盐穗木病程相关蛋白HcPR10抗血清的制备及鉴定

病程相关蛋白(PRs)在植物抗病抗逆过程中发挥重要作用。盐穗木病程相关蛋白基因HcPR10(GenBank:KF673356)来自盐穗木(Halostachys capsica)在600 mmol·L^-1 NaCl胁迫下的盐抑制差减文库。为探究盐穗木病程相关蛋白HcPR10发挥生物学功能的机制,该研究通过体外表达和纯化HcPR10重组蛋白制备特异性的HcPR10多克隆抗体。并采用双酶切构建原核重组表达载体pET28a-HcPR10,转化至大肠杆菌(Escherichia coli)BL21诱导表达,通过正交分析优化重组蛋白可溶性诱导表达的条件,利用Ni-NTA亲和层析柱纯化融合蛋白,免疫BALB/c小鼠制备多克隆抗体,基于纯化获得的His-HcPR10重组蛋白和转HcPR10拟南芥总蛋白,分别利用ELISA和Western Blotting检测抗血清效价和特异性。结果表明:成功构建重组表达载体pET28a-HcPR10; 正交结果显示诱导温度27 ℃,诱导转速200 r·min^-1,IPTG浓度0.7 mmol·L^-1,诱导时间6 h条件下可诱导表达大量可溶性目的蛋白; ELISA检测抗HcPR10血清效价达1:243 000,Western Blotting印迹结果显示制备的抗血清可以与重组蛋白和转基因拟南芥(Arabidopsis thaliana)中异源表达的HcPR10蛋白特异性结合。该研究获得了效价高、特异性强的盐穗木病程相关蛋白HcPR10抗血清,为进一步研究HcPR10的亚细胞定位及生物学功能奠定了基础。     

‘潘那利’番茄碱性螺旋环螺旋转录因子基因的克隆与表达分析

碱性螺旋环螺旋(basic/Helix Loop Helix, bHLH)转录因子是植物最大的转录因子家族之一,其广泛参与植物逆境胁迫响应。该研究从野生‘潘那利’番茄(Solanum pennellii Correll)中成功克隆出bHLH转录因子基因SpbHLH89(Sol Genomics登录号Sopen04g001150),采用qRT PCR分析其在干旱胁迫下的表达模式,并利用异源表达初步分析其对非生物胁迫的响应。结果表明：(1)SpbHLH89编码区包含684 bp,编码227个氨基酸,具有典型的碱性螺旋环螺旋区,主要定位于细胞核中;进化树结果显示,SpbHLH89转录因子高度保守,与拟绒毛烟草NtbHLH94(Nicotiana tomentosiformis)存在高度相似性。(2)qRT PCR结果显示,SpbHLH89在‘潘那利’番茄的茎、叶和花中均有表达,其表达量受干旱胁迫诱导。(3)SDS PAGE与Western bloting结果显示,pET 30a SpbHLH89重组蛋白大小约为31 kD。(4)在盐胁迫(400 mmol/L NaCl)和干旱胁迫(600 mmol/L甘露醇)条件下,异源表达重组蛋白的E. coli BL21(DE3)重组菌生长速度提高,说明异源表达SpbHLH89转录因子基因可提高细菌对非生物胁迫的耐受性。     

番茄转录因子基因SlWRKY6的克隆与原核表达分析

该研究基于番茄基因组数据库SGN(Sol Genomic Network)信息,利用RT PCR从栽培番茄‘M82’(Solanum lycopersicum)中成功克隆到番茄SlWRKY6基因(登录号：Solyc02g080890),通过qRT PCR方法和原核表达初步验证其生物学功能。结果表明：(1)生物信息学分析显示,番茄SlWRKY6基因ORF全长1 653 bp,编码550个氨基酸,其蛋白结构含有1个WRKYGQK保守结构域和C₂H₂锌指结构域,属于IIb类;其基因启动子上游1 500 bp含有多个激素响应元件和非生物胁迫响应元件。(2)进化树分析显示,SlWRKY6与潘那利番茄SpWRKY31 X1(NP_001352691.1)的相似性最高,且定位于细胞核内。(3)qRT PCR结果显示,SlWRKY6基因在番茄根、茎、叶中均有表达,在叶中的表达量最高,且受盐和干旱诱导表达。(4)SDS PAGE及Western blot结果显示,pET 30a SlWRKY6重组蛋白的大小约66 kDa,与预期大小一致。(5)原核表达分析显示,重组菌E. coli BL21∷pET 30a SlWRKY6在不同浓度盐(NaCl)和干旱(Mannitol)胁迫下生长速度显著低于对照菌E. coli BL21∷pET 30a,且在400 mmol/L NaCl、800 mmol/L甘露醇胁迫条件下最为显著;滴板实验初步验证SlWRKY6转录因子能提高重组菌E. coli BL21∷pET 30a SlWRKY6在ABA和pH 9(NaOH)胁迫的耐受性;在400 mmol/L NaCl、pH 5(HCl)、800 mmol/L甘露醇胁迫条件下耐受能力降低。研究表明,SlWRKY6转录因子可能通过参与ABA途径来响应非生物胁迫。     

