Toxicities of Phenoloxidase inhibitors against Pieris rapae L. and their toxicology

昆虫多酚氧化酶（EC.1.14.18.1，Phenoloxidase，简称PPO），又称酚氧化酶，酪氨酸酶，是昆虫体内酪氨酸代谢的关键酶，与黑色素的形成、昆虫蛹化、蜕皮时的鞣化、表皮硬化等直接相关，通过抑制害虫的多酚氧化酶活力将影响其幼虫的发育。该酶可以作为新型农药的作用靶标，而该酶的抑制剂则可发展成为具有应用前景的新型的病虫害杀虫剂。在本研究中我们以农作物重要鳞翅目害虫菜青虫的多酚氧化酶作为研究对象，检测合成的缩氨基硫脲化合物对其活力的影响，并在此基础上合成多种结构的缩氨基硫脲和3，4-二羟基苯甲酸酯类化合物，进一步验证其对菜青虫多酚氧化酶抑制作用及杀虫效能。同时克隆菜青虫的多酚氧化酶基因，...Phenoloxidase (PO, EC.1.14.18.1) was a key enzyme of the insect tyrosinase metabolism, which directly related with the melanin formation, insect pupation, tanning and cuticle sclerosis. Inhibition on PO could influent the development of insect larva, therefore, the enzyme could be considered as the target for new type pesticide. In this study, Pieris rapae (Lepidoptera: Pierididae), an important c...学位：博士后院系专业：生命科学学院生物化学与生物技术系_生物化学与分子生物学学号：200817011

Study on the rheology of xanthan and synergistic interaction with konjac gum and other gum

研究前期筛选获得一株多糖胶质高产菌Xanthomonas axonopodis所产的黄原胶FJAT-10151-DTJZ的品质,为该黄原胶的开发应用提供基础数据及参考。通过分析浓度、剪切速率、p H、加热温度、时间、冻融处理等对FJAT-10151-DTJZ粘度的影响研究其流变性,并研究其与结冷胶、黄原胶、凝胶多糖、瓜尔豆胶、刺槐豆胶、魔芋胶、果胶和壳聚糖8种胶的协效性。实验结果表明,FJAT-10151-DTJZ溶液的粘度随浓度的升高而升高,且为非牛顿流体;当FJAT-10151-DTJZ的浓度为1%时,其粘度为343 m Pa·s;p H、冻融对FJAT-10151-DTJZ的粘度影响不大;FJAT-10151-DTJZ的最佳加热温度为75℃;粘度随加热时间先增大后减小,当加热温度为75℃,加热时间为150 min,1%浓度的FJAT-10151-DTJZ溶液的粘度为808 m Pa·s。FJAT-10151-DTJZ只与魔芋胶有强烈的协同增效作用,与壳聚糖、结冷胶、黄原胶、凝胶多糖、瓜尔豆胶、刺槐豆胶、果胶无协效性。A Xanthomonas axonopodis strain producing xanthan gum had been screened,and researched the quality of xanthan gum FJAT-10151-DTJZ in order to provide basic data and reference for development and application. The rheology properties of FJAT-10151-DTJZ were discussed according to the viscosity variation with different conditions including concentration,shearing,p H,heating temperature,heating time and freezingthawing. The synergistic interactions of FJAT-10151-DTJZ with chitosan,gellan gum,xanthan gum,curdlan,guar gum,locust bean gum and pectin were also investigated. The results showed that polysaccharide gum solution FJAT-10151-DTJZ was non-Newton fluid. It's viscosity rised with its concentration and reached to343 m Pa·s when the concentration was 1%. The p H change and freezing-thawing played negligible effects on its viscosity. Its viscosity was 808 m Pa·s when it was heated at 75 ℃ for 150 min. Besides,FJAT-10151-DTJZ had no synergistic interaction with chitosan,gellan gum,xanthan gum,curdlan,guar gum,locust bean gum and pectin except konjac gum.国家948项目(2014-Z48);; 福建省公益类科研院所专项(2015R1018-2);; 福建省农业科学院科技创新项目(2015CX-7

生物藕合技术的原理及其应用

生物藕合是指将两种或多种分子进行键合的过程,此过程形成一个新的联合体,该联合体拥有其所有组分所具有的各特性。生物藕合技术自产生以来其应用不断推广,几乎已经涉及到生命科学领域的各个方面,也极大地推动了生物、化学、医学、农业等学科的发展。本文结合生物藕合技术的原理、特点,探讨了其发展与应用的推广与问题

