荒漠甲虫小胸鳖甲几丁质酶基因MpCht8c的克隆、鉴定与低温表达

为了挖掘荒漠昆虫小胸鳖甲Microdera punctipennis的耐寒性相关基因,从其4℃转录组数据库筛选出差异表达的几丁质酶基因片段394seq2,检测其编码蛋白的几丁质酶活性,研究该基因的表达对低温胁迫的响应情况。采用RACE技术扩增394seq2的5′端和3′端,克隆全长序列,将其ORF构建至原核表达载体pET28a,导入Transetta(DE3)感受态细胞,诱导表达融合蛋白,Western blot法检测表达蛋白的正确性;二硝基水杨酸法测定几丁质酶活性,qRT-PCR技术探究低温表达谱。结果表明,394seq2的5′-UTR为39 bp,3′-UTR为181 bp;ORF为1 140 bp。系统进化树显示该序列与赤拟谷盗几丁质酶8(TcCHT8)聚为一支,命名为MpCht8c。MpCht8c编码379个氨基酸,分子量为41.2 kDa,理论等电点pI为4.67。MpCHT8c含有完整的几丁质酶结构基序,其N端含有几丁质酶催化域,内有一个类18家族几丁质酶的保守基序KXXXXXGGW,中部是一段富PEST的连接区,C端是几丁质酶结合域,属于IV型昆虫几丁质酶。Western blot结果表明His-MpCHT8c在大肠杆菌中正确表达;融合蛋白粗酶液的几丁质酶活性为1.89 U/mL。在4℃冷胁迫0.5 h和5-9 h时Mpcht8c出现两个上调表达峰值,约为对照的2倍。研究表明荒漠昆虫小胸鳖甲的几丁质酶MpCHT8c具有几丁质酶活性,MpCht8c基因的表达可快速响应4℃低温胁迫。研究结果有助于深入研究几丁质酶在小胸鳖甲耐寒性方面的作用机理。     

蝴蝶兰PhTSJT1基因的克隆及在低温胁迫下的表达分析

本研究从蝴蝶兰叶片中克隆了茎部特异基因Ph TSJT1的全长序列(GenBank登录号为MF797883),并分析了其在不同组织及低温胁迫下的表达特性。结果表明,Ph TSJT1基因全长994 bp,编码239个氨基酸,属于Class-Ⅱ谷氨酰胺酰胺基转移酶超家族成员;同源性分析表明,该蛋白与多种植物的茎部特异蛋白和铝诱导蛋白有较高的同源性,进化上与小兰屿蝴蝶兰的茎部特异蛋白亲缘关系最近;该基因在营养器官中表达水平较高,在花器官中表达水平较低;13℃/8℃(昼/夜)的低温胁迫抑制PhTSJT1基因的转录表达,并随着低温胁迫时间的延长,Ph TSJT1基因的表达水平逐渐降低,在温度恢复正常时其表达水平升高;4℃冷胁迫低温条件下,PhTSJT1基因在处理1 h时,表达水平升高,处理8 h时表达水平最高,16 h后表达水平逐渐降低。由此推测,PhTSJT1参与4℃冷胁迫的分子调控。本研究不但有助于理解热带亚热带植物的耐冷机制,也为蝴蝶兰新品种的遗传改良提供帮助。     

