应用毕氏酵母高效表达耐高温植酸酶

自然界很难获得大量的耐高温植酸酶。采取连续延伸PCR方法 ,按照毕氏酵母的偏爱密码 ,合成 1.3kb烟熏曲霉耐高温植酸酶基因 fphy。合成基因与野生型基因核苷酸序列同源性为 74 % ,但编码的氨基酸序列一致。将耐高温植酸酶基因fphy插入毕氏酵母高效表达载体 pPIC9中 ,与分泌信号肽序列融合 ,通过同源重组将耐高温植酸酶基因整合到酵母染色体中 ,通过SDS PAGE检测和表达产物的酶活性筛选 ,得到重组转化子 ,证明植酸酶获得有效分泌和高效表达。经过 5L小罐高密度发酵 ,蛋白质表达量为 5 .6 g/L ;每毫升发酵液中植酸酶的活力单位达 130 0 0 0u ;表达产物耐高温性很强 ,90℃处理 80min后仍有 4 0 %的活性。     

黑曲霉Z6植酸酶基因phyA的克隆及表达

目的:克隆植酸酶基因phyA,构建毕赤酵母表达载体,转化毕赤酵母,并对重组工程菌的表达产物进行初步酶学性质研究.方法:以植酸酶高产菌株-黑曲霉Z6染色体DNA为模板,PCR扩增得到植酸酶基因phyA,序列鉴定后连接到毕赤酵母穿梭载体pPIC9K上,构建重组质粒pPIC9K-phyA,电击转化毕赤酵母KM71,筛选得到重组转化子.对重组工程菌表达产物进行SDS-PAGE分析和酶活性研究.结果:phyA序列分析表明该基因具有典型的植酸酶活性位点保守序列ArgHisGlyAlaArgTyrPro,与NC-BI已发表的植酸酶基因同源性较高,达到94%以上.该序列已提交GenBank,序列号为DQ318022.重组工程菌KM71-phyA7的PCR扩增证实了植酸酶基因已整合到酵母基因组中,植酸酶能有效分泌和表达,粗酶液酶活可达875U/mL.结论:植酸酶基因phyA在毕赤酵母中成功表达,为今后的定向改组奠定了基础.     

阿维链霉菌来源的磷脂酰丝氨酸合成酶基因的克隆及其在毕氏酵母中的异源表达

本文旨在构建阿维链霉菌(Streptomyces avermitilis)来源的磷脂酰丝氨酸合成酶基因(pss)的重组质粒,研究其在毕氏酵母中的异源分泌型表达。利用PCR技术克隆阿维链霉菌来源的pss基因,再通过电转化方法将重组质粒pOG-01转入毕氏酵母KM71中,构建重组工程菌KP1。实验结果表明,阿维链霉菌来源的磷酯酰丝氨酸合成酶基因在毕氏酵母KM71中成功表达,2 mL菌体上清催化50 mmol/L卵磷脂,转酯反应的转化率为58%,酶活为4.83 U/mL。     

人血管内皮细胞生长因子受体Flt-1胞外区cDNA在毕赤酵母中的表达和鉴定

将编码血管内皮细胞生长因子受体Flt-1胞外区1-3 loop 316个氨基酸残基的cDNA插入到含AOX1启动子和α分泌信号肽序列的Pichia pastoris酵母载体中,构建了重组表达质粒pPIC9K/Flt-1(1-3),转化酵母宿主菌GS115,筛选His⁺Mut^s表型转化子,经摇瓶培养,1%甲醇诱导表达4 d后,SDS-PAGE结果显示,培养上清中Flt1(13)表达量达总蛋白的30%以上。ELISA及Western blot实验表明,表达产物具有良好的抗原性和特异性。生物学活性检测证实其具有结合VEGF的能力和抑制VEGF对HUVEC细胞的促增殖功能。     