Cloning, prokaryotic expression and homology modeling analysis of midgut aminopeptidase gene PxAPN5 in Plutella xylostella (Lepidoptera:Plutellidae)

【目的】克隆和表达小菜蛾PluTEllA XylOSTEllA氨肽酶基因,并进行基因序列分析和同源建模分析。【方法】以小菜蛾中肠C dnA为模板克隆分析氨肽酶基因序列,原核表达氨肽酶蛋白并进行酶活性测定,应用配体印迹分析氨肽酶与Cry2Ab蛋白的结合,通过蛋白质建模对突变位点进行分析。【结果】从小菜蛾中肠C dnA扩增出氨肽酶基因,该基因全长2 853 bP,编码950个氨基酸,预测蛋白分子量为107.3871 k dA,等电点为5.24;进化树分析显示,克隆得到的氨肽酶基因属于APn家族5,将其命名为PX APn5(gEn bAnk登录号:kM034756)。PX APn5蛋白具有鳞翅目昆虫氨肽酶蛋白的保守性特征,即含有n-糖基化位点、O-糖基化位点和gPI锚定位点,具有“HEXXH“锌蛋白酶结构域和C端跨膜区域。在大肠杆菌ESCHErICHIA COlI中原核表达PX APn5,表达产物经SdS-PAgE电泳,在110 k dA附近出现特异性条带;酶活性测试显示菌体破碎上清液具有氨肽酶活性,比活力为1 047.2 u/g。配体印迹结果显示表达的PX APn5能与Cry2Ab蛋白特异性结合。多序列比对结果表明,与其他已报道的小菜蛾氨肽酶相比,PX APn5氨基酸序列有3个保守性位点发生了突变,并通过蛋白质建模的方式表征突变位点。【结论】本研究成功克隆和表达了具有氨肽酶活性的小菜蛾氨肽酶,并发现其能与Cry2Ab蛋白特异性结合;通过蛋白质建模对氨肽酶突变位点的特征及功能进行了预测。这些结果对小菜蛾氨肽酶的功能性研究提供了理论基础。【Aim】The aim of this study is to analyze the cloning,expression and homology modeling of midgut aminopeptidase gene Px APN5 in the diamondback moth,Plutella xylostella.【Methods 】 A aminopeptidase( APN) gene was cloned from the P.xylostella midgut,and bioinformatics analysis of the gene was performed.The APN protein was expressed using prokaryotic expression system,and its enzymatic activity was assayed.The interaction between APN and Cry2 Ab was determined by using ligand blot analysis.Homology modeling of APN gene was conducted for the prediction of characteristics and functions of mutation sites.【Results】The sequencing results showed that the cloned APN gene( NCBI accession no.: KM034756) is 2 853 bp in length and encodes 950 amino acids with the predicted molecular weight of 107.3871 k Da and isoelectric point of 5.24.Phylogenetic tree analysis indicated that this APN gene belongs to class 5 of APN family,and it was named as Px APN5.Px APN5 has the conservative features of the APN proteins in lepidopteran insects including N-glycosylation and Oglycosylation sites,GPI anchor point,C-transmembrane domains and zinc-metalloprotease domain(361HEXXH365).A 110 k Da specific protein band appeared when APN protein was inducibly expressed in Escherichia coli.Aminopeptidase activity assay showed that the supernatant of broken bacteria possessedaminopeptidase activity and its specific activity was 1 047.2 U / g.The ligand blot result indicated that Px APN5 could bind to Cry2 Ab specially.Multiple alignment of amino acid sequences demonstrated that there are three mutations in Px APN5 when compared to other known APN proteins from P.xylostella.【Conclusion】The Px APN5 protein with aminopeptidase activity was successfully cloned and expressed,and it could bind to Cry2 Ab.Prediction of characteristics and functions of mutation sites in Px APN5 was carried out by homology modeling.These results laid the foundation for the functional research of Px APN.国家自然科学基金项目(31371999;31370059

Effects of Medium on Sporification of Fusarium oxysporum Schl.

