喀斯特小流域栓皮栎林土壤螨类群落结构特征

为比较石漠化环境与喀斯特森林土壤螨类的群落结构差异,对贵州喀斯特地区朝营小流域栓皮栎林的土壤螨类群落结构本底进行了研究,经2014年各季节的4次调查,共发现土壤螨类3目54科83属.对螨类属数、个体数量、个体密度、Shannon多样性指数(H)、Margalef丰富度指数(SR)、Pielou均匀性指数(J)、捕食性螨类成熟度指数(MI)、甲螨MGP类群和甲螨营养结构等进行了分析.结果表明:土壤螨类在类群属数和个体数量上均以甲螨亚目的属占优势,夏季和秋季具有丰富的属数、较高的个体密度与多样性,春季和秋季具有丰富的个体数量,群落分布具有明显的表聚性.捕食性螨类夏季生态类群以K选择型为主,其他季节以r选择型为主;甲螨生态类群主要为P型和O型,缝甲螨属、异珠足甲螨属和合若甲螨属等属构成了栓皮栎林土壤螨类的营养功能集团.研究表明,该区山毛榉林与其他地区山毛榉林、其他不同类型森林的土壤螨类主要类群存在差异,其中含丰富属组成的派伦螨科、厉螨科、奥甲螨科和单翼甲螨科以及多奥甲螨属、派伦满属、菌甲螨属和单翼甲螨属等数量上占优势的类群属可作为山毛榉林土壤环境的生物指示.     

抗菌肽VIP在毕赤酵母中的高效表达及鉴定

基于人抗菌肽VIP(Vasoactive intestinal peptide)基因序列,按照毕赤酵母密码子偏好性设计引物;用SOE-PCR法扩增目的基因;然后将目的基因克隆至毕赤酵母分泌型表达载体pPICZαA上,构建VIP分泌表达菌株GS115-p PICZαA-vip。用甲醇诱导96 h收集上清,用质谱进行鉴定,结果显示分泌表达产物与人抗菌肽VIP理论值(3 326.82 Da)完全一致,表明人抗菌肽VIP成功得到分泌表达。琼脂糖凝胶扩散法实验结果显示,重组VIP对大肠杆菌Escherichia coli ATCC25922和金黄色葡萄球菌Staphylococcus aureus ATCC25923都有很强的抗菌活性,MIC(Minimal inhibitory concentration)分别为8 mmol/L和16 mmol/L。进一步细胞毒性和溶血性实验结果显示,重组VIP对正常细胞NCM460和IPEC-J2没有毒性,其对SD大鼠红细胞不具有溶血活性。通过透射电镜观察了VIP的抗菌机制,结果显示VIP主要通过破坏细胞膜的方式抑杀细菌。本研究为人抗菌肽VIP的开发应用和大量生产奠定了基础。     

巴氏杆菌糖酵解酶的黏附作用研究

巴氏杆菌（主要是多杀性巴氏杆菌）可以引起多种动物疫病（巴氏杆菌病）,同时也引起人类感染发病。[目的] 研究巴氏杆菌糖酵解酶对宿主细胞（兔肾细胞）和两种常见分子[纤连蛋白（fibronectin,Fn）和血浆纤维蛋白溶解酶原（plasminogen,Plg）]的黏附作用。[方法] 采用原核表达系统对多杀性巴氏杆菌的糖酵解酶进行表达并纯化及制备多克隆抗体,通过菌体表面蛋白定位检测、黏附与黏附抑制等实验探究巴氏杆菌糖酵解酶的黏附作用。[结果] 菌体表面蛋白检测结果显示除烯醇化酶和丙酮酸激酶外的7个糖酵解酶在多杀性巴氏杆菌表面存在。这7个糖酵解酶均能黏附兔肾细胞,但仅有磷酸葡萄糖异构酶的多克隆抗体能对多杀性巴氏杆菌黏附宿主细胞产生抑制作用。Far Western blotting结果显示9个糖酵解酶均能结合宿主Fn和Plg。招募抑制实验结果显示磷酸葡萄糖异构酶、醛缩酶、磷酸甘油酸变位酶的抗体对多杀性巴氏杆菌结合Fn和Plg都有抑制作用,磷酸果糖激酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶抗体仅对多杀性巴氏杆菌结合Fn或Plg有抑制作用。[结论] 多杀性巴氏杆菌糖酵解酶成员葡萄糖异构酶、磷酸果糖激酶、醛缩酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶、磷酸甘油酸变位酶在多杀性巴氏杆菌黏附宿主细胞或分子过程中发挥作用。该研究的完成将加深巴氏杆菌病分子发病机制的认识,并为巴氏杆菌病的诊断标识筛选、新型疫苗创制和药物靶标筛选等提供基础数据。     

Molecular characterization and expression analysis of a KIFC1-like kinesin gene in the testis of Eumeces chinensis