人三叶因子3在毕氏酵母中的表达及生物活性分析

利用PCR方法在人胎儿胎盘cDNA中扩增了人三叶因子3基因（hTFF3）,插入到含有AOX1启动子和α－因子信号肽序列的表达载体pPIC9K中,采用毕氏酵母表达系统对其进行了高效的分泌表达,并用G418筛选高拷贝整合转化子。2％甲醇诱导酵母表达48h后,上清经SDS－PAGE和Western印迹证明重组蛋白质以双体的形式分泌表达,占总蛋白质的45％,能被抗hTFF3抗体所识别。表达产物经S－Sepharose、Q-Sepharose离子交换及Sephacryl S-100纯化后纯度达到95％以上。体内生物学活性研究证实hTFF3可有效的防止盐酸诱导的大鼠胃溃疡。     

里氏木霉α-半乳糖苷酶基因agl2在毕氏酵母中高效表达

通过反转录PCR从里氏木霉RutC-30 基因中克隆到α-半乳糖苷酶基因agl2,将其构建到具有AOX1强启动子的表达载体pPIC9K中,线性化后转化到毕氏酵母GS115中进行表达。结果表明agl2基因在毕氏酵母GS115中得到了高效表达,在50 L发酵罐中高密度发酵168 h,酶活达最高值1 106 U/mL。经PCR产物鉴定,结果表明agl2基因已整合到毕氏酵母GS115基因组中。     

人血管抑素在丝氏酵母中的表达及其活性测定

利用RT－PCR方法从人肝细胞癌HepG2细胞株中扩增得到编码人血管抑素（angiostatin)的cDNA。将其插入毕氏酵母表达载体pPIC9K中,转化结氏酵母,获得了整合多拷贝血管抑素基因的菌株。了酵液经赖氨酸亲和柱纯化所得到的重组蛋白的产量为25mg/L。蛋白质氨基酸序列分析结果与国外报道一致。重组蛋白特异地抑制bFGF刺激的牛主动脉内皮细胞的增殖,ED50约为3mg/L。     

香蕉红素氧还蛋白酵母双杂交eDNA文库的构建及鉴定

结合改良的BD SMARTTMPCR cDNA Synthesis Kit User Manual cDNA合成试剂盒,反转录合成带有目的接头cDNA第一链,通过LD-PCR,扩增双链cDNA(ds cDNA).产物经双酶切后回收,与经同样双酶切的酵母双杂交系统中pGADT7载体连接转化,并进行了鉴定.结果表明:提取的RAN的A260/A280=1.82,表明所提RNA纯度高:双链cDNA文库大于0.2 kb,且弥散较长,成瀑布条带状;根据生长菌落计数,文库含有1.2×106转化子,重组子不同插入片段在0.3～2 kb之间.表明该文库达到建库要求,为下一步进行酵母双杂交试验奠定基础.此方法用于构建cDNA文库与酵母双杂交系统相结合的研究目前尚未见过相关报道.     

人食管癌cDNA酵母杂交文库的构建与鉴定及其应用

以往在研究由促癌物12-o-十四烷酰佛波醇-13-乙酯（12-o-tetradecanoylphorbol-13 -acetate,TPA）诱导的人永生化食管上皮细胞恶性变中基因的差异表达情况时,曾获得中性粒细胞明胶酶相关lipocalin（neutrophil gelatinase-associated lipocalin,NGAL）等新的食管癌癌变相关基因.为深入研究这些癌变相关基因在食管癌中以蛋白质-蛋白质或蛋白质-DNA相互作用为基础的功能网络调控关系,建立了一个食管癌cDNA酵母杂交文库.采用Trizol试剂从一种新的人食管癌细胞（SHEEC,由人永生化食管上皮细胞转化而来）中提取细胞总RNA,采用Oligotex mRNA Kit从细胞总RNA中制备PolyA⁺ mRNA,采用SuperScript^TM Choice System For cDNA Synthesis Kit合成cDNA,以PolyA⁺ mRNA为探针,化学发光法监测cDNA合成的质量,以pGADT₇为载体构建了SHEEC细胞的cDNA酵母杂交文库.文库的滴度为1.19×10⁹ cfu/ml,重组片段大小主要集中在0.5～6.0 kb,重组率为50％.在此基础上,应用酵母单杂交技术手段对该文库中的NF-κB元件结合因子进行了筛选,在SD/-his/-leu/［＋15 mmol/L 3-AT］缺陷培养基平板上共获得了约360个单克隆.按照平板上酵母单克隆直径的大小,选择直径大于2 mm者91个,提取质粒进行酶切鉴定和一对一酵母单杂交验证实验,结果获得了30个阳性重组子,并随机取其中的9个阳性重组子进行测序和GenBank/BLAST同源分析,发现某些基因编码的蛋白质产物在关键氨基酸残基位点上与p65和p50的NF-κB元件结合域公共序列具有高度一致性.这些实验结果表明,所构建的人食管癌cDNA酵母杂交文库是成功的.     