蝴蝶兰PhNAC1基因序列分析及对低温胁迫的响应

为探讨NAC转录因子在蝴蝶兰低温胁迫响应中的分子调控机理,该研究以蝴蝶兰的叶片为材料,运用RT-PCR及RACE技术克隆得到一条蝴蝶兰的NAC转录因子基因完整的cDNA序列,命名为PhNAC1(GenBank登录号MF797909),并分析了其在两种低温条件下的表达模式。结果表明:PhNAC1基因cDNA序列全长1 442 bp,ORF全长942 bp,编码313个氨基酸。预测其蛋白分子量为35.22 kDa,等电点为6.95,属于稳定亲水性蛋白。二级结构预测表明,无规则卷曲和延伸链为该蛋白的主要结构元件,与三级结构预测结果基本相符。PhNAC1编码的氨基酸序列与其他已登录的兰科植物NAC蛋白进行同源序列比对,表明与小兰屿蝴蝶兰(XP_0205763790)亲缘关系较近,序列一致性达97%,其次为铁皮石斛(XP_020695081),一致性为84%。实时荧光定量PCR分析表明,PhNAC1基因在营养器官和生殖器官中均有表达,在蕊柱中的表达量最高。在11℃/6℃低温条件下,PhNAC1基因的转录表达水平在前5天随着处理时间逐渐升高,到第7天开始下降;在4℃低温条件下,PhNAC1基因的表达水平在处理0.5 h时表达量有所下降,1 h后表达量上升至对照水平,之后无明显变化,在处理24至48 h又逐渐升高,推测PhNAC1基因参与蝴蝶兰低温胁迫响应。     

蝴蝶兰PhRCAβ基因的克隆及表达特性分析

为研究蝴蝶兰Rubisco活化酶(RCA)基因对低温胁迫的响应机制,以蝴蝶兰"满天红"为材料,分析了蝴蝶兰Rubisco活化酶基因PhRCAβ的全长序列(Gen Bank登录号为KU954548)及其表达特性。结果表明,PhRCAβ基因全长1 695 bp,编码440个氨基酸;其编码的蛋白具有RCA的特异位点,含叶绿体转运肽,定位于叶绿体中,其成熟蛋白的分子量为42.52 k D,等电点为蛋白5.54,属于RCA小亚基基因;该基因在绿色组织中转录表达;13℃/8℃(昼/夜)的低温胁迫抑制Ph RCAβ基因的转录表达,并随着低温胁迫时间的延长,PhRCAβ基因的表达水平逐渐降低,在温度恢复正常时其表达水平升高;4℃低温条件下,Ph RCAβ基因的表达水平随着低温处理时间的延长逐渐降低,并一直维持在较低水平。由此推测,PhRCAβ直接参与了蝴蝶兰叶片光合作用的调控。     

朝鲜淫羊藿EkCDPK基因的克隆及表达分析

钙依赖蛋白激酶(Calcium-Dependent Protein Kinase, CDPK)是在植物抗逆胁迫信号转导途径中起关键作用的酶。该研究采用RT-PCR结合RACE技术,从朝鲜淫羊藿(Epimedium koreanum Nakai)中克隆获得一个全长2 036 bp的钙依赖蛋白激酶基因(EkCDPK)。结果显示:(1)EkCDPK基因cDNA为1 410 bp,5′-UTR长387 bp, 3′-UTR长239 bp,编码469个氨基酸。EkCDPK蛋白保守结构域N端为S_TKc domain,C端为EF-hand domain,该蛋白具有1个CDPKs典型结构Ser/Thr蛋白激酶活性位点、1个ATP位点、4个Ca~(2+)结合位点、1个跨膜结构域、无信号肽的稳定亲水性蛋白质,二级结构主要由α-螺旋(43.71%)和无规则卷曲(37.10%)构成。(2)系统进化分析表明,EkCDPK与菘蓝CDPK蛋白表现出较高相似性(95%)。(3)实时荧光定量PCR分析表明,EkCDPK基因在根、茎、叶与花组织中均有表达,根中表达量最高,茎次之;干旱胁迫后15 h内表达量显著高于对照组,之后表达量开始下降;盐胁迫5、15和25 h后EkCDPK基因表达量均明显高于对照组。(4)成功构建了pET-28a-EkCDPK原核表达载体,诱导出约50 kD蛋白质,与预测大小一致,表明重组蛋白EkCDPK表达成功,为采用基因工程手段提高朝鲜淫羊藿抗逆性能研究奠定了基础。     