The member of the kinesin-14 subfamily, KIFC1, is a carboxyl-terminal motor protein that plays an important role in the elongation of nucleus and acrosome biogenesis during the spermiogenesis of mammals. Here, we had cloned and sequenced the cDNA of a mammalian KIFC1 homologue (termed ec-KIFC1) from the total RNA of the testis of the reptile Eumeces chinensis. The full-length sequence was 2,339 bp that contained a 216 bp 5′-untranslated region (5′UTR), a 194 bp 3′-untranslated region (3′UTR) and a 1,929 bp open reading frame that encoded a special protein of 643 amino acids (aa). The calculated molecular weight of the putative ec-KIFC1 was 71 kDa and its estimated isoelectric point was 9.47. The putative ec-KIFC1 protein owns a tail domain from 1 to 116 aa, a stalk domain from 117 to 291 aa and a conserved carboxyl motor domain from 292 to 642 aa. Protein alignment demonstrated that ec-KIFC1 had 45.6, 42.8, 44.6, 36.9, 43.7, 46.4, 45.1, 55.6 and 49.8 % identity with its homologues in Mus musculus, Salmo salar, Danio rerio, Eriocheir sinensis, Rattus norvegicus, Homo sapiens, Bos taurus, Gallus gallus and Xenopus laevis, respectively. Tissue expression analysis showed the presence of ovary, heart, liver, intestine, oviduct, testis and muscle. The phylogenetic tree revealed that ec-KIFC1 was more closely related to vertebrate KIFC1 than to invertebrate KIFC1. In situ hybridization showed that the ec-KIFC1 mRNA was localized in the periphery of the nuclear membrane and the center of the nucleus in early spermatids. In mid spermatids, the ec-KIFC1 had abundant expression in the center of nucleus, and was expressed in the tail and the anterior part of spermatids. In the late spermatid, the nucleus gradually became elongated, and the ec-KIFC1 mRNA signal was still centralized in the nucleus. In mature spermatids, the signal of the ec-KIFC1 gradually became weak, and was mainly located at the tail of spermatids. Therefore, the ec-KIFC1 probably plays a critical role in the spermatogenesis of E. chinensis.     

温度对茶尺蠖核型多角体病毒增殖动态的影响

多角体计数、对流免疫电泳、单向免疫扩散及火箭免疫电泳测定的结果表明:26℃适于茶尺蠖核型多角体病毒(EoNPV)的增殖。多角体含量或其相对值随着时间的推移而增长,并渐趋于平稳,两者间呈Logistic曲线关系。单位体重或单头幼虫所含的多角体数量(y_1或y_2)、扩散环直径(y_3)和火箭峰值(y_4)与时间(t)的关系式分别为:y_1=(8.1481)/(1 EXP(9.4210-0.0608t))×10~9PIB/克;y_2=(6.1596)/(1 EXP(5.4809-0.0376t))×10~8PIB/头,y_3=(1.4)/(1 EXP(2.710-0.015t))cm;y_4=(3.52)/(1 EXP(4.580-0.040t))cm。但30℃下,EoNPV增殖严重受抑制,饲毒后24～168小时内难以测出多角体,其后多角体含量也极显著低于26℃,乃至难以被三种免疫测定法测出。     

Association of the epithelial sodium channel with Apx and alpha-spectrin in A6 renal epithelial cells.

Recent molecular cloning of the epithelial sodium channel (ENaC) provides the opportunity to identify ENaC-associated proteins that function in regulating its cell surface expression and activity. We have examined whether ENaC is associated with Apx (apical protein Xenopus) and the spectrin-based membrane cytoskeleton in Xenopus A6 renal epithelial cells. We have also addressed whether Apx is required for the expression of amiloride-sensitive Na(+) currents by cloned ENaC. Sucrose density gradient centrifugation of A6 cell detergent extracts showed co-sedimentation of xENaC, alpha-spectrin, and Apx. Immunoblot analysis of proteins co-immunoprecipitating under high stringency conditions from peak Xenopus ENaC/Apx-containing gradient fractions indicate that ENaC, Apx, and alpha-spectrin are associated in a macromolecular complex. To examine whether Apx is required for the functional expression of ENaC, alphabetagamma mENaC cRNAs were coinjected into Xenopus oocytes with Apx sense or antisense oligodeoxynucleotides. The two-electrode voltage clamp technique showed there was a marked reduction in amiloride-sensitive current in oocytes coinjected with antisense oligonucleotides when to compared with oocytes coinjected with sense oligonucleotides. These studies indicate that ENaC is associated in a macromolecular complex with Apx and alpha-spectrin in A6 cells and suggest that Apx is required for the functional expression of ENaC in Xenopus epithelia.     

