大豆液泡膜H+-ATPase功能与构象关系的初步研究

大豆液泡膜V型H⁺-ATPase是ATPases中的一种,它在植物细胞的生长发育中有重要的作用.利用竹红菌乙素(HB）和KI这两种分别猝灭蛋白质疏水区域内源荧光和亲水区域内源荧光的荧光猝灭剂,在不同pH值、温度条件下对纯化的大豆液泡膜V型ATPase进行荧光猝灭实验,初步探讨了V型H⁺-ATPase的水解活性同其蛋白质折叠状态间的关系.研究表明,通过比较不同pH值、温度条件下蛋白质疏水区域和亲水区域内源荧光的荧光猝灭常数（K_SV）,发现当环境pH值、温度偏离酶的最适pH值和温度时,蛋白质的内源荧光强度降低且疏水区域和亲水区域内源荧光的荧光猝灭常数（K_SV）降低,说明伴随着酶的水解活性降低,蛋白质的折叠状态发生了变化.我们认为蛋白质在膜内的折叠状态变化是酶失活机制的一个重要方面,为植物的抗冻和抗盐研究提供了一定的参考.     

重组缣孢菌细胞色素P-450nor的性质研究

纯化的重组缣孢菌细胞色素P-450nor（recombinant fusarium oxysporum cytochrome P-450nor,rF·P-450nor）用于动力学研究. 测得米氏常数K_m(NO)和K_m(NADH)分别为0.128 mmol/L和0.208 mmol/L. V_max(N₂O)为11 363 min^-1. 光谱吸收特性研究表明：rF·P-450nor具有典型的血红素蛋白的特性,在413 nm有最大吸收峰. 加入还原剂Na₂S₂O₄时,最大吸收峰前移至405 nm. 与CO结合后,再加入还原剂Na₂S₂O₄时,在450 nm处表现最大吸收.与NO结合后,最大吸收峰移至430 nm附近. 这些光谱特征的变化与天然的F·P-450nor完全一致.     

鹅源新城疫病毒NP、P和L基因的克隆与P基因的表达鉴定

将鹅源新城疫病毒的NP、P和L基因通过RT PCR方法从尿囊液中扩增后分别克隆进pGEM-Teasy载体 ,再分别亚克隆到真核表达载体pCI neo上 ,通过酶切、PCR和测序验证克隆正确。利用P基因开放性阅读框 (ORF)上靠近终止密码上游的AgeI位点 ,将报告基因绿色荧光蛋白 (GFP)基因克隆进P基因真核表达重组质粒 ,分别转染COS-1细胞和CEF细胞 ,在倒置荧光显微镜下可见到绿色荧光 ,表明GFP基因已得到表达 ,由此证明P相似文献   

高纯度藻蓝蛋白分离纯化及光谱特性研究

藻蓝蛋白(PC)具有特征性吸收光谱和荧光光谱,是一种新颖的荧光分子探针.为了获取高纯度PC,经过反复探索研究建立了SephadexG-200、DEAE-SephadexA-25、HA、SephadexG-200的分离纯化程序。此法效果理想：PAGE显示为单条电泳带;纯度值(A₆₁₅／A₂₈₀)高达14,突破了国内外报告的最高值.     

重组APP-N端蛋白与神经节苷脂GM1相互作用后的构象变化

用基因重组方法制备人类β-淀粉样蛋白前体(APP)N端融合蛋白APP_28～123,应用荧光光谱和圆二色谱技术观测GM1对APP_28～123构象的影响.结果表明：APP_28～123与GM1水溶液和含GM1的脂质体孵育后,其荧光强度明显增强,最大发射峰位蓝移20 nm;APP_28～123在溶液中的二级结构以α螺旋为主,峰位在208 nm和222 nm,而APP_28～123与PC/GM1脂质体或GM1水溶液孵育后,其二级结构虽以α螺旋为主,但摩尔椭圆度(θ)值明显增强. 以上结果表明GM1改变了APP_28～123二级结构,提示GM1引起APP分子构象的改变,可能影响APP分子正常生理功能和跨膜转运过程.     

枸杞LbMYB103基因克隆及转化拟南芥的研究

以枸杞品种‘宁杞1号’花药为材料,采用 RT-PCR技术,分离了R2R3类MYB基因LbMYB103包含完整开放阅读框（ORF）的cDNA片段,碱基序列与已知基因HQ415755完全一致。运用Gateway技术构建LbMYB103基因植物过表达载体pMDC83-LbMYB103,利用基因枪法将融合有绿色荧光蛋白（GFP）的过表达载体转入洋葱表皮细胞,将LbMYB103基因定位在细胞核。实时荧光定量PCR分析发现,LbMYB103基因在花药中优势表达,果实中表达量较低,在根、茎和叶中均未检测到其转录本,推测LbMYB103基因可能在花药发育过程中起重要作用。通过根癌农杆菌介导法将pMDC83-LbMYB103转入拟南芥（Col-0）,经筛选获得T₁代抗性再生植株52棵,PCR鉴定有41棵阳性植株,收获T₁代种子,经抗性筛选获得T₂代抗性植株29棵,PCR鉴定有23棵阳性植株。实时荧光定量PCR分析表明,LbMYB103在拟南芥植株的基因组中正常表达。表型观察发现T₁和T₂代拟南芥花药发育异常,花发育迟缓,果荚短小无种子,进一步表明LbMYB103可能与植物的育性有关。该结果为进一步开展枸杞遗传转化,深入研究LbMYB103基因在枸杞花药发育过程中可能发挥的调控功能奠定了基础。     

