杂合抗菌肽CecA-mil的改造及在毕赤酵母中的分泌表达

参照毕赤氏巴斯德酵母(Pichia pastorts)偏好密码子,改造并化学合成杂合抗菌肽CecA-mil基因,改造后的CecA-mil基因克隆到pPICZα-A载体中,构建分泌型重组酵母表达载体pPICZα-A-CM,转化Pichia pastoris受体菌X-33。在醇氧化酶(AOX)启动子调控下,分子量约1．9kD的CecA-mil杂合抗菌肽获得表达,经表达条件优化,重组酵母菌的摇瓶发酵产率可达到245μg／mL。抗菌特性研究表明,该表达产物具有广谱抗菌活性,对多数G^-菌及G^ 菌均有较好的抑菌活性,特别是对氨苄青霉素抗性菌和卡那霉素抗性菌抑杀效果更好;具有热稳定性和酸稳定性。这些特点使得重组抗菌肽CecA-mil在食品防腐、疾病防治和动物饲料添加剂等方面显露出很好的应用前景。     

重组蛋白质在酵母表达体系中的高效表达策略

酵母由于其本身的一些优良特性和容易操作性,可以高水平表达重组蛋白,近年来已经有多个酵母表达的蛋白质多肽类药物上市。作为宿主的酵母最常用的是毕赤氏酵母和酿酒酵母。本文对酵母的一般特性、酵母表达操作、密码子、载体和表达策略、两种不同酵母表达系统的特点等进行了论述,供研究者进行酵母高效表达体系的选择与操作参考。     

稀有密码子影响人RNaseH-Apaf1融合蛋白原核表达

通过限制性内切酶克隆法构建两个pET-28a-TAT-RNaseH-Apaf1重组质粒,其中一个质粒经过稀有密码子的优化。将两个重组质粒分别转化原核表达宿主菌BL21(DE3)和Transetta(DE3),通过SDS-PAGE观察相应融合蛋白诱导表达的情况,并用His标签抗体和Apaf1抗体通过Western Blotting鉴定诱导蛋白。菌液PCR和测序证明经稀有密码子优化和未经稀有密码子优化的人源RNaseH-Apaf1序列成功重组到pET-28a-TAT载体中;经稀有密码子优化的重组质粒成功地在大肠杆菌宿主菌中表达RNaseH-Apaf1融合蛋白,并用Western Blotting检测到该融合蛋白。结果表明,稀有密码子可影响人RNaseH-Apaf1融合蛋白的原核表达。     

带有木糖还原酶基因和木糖醇脱氢酶基因的重组酿酒酵母的构建

采用双载体系统,将携带有瑞氏木霉木糖醇脱氢酶基因的表达质粒pAJ401－Xdh1转化已带有树干毕赤氏酵母木糖还原酶基因的重组酿酒酵母H475,构建了同时带有毕赤氏酵母木糖还原酶基因和瑞氏木霉木糖醇脱氢酶基因的重组酿酒酵母HX1。研究了重组酿酒酵母HX1对木糖的转化利用情况。     

基因设计对重组蛋白表达的影响研究进展

利用异源表达系统生产重组蛋白已成为现代基因工程和生物工程研究热点和重点。但是研究者发现并非所有的基因都能在异源宿主中高效表达,除了宿主、分泌途径、启动子等因素外,基因自身的序列也蕴含了多种影响蛋白表达的因素,如密码子偏爱性,密码子对偏爱性,GC含量,mRNA二级结构,mRNA稳定性等。从基因设计的角度对影响蛋白表达的因素和方法进行了综述,尤其是对密码子优化和密码子对优化,详细讨论了与传统基因优化理念截然不同的密码子协调化及密码子对协调化等最新进展。     

带有木糖还原酶基因和木糖醇酶基因的重组酿酒酵母的构建

采用双载体系统,将携带有瑞氏木霉木糖醇脱氢酶基因的表达质粒ｐＡＪ４０１－ｘｄｈ１转化已带有树干毕赤氏酵母木糖还原酶基因的重组酿酒酵母Ｈ４７５,构建了同时带有毕赤氏酵母木糖还原酶基因和瑞氏木霉木产基因的重组酿酒酵母ＨＸ１,研究了重组酿酒酵母ＨＸ１对木听转化利用情况。     

杂合抗菌肽CecA_mil的改造及在毕赤酵母中的分泌表达

参照毕赤氏巴斯德酵母（Pichia pastoris）偏好密码子,改造并化学合成杂合抗菌肽CecA_mil基因,改造后的CecA_mil基因克隆到pPICZα_A载体中,构建分泌型重组酵母表达载体pPICZα_A_CM,转化Pichia pastoris受体菌X_33。在醇氧化酶(AOX)启动子调控下,分子量约1.9 kD的CecA_mil杂合抗菌肽获得表达,经表达条件优化,重组酵母菌的摇瓶发酵产率可达到245μg/mL。抗菌特性研究表明,该表达产物具有广谱抗菌活性,对多数G－菌及G＋菌均有较好的抑菌活性,特别是对氨苄青霉素抗性菌和卡那霉素抗性菌抑杀效果更好;具有热稳定性和酸稳定性。这些特点使得重组抗菌肽CecA_mil在食品防腐、疾病防治和动物饲料添加剂等方面显露出很好的应用前景。     