生物素对地衣芽孢杆菌生长和乳酸形成的影响

生物素对地衣芽孢杆菌生长和乳酸形成的影响胡尚勤（重庆师范学院生物系重庆６３００４７）地衣芽孢杆菌（Ｂａｃｉｌｕｓｌｉｃｇｅｎｉｆｏｒｍｉｓ）是我国９０年代首次从正常待产妇阴道拭子分离出来的一个无毒新菌株。现在国内已经用它来生产“整肠生”活菌剂。它是一...     

农杆菌介导的单子叶植物早熟禾的转化

利用种子和胚分别在两种培养基K3和K5诱导产生了早熟禾(Poa pratensis L.)一个品种Mado的胚性愈伤组织.K3培养基含有10.0μmol/L的二氯苯氧乙酸(2,4-D)、0.5μmol/L的苄氨基嘌呤(BAP).K5培养基是K3另加0.5μmol/L的硫酸铜.光照条件为20～30 μmol.m-2.s、16 h光照、8 h黑暗.温度保持在24℃.用携有bar基因和gus基因的pDM805质粒转化的农杆菌AGL1对胚性愈伤组织进行转化.共得到4个转基因株系.影响转基因效率的主要因素有愈伤组织的胚性、光照条件、共转化时间、抗生素浓度、选择压力.本研究建立了单子叶早熟禾农杆菌介导的转基因方案.     

灰葡萄孢丝裂原活化蛋白激酶编码基因bmp1和bmp3的功能

【背景】植物病原真菌丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号途径参与病菌有性生殖、细胞壁完整、菌丝侵染、致病力、胁迫响应等过程,灰葡萄孢MAPK信号途径参与病菌生长发育、致病力以及胁迫响应,但MAPK信号途径基因在灰葡萄孢中的功能尚未完全阐明,该信号途径对灰葡萄孢的生长发育和致病力的调控机制尚不明确。【目的】明确灰葡萄孢MAPK编码基因bmp1、bmp3在病菌生长发育、致病力以及氧化胁迫响应过程的功能,为进一步阐明MAPK信号途径调控灰葡萄孢生长发育和致病力的分子机制奠定基础。【方法】利用RNAi技术构建灰葡萄孢MAPK编码基因bmp1和bmp3的RNAi突变体,并以野生型BC22菌株为对照,对bmp1和bmp3基因的RNAi突变体的表型、致病力以及对氧化胁迫的敏感性进行分析。【结果】灰葡萄孢bmp1和bmp3基因的RNAi突变体其菌落形态、菌丝形态均与野生型BC22菌株没有明显差别;bmp1基因的RNAi突变体生长速率明显减慢,分生孢子产量明显降低;bmp3基因的RNAi突变体的生长速率与野生型BC22菌株没有明显差别,不能产生分生孢子。bmp1和bmp3基因的RNAi突变体在番茄果实的表面均不能产生明显的致病症状,而且不能穿透玻璃纸。bmp1基因的RNAi突变体在含有H_2O_2的培养基上受抑制的程度显著低于野生型,而在含甲萘醌的培养基上受抑制的程度显著高于野生型;bmp3基因的RNAi突变体在含有H_2O_2和甲萘醌的培养基受抑制的程度均显著高于野生型。【结论】灰葡萄孢bmp1基因正调控病菌生长、分生孢子形成、致病力和穿透能力,参与调控病菌对氧化胁迫的响应;灰葡萄孢bmp3基因正调控病菌分生孢子形成、致病力、穿透能力以及对氧化胁迫的响应。     

地上部植食者褐飞虱对不同水稻品种土壤线虫群落的影响

地上和地下部生物群落的交互作用对于调控陆地生态过程具有重要作用。在盆栽条件下利用2×2析因设计研究了褐飞虱(Nilaparvata lugens)取食不同水稻品种后对土壤线虫群落的影响。结果表明, 褐飞虱侵害水稻9 d后, 感虫品种(广四和汕优63)的土壤线虫总数、属数及自生线虫(食细菌线虫、食真菌线虫和捕食性线虫)数量增加, 并且一般达到显著水平(P<0.05); 而上述指标在抗虫品种(汕优559和IR36)土壤中则呈现相反的趋势。植食性线虫数量在强感虫品种广四上显著增加(P<0.05), 而在强抗虫品种IR36上显著减少(P<0.05)。褐飞虱和水稻品种对土壤线虫的生态指数(线虫通道指数、Shannon-Wiener指数、成熟度指数、富集指数和结构指数)没有明显影响, 可能与供试土壤线虫群落组成单一及褐飞虱作用时间较短有关。总之, 褐飞虱强烈影响土壤线虫数量、群落组成和营养结构, 并且作用的方向(促进或抑制)和程度依赖于水稻的品种特性, 揭示出地上部植食者的短期侵害将对稻田土壤生态系统的结构和功能产生深远影响。