昆虫神经毒素LqhIT2的表达、抗血清制备及活性分析

根据毕赤酵母密码子偏爱性,不改变毒素蛋白质一级结构,设计合成了昆虫神经毒素LqhIT2基因,并分别克隆至大肠杆菌融合表达载体pPET30-a( )和毕赤酵母分泌表达载体pPIC9K.在IPTG的诱导下,神经毒素在大肠杆菌中融合表达,表达产物经镍亲和层析纯化后,用于免疫BALB/c小鼠,制备了特异性较高的抗血清,抗体滴度超过1:128 000.利用制备的抗血清,采用斑点杂交,筛选得到了较高水平分泌表达重组LqhIT2的酵母转化子,摇瓶条件下,毒素表达量约9 mg/L.大肠杆菌表达产物没有生物活性,酵母表达产物经注射蝗虫表现出杀虫活性.     

Astragalar morphology of late Eocene anthropoids from the Fayum Depression (Egypt) and the origin of catarrhine primates

The phylogenetic relationships of the late Eocene anthropoids Catopithecus browni and Proteopithecus sylviae are currently a matter of debate, with opinion divided as to whether these taxa are stem or crown anthropoids. The phylogenetic position of Catopithecus is of particular interest, for, unlike the highly generalized genus Proteopithecus, this taxon shares apomorphic dental and postcranial features with more derived undoubted catarrhines that appear in the same region 1-2 Ma later. If these apomorphies are homologous and Catopithecus is a stem catarrhine, the unique combination of plesiomorphic and apomorphic features preserved in this anthropoid would have important implications for our understanding of the crown anthropoid morphotype and the pattern of morphological character transformations that occurred during the early phases of stem catarrhine evolution.Well-preserved astragali referrable to Proteopithecus, Catopithecus, and the undoubted early Oligocene stem catarrhine Aegyptopithecus have provided additional morphological evidence that allows us to further evaluate competing hypotheses of interrelationships among Eocene-Oligocene Afro-Arabian anthropoids. Qualitative observations and multivariate morphometric analyses reveal that the astragalar morphology of Proteopithecus is very similar to that of early Oligocene parapithecids and living and extinct small-bodied platyrrhines, and strengthens the hypothesis that the morphological pattern shared by these taxa is primitive within crown Anthropoidea. In contrast, Catopithecus departs markedly from the predicted crown anthropoid astragalar morphotype and shares a number of apomorphic features (e.g., deep cotylar fossa, laterally projecting fibular facet, trochlear asymmetry, mediolaterally wide astragalar head) with Aegyptopithecus and Miocene-Recent catarrhines. The evidence from the astragalus complements other independent data from the dentition, humerus and femur of Catopithecus that support this taxon's stem catarrhine status, and we continue to maintain that oligopithecines are stem catarrhines that constitute the sister group of a clade containing propliopithecines and Miocene-Recent catarrhines.     

中国异极衣科地衣二新记录种

该文综合运用形态学、解剖学和化学等方法对山东大型地衣进行分类研究,发现了两个中国新记录种,即德氏蜂窝衣(Heppia despreauxii)和多孢小极衣(Lichinella myriospora).德氏蜂窝衣隶属于蜂窝衣属(Heppia),生于光线充足且裸露的土壤上,主要识别特征为下皮层缺失、子实层IKI+蓝色;多...     