选择8种培养基,采用液体培养法,研究培养基质对尖孢镰刀菌(Fusarium oxysporum Schl.)281菌株小孢子产生量的影响,结果表明,在PDA培养基上培养144h得到最大小孢子产量1.283×109cfu/ml,与其它培养基产孢量差异显著(P=0.05)。培养至192h时,ATCC培养基的小孢子数量达到1.325×109cfu/ml,与PDA培养基之间无显著差异(P=0.05),与其它6种培养基之间差异显著(P=0.05)。综合比较8种培养基,PDA培养基培养时间短,配制简单,是较为理想的产小孢子培养基。The results of eight mediums effect on sporification of Fusarium oxysporum 281 showed that the number of sporification is 1.283×109cfu/ml in PDA medium after 144h,Significant difference were observed between PDA medium and others(P=0.05).The number of sporification is 1.325×109cfu/ml in ATCC medium after 192h,there are no significant difference between ATCC and PDA medium(P=0.05).PDA medium is suitable for sporification.福建省自然科学基金“内生生防菌短芽孢杆菌GFP标记及其对黄瓜枯萎病生防机理的研究”(2006J0068);; 福建省发改委重点项目“生物农药高毒力菌株筛选利用与菌种基因资源库的建立”(闽发改投资[2006]781号

Polymorphism of Fatty Acid of Ralstonia solanacearum in Fujian Province

【目的】利用气相色谱技术检测福建省的40株青枯雷尔氏菌(Ralstonia solanacearum)菌株细胞内的脂肪酸,分析其脂肪酸分布的多态性;研究青枯雷尔氏菌脂肪酸多态性与青枯雷尔氏菌现有种下分化方法之间的关系。【方法】对40株青枯雷尔氏菌脂肪酸进行气相色谱分析,比较同一寄主分离的青枯雷尔氏菌和不同寄主分离的青枯雷尔氏菌脂肪酸的分布;对40株青枯雷尔氏菌脂肪酸进行聚类分析,分析聚成的各类青枯雷尔氏菌脂肪酸的特点以及脂肪酸多态性与其生理小种、生化型和致病性之间的关系。【结果】同一寄主分离的青枯雷尔氏菌和不同寄主分离的青枯雷尔氏菌,其脂肪酸都存在着明显的多态性;对40株青枯雷尔氏菌的脂肪酸进行聚类分析,可以聚成3类,即groupⅠ、groupⅡ和groupⅢ;青枯雷尔氏菌生理小种1存在着不同的脂肪酸类群,青枯雷尔氏菌脂肪酸多态性与其生化型之间不存在相关性,但是脂肪酸和致病性之间存在一定的相关性:groupⅠ为无致病性菌株,groupⅡ为过渡性菌株,groupⅢ为强致病性菌株。【结论】福建省青枯雷尔氏菌脂肪酸分布存在着明显的多态性;青枯雷尔氏菌脂肪酸多态性与致病性之间存在一定的相关性,脂肪酸有望成为青枯雷尔氏菌小种鉴定的新指标。【Objective】The fatty acids of 40 strains of Ralstonia solanacearum isolated from different hosts in the fields in Fujian Province were detected by gas chromatography (GC). The polymorphism of R. solanacearum fatty acids relating to the pathogenicity was observed. 【Method】 The MIDI system and cluster analysis were introduced in analyzing fatty acids to display the relations among the polymorphism, race, biovartype and pathogenicity. 【Result】 The results showed that the patterns of fatty acids were significant different in R. solanacearum strains both isolated from the different hosts and the different body parts of the same hosts. According to the fatty acids the strains were clustered into three groups, e.g. group Ⅰ relating to the strains with non-pathogenicity, group Ⅱ in which the strain pathogenicity was changeable with some virulent and avirulent ones, and group Ⅲ respondent to high pathogenicity. It was proved that the model of fatty acids has no relations to races and biovartypes in R. solanacearum. 【Conclusion】It is the fist time to describe the polymorphism of fatty acids in R. solanacearum in this paper. The pathogenicity could be grouped by the models of fatty acids to distinguish the pathogenicity, which could be used in the identification of R. solanacearum under species differentiation.国家“863”项目(2002AA244031);; 福建省青年科技人才创新项目(2003.J046

Effects of Initial p H Value for the Fermentation of Bacillus subtilis FJAT-14254 on the Generation of Metabolites Substances