烟草NtNHX1 3基因的克隆及表达特性

以栽培烟草‘云烟87’为材料,通过同源克隆方法分离了烟草NHX（Na⁺,K⁺/H⁺ exchanger）基因NtNHX1 3。结果表明：NtNHX1 3基因CDS长度1 617 bp,编码蛋白质长度为538 aa,蛋白理论等电点为8.67,分子量为59.34 kD。预测NtNHX1 3属于膜蛋白,含有11个跨膜区,且含有NHX类蛋白保守位点氨氯吡嗪咪结合位点。进化树分析显示,NtNHX1 3与菊苣、野菊花NHX遗传距离最近。qRT PCR组织特异性表达分析表明,NtNHX1 3在烟草叶片中表达量最高,根、茎、花中也有表达;盐胁迫处理后NtNHX1 3基因的表达上调,表明该基因参与了盐胁迫反应;打顶初期NtNHX1 3基因的表达呈逐渐上调的趋势,与该时期钾含量逐渐升高相吻合。研究推测,NtNHX1 3具有将钾离子从细胞质转运至液泡的功能。     

异子蓬PEPC基因原核表达及其重组菌在非生物胁迫下的耐受力解析

该研究在Escherichia coli Transetta（DE3）中表达了C₄盐生植物异子蓬的磷酸烯醇式丙酮酸羧化酶基因（PEPC）,并进行了酶学特性及非生物胁迫响应分析,以期初步阐明PEPC基因在非生物胁迫下的生物学功能,为异子蓬PEPC的非生物胁迫的抗性生理研究提供参考依据。基于5′-RACE技术获得异子蓬PEPC全长基因（GenBank登录号为KP985714）,构建了E.coli Transetta::pGEX-4T-1-PEPC重组菌株。通过分光光度法和酶联免疫吸附技术（ELISA）分别测定了PEPC重组蛋白的酶活和含量,并检测E.coli Transetta::pGEX-4T-1-PEPC重组菌株在非生物胁迫下的耐受性。生物信息学分析发现,该PEPC基因的cDNA全长为2 901 bp,编码966个氨基酸,与甜菜（Beta vulgaris）PEPC的氨基酸序列一致性达90%,具有PEPC的典型保守结构域（PEPcase）以及VlTAHPTQsiRR和VMIGYSDSgKDAG活性位点;PEPC蛋白属PEPC-1型的不含信号肽的非分泌型亲水蛋白。Western blot结果显示,融合GST的PEPC蛋白的分子量约130~140 kD;在37 ℃用0.8 mmol/L IPTG诱导E.coli Transetta::pGEX-4T-1-PEPC重组菌株显示出更高的PEPC酶活及含量,而且E.coli Transetta::pGEX-4T-1-PEPC重组菌在200~800 mmol/L NaCl、5%~20% 聚乙二醇（PEG） 6 000、25 ℃~52 ℃温度范围、50~400 μmol/L甲基紫精和pH 3.0~11.0的非生物胁迫下生长均明显优于对照。研究表明,异子蓬PEPC基因的表达提高了E.coli耐受非生物胁迫的能力。     

草菇过氧化氢酶基因(VvCAT1)异源表达及耐温度胁迫功能分析

[背景] 过氧化氢酶（catalase,CAT）参与真菌的生长发育,逆境胁迫时保护真菌免受氧化损伤。[目的] 实现草菇过氧化氢酶基因（VvCAT1）的异源表达,分析VvCAT1耐温度胁迫的功能。[方法] 克隆VvCAT1,构建过表达载体pBAR GPE1/VvCAT1,转化到大肠杆菌（Escherichia coli）菌株Stbl3中,异源表达草菇过氧化氢酶。测定温度胁迫后重组菌（pBAR GPE1/VvCAT1/Stbl3）与对照菌（pBAR GPE1/Stbl3）的过氧化氢酶活性和生长情况,验证VvCAT1的功能。[结果] 重组菌的CAT酶活性显著提高,生长情况显著优于对照菌。[结论] VvCAT1的导入及表达显著提高了大肠杆菌Stbl3的耐温度胁迫功能。     