藏药多刺绿绒蒿种子萌发特性研究

多刺绿绒蒿(Meconopsis horridula)为罂粟科绿绒蒿属一年生草本植物,是一种极具观赏价值和药用价值的高山植物,目前处于濒危状态,因此研究多刺绿绒蒿种子的萌发特性对其种子育苗及人工栽培具有重要意义。为了提高多刺绿绒蒿的种子发芽率,该研究以多刺绿绒蒿的种子为材料,分析了不同消毒剂、浸种时间、温度和外源植物激素对种子萌发特性的影响。结果表明:(1)最适消毒方法为75%乙醇1 min+3%H2O25 min,最适浸种时间为24 h,最适温度和光照条件为20℃/10℃(光照12 h/黑暗12 h),用无菌水浸种后的种子发芽率为49.67%。(2) GA_3100～600 mg·L~(-1)和NAA 5～30 mg·L~(-1)可以提高种子的发芽率、发芽势和发芽指数,缩短发芽启动时间和发芽持续时间,对种子的萌发有促进作用。(3) 6-BA 5 mg·L~(-1)和10 mg·L~(-1)对种子的萌发有一定的促进作用,但不显著,6-BA浓度≥15 mg·L~(-1)则抑制种子的萌发。(4)用GA3500 mg·L~(-1)浸种后的种子发芽指标最好,发芽率、发芽势和发芽指数分别为69.67%、33.00%、4.51,种子的发芽起始时间和发芽持续时间分别为10.67 d、11.67 d。     

scabrous Modifies Epithelial Cell Adhesion and Extends the Range of Lateral Signalling during Development of the Spaced Bristle Pattern in Drosophila

The role of scabrous (sca) in the evenly spaced bristle pattern of Drosophila is explored. Loss-of-function of sca results in development of an excess of bristles. Segregation of alternately spaced bristle precursors and epidermal cells from a group of equipotential cells relies on lateral inhibition mediated by Notch and Delta (Dl). In this process, presumptive bristle precursors inhibit the neural fate of neighbouring cells, causing them to adopt the epidermal fate. We show that Dl, a membrane-bound ligand for Notch, can inhibit adjacent cells, in direct contact with the precursor, in the absence of Sca. In contrast, inhibition of cells not adjacent to the precursor requires, in addition, Sca, a secreted molecule with a fibrinogen-related domain. Over-expression of Sca in a wild-type background, leads to increased spacing between bristles, suggesting that the range of signalling has been increased. scabrous acts nonautonomously, and we present evidence that, during bristle precursor segregation, Sca is required to maintain the normal adhesive properties of epithelial cells. The possible effects of such changes on the range of signalling are discussed. We also show that the sensory organ precursors extend numerous fine cytoplasmic extensions bearing Dl molecules, and speculate on a possible role for these structures during signalling.     

不同光质对白及组培苗生长及光合特性的影响

白及的自然繁殖率极低,组培育苗是其种苗繁殖的主要方式之一.为探索提高白及组培育苗质量及缩短育苗周期的高效人工光环境,该文以紫花白及(Bletilla striata)为试验材料,研究LED光质对白及组培苗的生长和光合特性的影响.结果表明:提高红蓝光组合中的蓝光占比,有利于促进白及组培苗的生长和生物量的积累,而白及的球茎...     

中国兰科二新记录种

兰科植物资源调查是掌握地区兰科本底资料的基础,对研究兰科植物的地理分布和资源多样性具有重要意义.该文报道了分别产自中国西藏自治区墨脱县和巴宜区的石豆兰属(Bulbophyllum Thou.)中国分布新记录种——尼泊尔大苞兰(B.raskotii J.J.Verm.,Schuit.&de Vogel)和曲唇兰属[Pan...     

Stratigraphy, artefact industries and hominid associations for Sterkfontein, member 5

A revised stratigraphy for the early hominid site of Sterkfontein (Gauteng Province, South Africa) reveals a complex distribution of infills in the main excavation area between 2.8 and 1.4 m.y.a, as well as deposits dating to the mid to late Pleistocene. New research now shows that the Member 4 australopithecine breccia (2.8-2.6 Ma) extends further west than was previously thought, while a late phase of Member 4 is recognized in a southern area. The artefact-bearing breccias were defined sedimentologically as Member 5, but one supposed part of these younger breccias, the StW 53 infill, lacks in situ stone tools, although it does appear to post-date 2.6 Ma when artefacts first appear in the archaeological record. The StW 53 hominid, previously referred to Homo habilis, is here argued to be Australopithecus. The first artefact-bearing breccia of Member 5 is the Oldowan Infill, estimated at 2-1.7 Ma. It occupies a restricted distribution in Member 5 east and contains an expedient, flake-based tool industry associated with a few fossils of Paranthropos robustus. An enlarged cave opening subsequently admitted one or more Early Acheulean infills associated in Member 5 west with Homo ergaster. The artefacts attest to a larger site accumulation between ca. 1.7 and 1.4 Ma, with more intensive use of quartzite over quartz and a subtle but important shift to large flakes and heavier-duty tools. The available information on palaeoenvironments is summarized, showing an overall change from tropical to sub-tropical gallery forest, forest fringe and woodland conditions in Member 4 to more open woodland and grassland habitats in the later units, but with suggestions of a wet localized topography in the Paranthropus -bearing Oldowan Infill.     