荒漠甲虫小胸鳖甲低温响应几丁质酶基因Mpcht19的克隆及表达模式分析

【目的】探索荒漠昆虫小胸鳖甲Microdera punctipennis低温响应的分子机理并挖掘其耐寒基因。【方法】根据低温转录组数据筛选并克隆小胸鳖甲几丁质酶基因,并进行生物信息学分析;通过实时荧光定量PCR和免疫组织化学法分析该基因在成虫不同组织中和4℃低温胁迫下的表达模式分析。【结果】从小胸鳖甲成虫克隆获得一个几丁质酶基因Mpcht19(Gen Bank登录号:KY126382)。生物信息学分析表明,该基因编码的蛋白Mp CHT19属于Ⅳ型昆虫几丁质酶,理论分子量约为27.18 k D,无信号肽,具亲水性。系统进化分析表明,Mp CHT19与赤拟谷盗Tribolium castaneumⅣ型几丁质酶Tc CHT19同源性最高,氨基酸序列一致性为34.05%。实时荧光定量PCR结果显示,Mpcht19在成虫脂肪体和后肠中高表达,具有组织特异性;该基因的表达还受4℃低温诱导。免疫组织化学分析结果显示,在4℃低温下Mp CHT19蛋白在脂肪体和后肠中表达。【结论】Mpcht19基因的表达具有组织特异性,并受低温诱导。研究结果有助于深入研究几丁质酶与小胸鳖甲耐寒性的关系。     

嗜热子囊菌原变种Thermoascus aurantiacus var.aurantiacus热稳定几丁质酶的克隆表达及活性研究

研究通过RT-PCR和Tail-PCR技术从嗜热子囊菌原变种Thermoascus aurantiacus var.aurantiacus中克隆了一个几丁质酶同源基因。该基因全长1,253bp,包含一个由1,197个碱基构成的开放阅读框,编码398个氨基酸。序列比对分析表明,该基因编码蛋白属于糖苷水解酶18家族的几丁质酶。利用基因重组的方法构建酵母分泌型表达载体,并转化毕赤酵母。在甲醇的诱导下,重组蛋白得到了高效表达,第6天的表达量最高,达到0.433g/L,酶活力为28.96U/mg,同时对表达的几丁质酶进行了纯化,SDS-PAGE检测该蛋白的分子量为43.9kDa。该几丁质酶的最适反应温度为60℃,最适反应pH值为8.0,70℃处理30min仍有45%的相对酶活,具有较好的热稳定性及工业应用价值。     

玉米亚硫酸氧化酶基因的克隆及其结构与表达分析

为明确亚硫酸氧化酶(sulfite oxidase,SO)基因的结构特征和进化关系及其在玉米不同组织器官发育过程中的表达和分布特性,采用RACE技术克隆了玉米SO基因(ZmSO)的全长cDNA。序列分析表明,获得的ZmSO全长1 492bp,其中5′-UTR 160bp,3′-UTR 138bp,开放阅读框为1 194bp,编码397个氨基酸组成的蛋白质。对该基因编码氨基酸保守结构域的分析发现,ZmSO包含1个钼辅因子结合域、1个自身二聚化域和1个过氧化物体靶信号序列。系统进化分析显示,SO在进化上较为保守,玉米与其它植物的SO相似性较高。荧光定量RT-PCR分析表明,在玉米成株期,根、茎、叶、雄花和幼穗中,ZmSO在根部表达丰度最低,在叶片和幼穗中表达量较高。酶活性测定结果显示,不同器官中SO活性与其mRNA转录水平上的表达趋势相似。     