为考察酸碱条件对枯草芽胞杆菌fJAT-14254代谢物产生的影响,采用气相色谱-质谱联用分析枯草芽胞杆菌fJAT-14254菌株的胞内成分。结果表明:通过谱库扫描得到32种高匹配率代谢物的初步鉴定结果,主要包括氨基酸类、酸类、烷烃类等,其中含量相对较高的有7种;枯草芽胞杆菌fJAT-14254代谢物的种类和含量与其培养环境的酸碱度变化呈一定的相关性,强酸性(P H=3)条件下培养的胞内代谢物具有特异性,而接近中性(P H=5、7)条件及碱性(P H=9、11)条件下培养胞内代谢物则相似。由此推断,不同P H条件影响发酵终点的P H,进而影响枯草芽胞杆菌fJAT-14254代谢物的产生。To study the relationship between p H of culture medium and intracellular metabolic of Bacillus subtilis FJAT-14254.Intracellular metabolites of B.subtilis FJAT-14254 were analyzed by GC-MS.564 metabolites were detected.31 metabolic markers including hexadecanoic acid, was obtained by data mining.The biochemical substances of B.subtilis FJAT-14254, produce in different p H conditions, had high specificity.The change of metabolites in different p H conditions was complex, not only linear relation.This research provided preliminary study of stress resistance of B.subtilis in metabolic level.公益性行业(农业)科研专项经费(No.201303094); 福建省农科院院青年创新基金(No.2013dqd-6); 福建省农科院杰出青年人才基金(No.2014JQ-2

Fingerprint analysis on methyl fatty acid and its applications in microbial study

脂肪酸甲酯谱图分析方法(Fatty Acid Methyl Ester,FAME)是鉴于脂肪酸可作为生物标记物而发展起来的分析技术。本文介绍了FAME谱图分析方法及其在微生物学领域的应用,包括在微生物检测、鉴定和微生物多样性研究中的应用。Fatty acid is an important biomarker.Its application in biology research has become increasingly popular.The method of FAME fingerprint analysis is based on fatty acid's utilization as a biomarker.This paper describes the FAME fingerprint analysis and its applications for microbial studies,including identification of unknown microbes and assessment of microbial diversity.国家863计划项目(2006AA10A211);; 福建省发展和改革委员会重点项目[闽农产(2006)10号];; 福建省青年科技人才创新项目(2003.J046

Effect of Bacillus cereus on Virulence of Ralstonia solanacearum for Tomato Bacterial Wilt

青枯雷尔氏菌(Ralstonia solanacearum)从其致病性上可分为能使植株发病的强致病力菌株和不能使植株发病的弱致病力菌株.利用蜡状芽孢杆菌(Bacillus cereus)与致病性稳定的青枯雷尔氏菌强致病力菌株进行共培养,处理后的青枯雷尔氏菌在TTC平板上表现为弱毒株的菌落特征,出现了致弱现象.经过16S rDNA基因序列测定,证明了用蜡状芽孢杆菌处理前后的菌株为青枯雷尔氏菌,并非是其它的污染菌株.通过回接番茄盆栽苗发现致弱前的青枯雷尔氏菌能有效引起番茄发生青枯病,而致弱后的菌株丧失致病力,不能引起番茄发病.Ralstonia solanacearum is divided into two strains from its pathogenicity,one is virulent strain which can induce plants disease,and the other is avirulent strain which can not.After cocultivated with Bacillus cereus,the virulent strain of Ralstonia solanacearum was attenuated and showed the characters of weak pathogenicity strain on the TTC medium.It is improved that the strains before and after cocultivated with Bacillus cereus are Ralstonia solanacearum,not other pollutional bacteria,by sequencing the 16S rDNA.The results showd that the original Ralstonia solanacearum could induce bacterial wilt when inoculated to tomato and the attenuate strain can not.福建省科技重点项目(2005N045);; 细胞生物学与肿瘤细胞工程教育部重点实验室开放基金资

Study on the Growing Competition Relationship Between Virulent and Avirulent Strains of Ralstonia solanacearum Isolated from Tomato

在单独和混合培养条件下,研究了番茄青枯雷尔氏菌的强致病力(RV)与无致病力菌株(RA)的生长能力的差异.单独培养时,24h前无致病力菌株的菌体生长量超过强致病力菌株,24h后强致病力菌株菌体生长量超过无致病力菌株,12h时无致病力菌株的生长速率是强致病力菌株的5.6倍,60h时无致病力菌株的菌量仅为强致病力菌株的0.37倍.混合培养时,混合比例RV RA1 and reduced when RVRA=1 or <1;the revised increased ration of the virulent in five mixture treatments were -119.57%～-33.57%.All the amounts of the avirulent strain in five mixture treatments increased,the revised increased ration were 32.03%～346.09%.It was concluded that the avirulent had higher growth ability than the virulent in short time under sole culture condition,and could inhibit the growth of the virulent under mixture culture condition,which could be used as an biocontrol agent for the short-term control efficiency of ABPS to bacterial wilt disease of tomato in the field.国家863计划项目(2002AA244031 2);; 福建省科技厅重大项目(2000Z031);; 福建省计委项目(闽计农经[2002]48号)资