猪PR-39抗菌肽基因在毕赤酵母中的表达

[目的]在毕赤酵母中表达抗菌肽PR-39基因,获得有抗菌活性的PR-39。[方法]根据酵母和猪密码子偏好性,对其密码子进行优化改造。将经SOE-PCR获得的PR-39基因与毕赤酵母表达载体pPIC9K连接,构建重组载体pPIC9K-PR-39。经SacⅠ线性化电击转化毕赤酵母GS115,取阳性克隆进行髙拷贝转化子筛选和诱导表达。[结果]pPIC9K-PR-39重组质粒构建成功,pPIC9K-PR-39菌株发酵产物检测结果对DH5α大肠杆菌和金黄色葡萄球菌都有抑菌效果。[结论]获得了PR-39基因的重组酵母,并用毕赤酵母系统成功地分泌表达了具有明显抗菌活性的抗菌肽PR-39。     

斑点叉尾鮰LEAP-2和NK-lysin抗菌肽在毕赤酵母中的串联表达

[目的]合成密码子优化的斑点叉尾鮰(Ictalurus punctatus)LEAP-2与NK-lysin成熟肽的串联基因(LN),构建真核重组表达载体p PICZαA-LN,实现LN在毕赤酵母中的重组DNA表达并对其纯化。[方法]参考编码斑点叉尾鮰LEAP-2和NK-lysin成熟肽的c DNA序列,根据毕赤酵母(Pichia pastoris)的密码子偏爱性,优化合成LEAP-2与NK-lysin成熟肽的串联基因LN,两者之间通过α因子信号肽酶切位点对应的核苷酸序列连接;选择表达载体p PICZαA,构建重组表达载体p PICZαA-LN;电转化至毕赤酵母X-33,29℃、p H 6.0条件下,采用1.0%甲醇诱导表达目的蛋白;采用阳离子交换层析对表达产物进行分离纯化。[结果]Tricine-SDS-PAGE分析显示,培养72 h的表达产物经阳离子交换层析,获得了重组体LEAP-2成熟肽、NK-lysin成熟肽和两者的串连表达物;LEAP-2成熟肽的分泌表达效率较NK-lysin成熟肽的高。[结论]实验构建了斑点叉尾鮰LEAP-2与NK-lysin串联基因的真核重组表达载体,并在毕赤酵母X-33中成功表达。     

Hepcidin的基因克隆及其在毕赤酵母中的分泌表达

根据已知hepcidin氨基酸序列,参照毕赤氏巴斯德酵母(Pichia pastoris)密码子偏好性,设计合成了hepcidin目的基因。所合成的hepcidin基因全长96bp,其5′端引入KEX2基因产物(Kex2)的特异性识别位点序列,以保证表达产物具有天然N端。通过基因重组的方法将hepcidin基因克隆到pPicZαA载体中,构建了分泌型重组酵母表达载体pPICZαA-Hepc,经电转至毕赤酵母GS115中表达。使用浓度高达1500μg/mL的Zeocin筛选得到高拷贝插入GS115菌株,经摇瓶发酵和甲醇诱导,上清液有明显的hepcidin表达,表达量达到100mg/L。初步抗菌特性研究表明,该表达产物对枯草芽孢杆菌有明显的抑菌作用,而对大肠杆菌抑菌效果不明显。     

Foreign exploration for Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) and associated natural enemies on avocado (Persea americana Miller)

Scirtothrips perseae Nakahara was discovered attacking avocados in California, USA, in 1996. Host plant surveys in California indicated that S. perseae has a highly restricted host range with larvae being found only on avocados, while adults were collected from 11 different plant species. As part of a management program for this pest, a “classical” biological control program was initiated and foreign exploration was conducted to delineate the home range of S. perseae, to survey for associated natural enemies and inventory other species of phytophagous thrips on avocados grown in Mexico, Guatemala, Costa Rica, the Dominican Republic, Trinidad, and Brazil. Foreign exploration efforts indicate that S. perseae occurs on avocados grown at high altitudes (>1500 m) from Uruapan in Mexico south to areas around Guatemala City in Guatemala. In Costa Rica, S. perseae is replaced by an undescribed congener as the dominant phytophagous thrips on avocados grown at high altitudes (>1300 m). No species of Scirtothrips were found on avocados in the Dominican Republic, Trinidad, or Brazil. In total, 2136 phytophagous thrips were collected and identified, representing over 47 identified species from at least 19 genera. The significance of these species records is discussed. Of collected material 4% were potential thrips biological control agents. Natural enemies were dominated by six genera of predatory thrips (Aeolothrips, Aleurodothrips, Franklinothrips, Leptothrips, Scolothrips, and Karnyothrips). One genus each of parasitoid (Ceranisus) and predatory mite (Balaustium) were found. Based on the results of our sampling techniques, prospects for the importation of thrips natural enemies for use in a “classical” biological control program in California against S. perseae are not promising.     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

