生于术后5~7 d。轻度和中度患者可无症状,也可表现为恶心、呕吐、头痛等;重度患者可出现严重神经精神症状、脑水肿甚至死亡。术后迟发性低钠血症危险因素众多且复杂,目前尚缺乏预防和监测标准。本文从经蝶窦鞍区病变手术后迟发性低钠血症的发生机制、人群特点、监测与诊断、预防与处理等方面进行综述,为临床实践提供依据。

玉米大斑病菌效应因子StSse19基因的克隆、表达模式分析及在原核系统中的表达

玉米大斑病是由玉米大斑病菌引起的玉米叶部真菌性病害。本课题组前期通过对病菌侵染玉米的转录组分析发现,效应因子StSse19为重要的致病因子,但对其结构及功能尚不清晰。本研究以野生型菌株01-23的cDNA为模版,克隆StSse19并对其结构进行生物信息学预测;结合对病菌侵染玉米的RNA-Seq数据分析明确该基因的表达模式,进而利用qRT-PCR技术证实其表达模式;构建StSse19的融合表达载体pET28a-StSse19,在大肠杆菌BL21(DE3)中以IPTG诱导表达该目的基因,通过SDS-PAGE技术检测其表达情况。结果表明,StSse19的CDS序列由330个核苷酸组成,编码109个氨基酸残基,序列比对发现该蛋白无同源蛋白,表明其为大斑病菌的特异效应蛋白;对StSse19的表达模式分析发现,该基因在侵染玉米24 h的表达量显著提高,72 h表达量下降,推测该基因参与病菌的侵染过程;将StSse19在原核系统中进行表达,SDS-PAGE分析结果显示,该蛋白大小为28 kD,用Ni柱层析法结合Western blot检测显示获得了纯化的目的蛋白。该研究明确了StSse19基因的结构特征及表达模式、获得了StSse19在原核体系中的表达蛋白,为深入揭示其功能及作用机制奠定了基础。

基于CBCT观察上颌阻生尖牙患者蝶鞍形态变异情况

目的 利用锥形束CT(cone-beam computed tomography,CBCT)探讨上颌尖牙阻生与蝶鞍形态变异的相关性,从三维方向上评价蝶鞍变异对上颌尖牙阻生的临床预判。方法 收集125例上颌阻生尖牙患者(研究组)和125例与之相匹配的尖牙正常萌出者(对照组)。将所有样本CBCT数据导入Dolphin软件,进行重建头颅侧位片和三维图像,记录观察各组蝶鞍桥接(Ⅰ、Ⅱ、Ⅲ型)的类型分布和发生情况。使用SPSS 25.0软件对各组结果进行统计学分析。结果 研究组蝶鞍桥接的发生率显著高于对照组(P=0.004),PIC(腭侧尖牙阻生)组蝶鞍桥接的发生率显著高于对照组(P=0.007),Ⅱ型和Ⅲ型蝶鞍桥接分布和对照组存在统计学差异(P=0.012)。而BIC(唇侧尖牙阻生)组和对照组蝶鞍桥接的发生情况无统计学差异。三维重建图像上各组间蝶鞍桥接发生率无统计学差异(P>0.05)。结论 上颌尖牙阻生与蝶鞍桥接的发生情况有相关性,但仅上颌尖牙腭侧阻生患者更易发生蝶鞍桥接,而上颌尖牙唇侧阻生患者蝶鞍桥接的发生情况无变化。

玉米大斑病菌细胞周期蛋白依赖性激酶结构亚基StCks1鉴定及其基因功能分析

【目的】Cks1(cyclin-dependent kinase subunit 1)是细胞周期蛋白依赖性激酶复合体CDK(cyclin-dependent kinase)的结构亚基,是细胞周期调控过程中的关键基因。论文旨在鉴定玉米大斑病菌(Setosphaeria turcica)StCks1,分析玉米大斑病菌附着胞发育过程中StCks1表达量差异及其互作蛋白,为进一步研究StCks1在附着胞发育中的作用打下基础。【方法】通过对玉米大斑病菌野生型菌株01-23全基因组数据分析,获取StCks1蛋白序列;利用软件MEGA 5.0和在线数据库Pfam、SMART对酿酒酵母、裂殖酵母和拟南芥等物种Cks1蛋白进行系统发育树构建和保守结构域分析;收集玉米大斑病菌附着胞发育不同时期样品进行转录组测序以及表达量分析。利用原核诱导表达系统,构建原核表达载体pGEX-6p-3-StCks1,并转化大肠杆菌表达菌株BL21,对重组蛋白GST-StCks1诱导表达纯化;通过GST pull-down、Western blot技术和酵母双杂交试验,鉴定StCks1的互作蛋白。【结果】玉米大斑病菌全基因组中鉴定到唯一StCks1蛋白编码基因,该蛋白由130个氨基酸组成;其三级结构主要由4股β折叠和3个α螺旋构成,含有HxPEPH(His-anyPro-Glu-Pro-His)保守位点和Cks保守结构域;通过转录组测序和表达量分析发现,StCks1在附着胞发育不同时期表达量显著上调;重组蛋白GST-StCks1在1 mmol·L^(-1) IPTG,30℃诱导2 h可获得大量可溶性蛋白;通过GST pull-down技术获取大量互作蛋白并进行质谱鉴定,通过Western blot和酵母双杂交试验,验证StCks1与细胞周期蛋白依赖性激酶CDK1存在互作。【结论】玉米大斑病菌中含有唯一的StCks1,通过与细胞周期蛋白依赖性激酶CDK1互作调控附着胞的发育。