盐穗木HcUKPP原核表达及重组菌非生物胁迫耐受性研究

该研究采用RT-PCR技术,从盐穗木cDNA文库中克隆获得未知功能多肽HcUKPP基因,构建了大肠杆菌Escherichia coli BL21∷pET30a-HcUKPP重组菌株,并检测了重组菌株在不同非生物胁迫下的耐受性。结果显示:HcUKPP基因开放阅读框为243bp,融合His的HcUKPP蛋白的分子量约为15kD。在37℃条件下,不同浓度的异丙基-β-D硫代半乳糖苷(IPTG)诱导4h后His-HcUKPP融合蛋白均可表达,且E.coli BL21∷pET30aHcUKPP重组菌在不同浓度NaCl(100~900mmol/L)、聚乙二醇(2.5%~20%,PEG 6000)和甲基紫精(25~200μmol/L)胁迫处理下,其生长均具有明显优势。尤其是在500mmol/L NaCl、10%PEG 6000和75μmol/L甲基紫精胁迫12h后,重组大肠杆菌BL21呈现出极显著的优势,分别达到了对照菌的1.81、1.47和3.48倍。研究表明,盐穗木HcUKPP可以显著提高重组大肠杆菌对不同非生物胁迫的耐受性,证明HcUKPP是一类新发现的能够响应非生物胁迫的多肽。     

蒺藜苜蓿MtbHLH148转录因子的克隆与转化及其功能分析

bHLH(Basic helix loop helix, bHLH)转录因子家族是植物最大的转录因子家族之一,广泛参与植物生长发育和盐胁迫应答机制。该研究利用同源克隆的方法克隆蒺藜苜蓿(Medicago truncatula)的MtbHLH148基因,采用qRT PCR方法分析MtbHLH148基因在蒺藜苜蓿中的表达特性,构建超表达载体并通过农杆菌侵染法转化拟南芥(Arabidopsis thaliana),对转基因拟南芥的耐盐性相关功能进行分析研究。结果显示：(1)从蒺藜苜蓿中获得MtbHLH148基因,该基因cDNA全长1 343 bp,包含开放阅读框为603 bp,编码 201 个氨基酸,蛋白分子量22.7 kD,等电点为11.76;蛋白结构分析显示,该蛋白无跨膜结构域,无信号肽,为亲水性蛋白;含有精氨酸／赖氨酸残基的保守结构域和典型的bHLH结构域;二级结构以α 螺旋和无规则卷曲为主。(2)亚细胞定位表明,MtbHLH148蛋白定位在细胞核。(3)进化树分析表明,MtbHLH148与大豆(Glycine max)的亲缘性最近;启动子分析发现,该基因启动子区域含有光响应元件、MYB结合位点以及ABA应答元件ABRE,可能参与非生物胁迫。(4)qRT PCR分析发现,MtbHLH148基因在蒺藜苜蓿的茎中表达量最高,叶中表达量最低,且MtbHLH148基因受ABA(100 μmol/L)诱导并在盐胁迫(200 mmol/L NaCl)处理8 h内表达量上调,而在低温(4 ℃)处理时表达量明显下调。(5)成功构建超表达载体pCAMBIA3301 MtbHLH148并转化拟南芥获得16个抗性株系,经鉴定有12个过表达株系,其中表达量最高的转基因株系为OE8;对OE8株系耐盐性功能分析发现,转基因拟南芥植株的发芽率明显高于野生型,盐胁迫下转基因拟南芥的根长是野生型的1.5倍,表明其耐盐性得到了增强。研究表明,MtbHLH148基因可能在盐胁迫调节机制中具有一定的调控作用。     

耐辐射奇球菌dps突变株的构建和蛋白功能初步研究

Dps(DNAprotection during starvation)蛋白是原核生物中特有的一类具有铁离子结合和抗氧化损伤功能的重要蛋白。利用体外PCR扩增技术和体内同源重组方法,获得了耐辐射奇球菌(Deinococcus radiodurans)dps全基因(DRB0092)缺失突变株。对突变株和野生型分别进行不同浓度过氧化氢(H₂O₂)处理,结果表明:与野生型菌株R1相比,dps突变株在低浓度H₂O₂(≤10mmol/L)条件下存活率急剧下降,而高浓度(≥30mmol/L)下则完全致死。Native-PAGE活性染色结果显示,稳定生长期dps突变株体内两种过氧化氢酶(KatA和KatB)的活性较野生型R1分别上调2.3倍和2.6倍。通过质粒构建和大肠杆菌诱导表达,获得可溶性Dps蛋白。体外结合和DNA保护实验结果显示:Dps具有明显的DNA结合功能,并能保护质粒DNA免受羟自由基攻击。本研究证明,Dps蛋白在耐辐射奇球菌抗氧化体系中发挥重要作用,可能对该菌极端抗性机制有重要贡献。     

里氏木霉内切葡萄糖苷酶Ⅳ在毕赤酵母中的表达*

进行了内切葡萄糖苷酶Ⅳ(EGⅣ)在毕赤酵母(Pichia pastoris)表达系统中的表达。采用RT-PCR的方法从里氏木霉(Trichoderma reesei)中分离到eg4基因。将eg4基因与毕赤酵母表达载体pPICZαA连接,得到重组质粒pPICZαA-eg4。将该重组质粒线性化后转化毕赤酵母GS115,eg4基因通过同源重组被整合到毕赤酵母的染色体上,并处于酵母α因子的下游,得到重组菌株P.pastoris-EGⅣ1。在甲醇     

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.     

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.

     

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.     

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.