甜荞FeFUL2基因的克隆与表达分析

为了探索甜荞FUL同源基因参与花与籽粒发育调控的分子机制,该文采用同源克隆的方法从甜荞(Fagopyrum esculentum)长花柱和长雄蕊突变体(lpls)中克隆到1个长837 bp的FeFUL2基因(GenBank登录号为MG779493.1),其包含长690 bp的完整开放阅读框,编码1个由229个氨基酸残基组成的MADS-box转录因子。通过对FeFUL2进行分子系统发生、同源蛋白比对与转录因子结构分析,结果显示FeFUL2与核心真双子叶植物AP1/FUL亚家族转录因子中的euFUL进化系聚于1个进化分支,属甜荞euFUL型MADS-box转录因子,且包含1个57个氨基酸残基长的高度保守的MADS结构域、1个69个氨基酸残基长的次级保守的K结构域,其C末端转录激活区在序列长度和氨基酸残基组成上与其他euFUL型转录因子差异较大,但仍含有2个euFUL型转录因子特有的保守基元:FUL motif和paleo AP1 motif。用qPCR检测基因表达的组织特异性显示:FeFUL2基因在甜荞lpls突变体的根、茎、叶、花被片、雄蕊、雌蕊和发育4 d的幼果中均有表达,但其在花被片中表达量极显著高于该基因在其他器官中的表达量(LSD,P0.01)。综合转录因子的结构与基因的表达模式推测,FeFUL2基因与其他euFUL型基因的功能可能存在一定差异,其在花发育过程中可能主要参与甜荞花被片的发育调控。     

四川卧龙国家级自然保护区马先蒿属一新种——熊猫马先蒿

熊猫马先蒿(Pedicularis pandania)是在四川卧龙国家级自然保护区发现的马先蒿属一新种。该新种属于互生叶类群,其基生叶成丛,花冠属于短管有喙型,且下唇将喙包裹住,花管在近萼端发生约180度的扭旋使得花冠下唇在侧上方,这一特征明显区别于其他国产马先蒿物种。分子系统发育分析结果表明,熊猫马先蒿是Clade 7 成员,虽然与火焰系、假欧氏系、喙齿系、拟蕨系和长喙系等成员构成姐妹关系,但熊猫马先蒿的花冠形态特征明显区别于近缘物种。     

千斤拔多糖对小鼠免疫功能的调节作用

为研究千斤拔多糖对正常及免疫低下小鼠免疫功能的调节作用,该研究选用SPF级BALB/c小鼠,免疫抑制小鼠采用隔天皮下注射环磷酰胺(40 mg·kg~(-1))5次,通过测定脾脏、胸腺质量及计算脏器指数,采用碳粒廓清法计算单核巨噬细胞吞噬功能,在鸡红细胞免疫后测定小鼠血清溶血素抗体水平,同时观察高、低剂量千斤拔多糖(剂量分别为500、1 000 kg·d~(-1))对正常小鼠及免疫低下小鼠免疫调节的影响。结果表明:千斤拔多糖高、低剂量组对正常及免疫低下小鼠均有不同程度的增加脾脏指数、胸腺指数的作用;千斤拔多糖高、低剂量组对正常及免疫低下小鼠均能提高廓清指数,但没有统计学意义;千斤拔多糖高剂量组对正常及免疫低下小鼠的血清溶血素抗体水平都有显著性提高。这说明千斤拔多糖能有效提高正常及免疫低下小鼠免疫功能的作用。