珙桐中一个与低温相关基因的克隆及其表达研究

低温诱导膜蛋白是由低温诱导表达蛋白基因编码的一类疏水性蛋白,在植物抵御寒冷环境时起着一定的保护作用。从珙桐cDNA文库中获得一个未知基因（DiRCI）,该基因长539bp,其中包括174bp的开放阅读框,92bp的5′末端非编码区和273bp的3′末端非编码区,编码57个氨基酸残基蛋白,在氨基酸水平上同源性较高的是车前草的低温诱导膜蛋白（登入号：ACA66247.1）,其相似性为89.5%。半定量RT-PCR分析发现,25℃以上,该基因在珙桐各器官中基本上均未表达,但经8℃低温处理时,在成熟叶片、叶柄和成熟未萌发的种子中均有表达,但是在根中基本没有表达,进一步研究发现该基因在24h～48h内表达量增多并达到最高值,48h后其表达量逐渐减弱直至消失,说明该基因确实与低温诱导相关,从而初步推测该基因为低温诱导膜蛋白基因。该基因的克隆丰富和保存了珙桐基因资源,并为进一步研究冷胁迫的分子机制奠定了基础。     

珙桐中一个与低温相关基因的克隆及其表达研究(英文)

低温诱导膜蛋白是由低温诱导表达蛋白基因编码的一类疏水性蛋白,在植物抵御寒冷环境时起着一定的保护作用。从珙桐cDNA文库中获得一个未知基因(DiRCI),该基因长539bp,其中包括174bp的开放阅读框,92bp的5′末端非编码区和273bp的3′末端非编码区,编码57个氨基酸残基蛋白,在氨基酸水平上同源性较高的是车前草的低温诱导膜蛋白(登入号:ACA66247.1),其相似性为89.5%。半定量RT-PCR分析发现,25℃以上,该基因在珙桐各器官中基本上均未表达,但经8℃低温处理时,在成熟叶片、叶柄和成熟未萌发的种子中均有表达,但是在根中基本没有表达,进一步研究发现该基因在24h～48h内表达量增多并达到最高值,48h后其表达量逐渐减弱直至消失,说明该基因确实与低温诱导相关,从而初步推测该基因为低温诱导膜蛋白基因。该基因的克隆丰富和保存了珙桐基因资源,并为进一步研究冷胁迫的分子机制奠定了基础。     

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.     

Molecular phylogeny of Phebalium (Rutaceae: Boronieae) and related genera based on the nrDNA regions ITS 1+2

Parsimony analyses of the internal transcribed spacer regions of nuclear ribosomal DNA (ITS 1 & ITS 2) for 38 taxa sampled from the Phebalium group (Rutaceae: Boronieae) and two outgroups confirm that, with the exception of Phebalium sensu stricto and Rhadinothamnus, six of the currently recognised genera within the group are monophyletic. The data indicate that Phebaliums. str. is paraphyletic with respect to Microcybe, and Rhadinothamnus is paraphyletic with respect to Chorilaena. Rhadinothamnus and Chorilaena together are the sister group to Nematolepis. Drummondita, included as an outgroup taxon, clustered within the ingroup as sister to Muiriantha and related to Asterolasia.The phylogeny suggests that the evolution of major clades within a number of these genera (e.g. Phebalium) relates to vicariance events between eastern and south-western Australia. Leionema is an eastern genus, with the most basal taxon being the morphologically distinct Leionema ellipticum from northern Queensland. Leionema also includes one species from New Zealand, but this species (as with some others) proved difficult to sequence and its phylogenetic position remains unknown. Taxonomic changes at the generic level are recommended.The authors wish to thank Paul G.Wilson, PERTH, for advice and discussion, and Paul Forster, BRI, for collecting and providing material of Leionema ellipticum. The project was supported by a Melbourne University Postgraduate Award (to BM), the Australian Biological Resources Study (ABRS), Australian Systematic Botany Society and Wolf Den (Australia) Investments.     