Biological control of three floating water weeds,<Emphasis Type="Italic">Eichhornia crassipes,Pistia stratiotes</Emphasis>, and <Emphasis Type="Italic">Salviniamolesta</Emphasis> in the Republic of Congo

Since 1999, four specific weevils (Coleoptera, Curculionidae) were released in the Republic of Congo against three exotic floating water weeds: Neochetina eichhorniae Warner and N. bruchi Hustache against water hyacinth, Neohydronomus affinis Hustache against water lettuce, and Cyrtobagous salviniae Calder and Sands against water fern. Recoveries of exotic weevils were made from all 24 release sites except one, and all four species have established and spread (up to 800 km for water hyacinth weevils). Within a few years of releases, control of water fern and water lettuce was such that fishing and navigation could be resumed, while reductions of water hyacinth populations were only beginning.     

A molecular phylogeny of Hebeloma species from Europe

In order to widen the scope of existing phylogenies of the ectomycorrhizal agaric genus Hebeloma a total of 53 new rDNA ITS sequences from that genus was generated, augmented by sequences retrieved from GenBank, and analysed using Bayesian, strict consensus and neighbour joining methods. The lignicolous Hebelomina neerlandica, Gymnopilus penetrans, and two species of Galerina served as outgroup taxa. Anamika indica, as well as representatives of the genera Hymenogaster and Naucoria, were included to test the monophyly of Hebeloma, which is confirmed by the results. Hebeloma, Naucoria, Hymenogaster and Anamika indica cluster in a strongly supported monophyletic hebelomatoid clade. All trees largely reflect the current infrageneric classification within Hebeloma, and divide the genus into mostly well-supported monophyletic groups surrounding H. crustuliniforme, H. velutipes, H. sacchariolens, H. sinapizans, and H. radicosum, with H. sarcophyllum being shown at an independent position; however this is not well supported. The section Indusiata divides with strong support into three groups, the position of the pleurocystidiate Hebeloma cistophilum suggests the possible existence of a third subsection within sect. Indusiata. Subsection Sacchariolentia is raised to the rank of section.     

Taxonomic status of the species of the green algal genusNeochloris

Komárek has recently reviewed the various species assigned to the green algal genusNeochloris Starr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia. Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.     

Die GattungKarschia — Bindeglied zwischen bitunicaten Ascomyceten und lecanoralen Flechtenpilzen?

The genusKarschia, in the earlier sense, including saprophytes and parasites on lichens, has been thought to be a non-lichenized parallel genus of the lichen genusBuellia. Modern workers included it on the one hand inBuellia, on the other hand combined it with bitunicate ascomycetes. It is now proved thatKarschia is heterogeneous and contains but superficially similar members both of the genusBuellia of theLecanorales and of typical or masked bitunicateAscomycetes. Therefore, it can not be regarded as a link betweenLecanorales andDothideales. The type species ofKarschia belongs to theDothideales.

     

Demographic characteristics of an intertidal bay goby (Lepidogobius lepidus)

Synopsis In a fourteen month study (May 1976 – June 1977) I examined the following characteristics of an intertidal bay goby (Lepidogobius lepidus) in Morro Bay, California, U.S.A.: annual and seasonal patterns of abundance, age composition and growth rates, survivorship and mortality patterns, and the reproductive cycle for female gobies. Fishes were collected with the aid of quinaldine and otoliths and ovaries removed. Age and growth rates were estimated from otolith annuli using a back calculation formula and a Brody-Bertalanffy growth curve. Mortality rates were derived using the methods of Heincke (1913), Robson & Chapman (1960), mean age, and a catch curve (Ricker 1975). A gonad index was used to describe the annual reproductive cycle. Results indicated that abundance fluctuated seasonally and that these fluctuations appeared to be caused by reproductive emigrations. Bay gobies reached an age of 7+ and a standard length of 87 mm. Growth was relatively constant (6 mm yr⁻¹) until age 5, at which point it began to decline. The mean rates of survivorship, mortality, and instantaneous mortality were 0.75, 0.25, and 0.29 respectively. Mortality rates for individual age classes ranged from 0.13 to 0.51 and increased with age. This stock appears to reproduce mainly during the winter.     

Systematic significance of mature embryo of bamboos

The mature embryo of seven species belonging to five genera of Indian bamboos is described. In all these the basic pattern of embryo organisation is same: the scutellar and coleoptilar bundles are not separated by an internode, the epiblast is absent, the lower portion of the scutellum and the coleorhiza are separated by a cleft and the margins of embryonic leaves overlap. The features unique to fleshy fruited bamboos are: presence of a massive scutellum, the juxtaposition of plumule and radicle and the occurrence of a bud in the axil of the coleoptile. The fleshy fruit bearing bamboos should be classified into one group, the tribeMelocanneae. Evidence is provided to recognise additional groups in the subfamilyBambusoideae.