New species and new combinations in Peruvian Orchidaceae

seventeen new species and combinations are proposed in the generaChondrorhyncha, Cischweinfia, Cochlioda, Eloyella, Encyclia, Kefersteinia, Koellensteinia, Macroclinium, Rodriguezia, Solenidiopsis, andStenia. All new species are illustrated. A key is provided for 2-flowered species ofMacroclinium, PeruvianSigmatostalix, and PeruvianStenia. Solenidium (Solenidiopsis) peruvianum Schltr. is lectotypified.     

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.     

Phylogenetic investigations of Sordariaceae based on multiple gene sequences and morphology

The family Sordariaceae incorporates a number of fungi that are excellent model organisms for various biological, biochemical, ecological, genetic and evolutionary studies. To determine the evolutionary relationships within this group and their respective phylogenetic placements, multiple-gene sequences (partial nuclear 28S ribosomal DNA, nuclear ITS ribosomal DNA and partial nuclear β-tubulin) were analysed using maximum parsimony and Bayesian analyses. Analyses of different gene datasets were performed individually and then combined to generate phylogenies. We report that Sordariaceae, with the exclusion Apodus and Diplogelasinospora, is a monophyletic group. Apodus and Diplogelasinospora are related to Lasiosphaeriaceae. Multiple gene analyses suggest that the spore sheath is not a phylogenetically significant character to segregate Asordaria from Sordaria. Smooth-spored Sordaria species (including so-called Asordaria species) constitute a natural group. Asordaria is therefore congeneric with Sordaria. Anixiella species nested among Gelasinospora species, providing further evidence that non-ostiolate ascomata have evolved from ostiolate ascomata on several independent occasions. This study agrees with previous studies that show heterothallic Neurospora species to be monophyletic, but that homothallic ones may have a multiple origins. Although Gelasinospora and Neurospora are closely related and not resolved as monophyletic groups, there is insufficient evidence to place currently accepted Gelasinospora and Neurospora species into the same genus.     

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.

     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Changes of dung beetle communities from rainforests towards agroforestry systems and annual cultures in Sulawesi (Indonesia)

Little is known about how tropical land-use systems contribute to the conservation of functionally important insect groups, including dung beetles. In a study at the margin of Lore Lindu National Park (a biodiversity hotspot in Central Sulawesi, Indonesia) dung-beetle communities were sampled in natural forest, young secondary forest, agroforestry systems (cacao plantations with shade trees) and annual cultures (maize fields), each with four replicates (n = 16 sites). At each site we used 10 pitfall traps, baited with cattle dung, along a 100 m transect for six 3-day periods. The number of trapped specimens and species richness at the natural forest sites was higher than in all land-use systems, which did not significantly differ. Each land-use system contained, on average, 75% of the species richness of the natural forest, thereby indicating their importance for conservation. However, a two-dimensional scaling plot based on NESS indices (m = 6) indicated distinct dung beetle communities for both forest types, while agroforestry systems and annual cultures exhibited a pronounced overlap. Mean body size of dung beetles was not significantly influenced by land-use intensity. Five of the six most abundant dung beetle species were recorded in all habitats, whereas the abundance of five other species was significantly related to habitat type. Mean local abundance and number of occupied sites were closely correlated, further indicating little habitat specialisation. The low dung beetle diversity (total of 18 recorded species) may be due to the absence of larger mammals in Sulawesi during historical times, even though Sulawesi is the largest island of Wallacea. In conclusion, the dung beetle fauna of the lower montane forest zone in Central Sulawesi appears to be relatively robust to man-made habitat changes and the majority of species did not exhibit strong habitat preferences.     

安徽牯牛降的丝孢菌Ⅰ

本文报导作者采自安徽枯牛降自然保护区的18种丝孢菌,分属于5个属,其中有3个新种:牛皮冻生尾孢(Cercospora paederiicola),山鸡椒假尾饱(Pseudocercospora litseae-cubebae),鸡血藤生假尾孢(P. millettiicola)和2个中国新纪录。文中对新种进行了描述及绘图,新记录种作了简要说明。研究的标本保存在中国科学院微生物研究所真菌标本室(HMAS)。