EB病毒LMP1-CTAR3对NP69细胞增殖和蛋白质表达的影响

为了探讨EB病毒潜伏性膜蛋白 1(LMP1)第三个功能活性区域(CTAR₃)在鼻咽上皮细胞NP69中的转化作用机制,采用逆病毒感染的方法,将浓缩的逆病毒RV-LMP1和RV-LMP1^Δ232～351分别感染鼻咽上皮细胞NP69,建立NP69-LMP1与NP69-LMP1^Δ232～351稳定表达细胞系．通过绘制生长曲线、平皿克隆形成试验和软琼脂集落形成试验比较野生型和突变型LMP1对NP69细胞增殖的影响,运用蛋白质组学方法鉴定NP69-LMP1与NP69-LMP1^Δ232～351细胞间的差异表达蛋白,选用实时荧光定量RT-PCR与Western blot对其中部分蛋白质点差异表达进行验证．结果发现：a．突变型LMP1^Δ232～351促NP69细胞增殖的能力较野生型LMP1明显降低(n=3,P < 0.05);b．鉴定了LMP1-CTAR₃在NP69细胞中参与调节的16个蛋白质(表达上调的蛋白质8个,下调的8个)．c．实时荧光定量RT-PCR和Western blot证实了部分上述蛋白质的差异表达．以上结果说明,LMP1-CTAR₃是其发挥促细胞增殖的重要活性部位,可能通过参与调节G蛋白和异柠檬酸脱氢酶等蛋白质的表达而起作用．     

小麦TaWRKY47基因的克隆与表达分析

为了探究小麦WRKY基因的功能,该研究采用RT PCR方法,在小麦叶片组织中克隆WRKY基因,并对其进行生物信息学和不同逆境胁迫下的表达分析。结果表明：(1)成功克隆得到1个小麦WRKY基因,命名为TaWRKY47。(2)TaWRKY47基因开放阅读框长度为900 bp,编码299个氨基酸,含有一个WRKY保守结构域和一个C₂HC锌指结构域,属于WRKY基因家族的第Ⅲ类成员。(3)亚细胞定位分析结果显示,TaWRKY47蛋白定位于细胞核。(4)荧光定量PCR结果表明,TaWRKY47基因在小麦根、茎、叶、雄蕊和雌蕊中均有表达,其中在雌蕊中表达量最高,且受低温、干旱、盐、ABA和H₂O₂等胁迫表达增强,推测TaWRKY47基因参与了小麦的逆境胁迫过程。该研究结果为进一步研究TaWRKY47基因功能与抗逆机制奠定了理论基础。     

细菌荧光酶报告基因载体构建及在Hela细胞中的表达*

采用ＰＣＲ点突变技术,构建含有细菌荧光酶融合ｌｕｘＡＢ报告基因的重组哺乳动物载体ｐｃＤＮＡ３－ｌｕｘＡＢ,脂质体ｌｉｐｏｆｅｃｔｉｎ转染和Ｇ４１８抗性筛选稳定转染的Ｈｅｌａ细胞。ＰＣＲ和酶切电泳证明构建的正确性;经稳定转染ｐｃＤＮＡ_３－ｌｕｘＡＢ的Ｈｅｌａ细胞,用发光仪可测得较强的发光强度（最大值为４．１２ｍＶ／４０μｇ细胞总蛋白粗提物）。而亲本及ｐｃＤＮＡ_３稳定转染的Ｈｅｌａ细胞则在本底值范围,ｐｃＤＮＡ_３<     

O-GlcNAc糖基转移酶(OGT)基因表达的抑制对 tau蛋白磷酸化作用的影响

研究HEK293T细胞中O?鄄GlcNAc糖基转移酶(OGT)基因表达抑制对与AD密切相关的tau蛋白磷酸化与糖基化修饰的影响．以OGT基因为靶点设计三段siRNA(OGT-siRNA_1～3),用脂质体的方法转染OGT-siRNA_1～3进入HEK293T细胞,通过RT-PCR检测OGT-siRNA_1～3对OGT mRNA的抑制．将pEGFP/OGT与OGT-siRNA_1～3共转染HEK293T细胞,24 h后在倒置荧光显微镜下观察各组GFP/OGT的表达,根据GFP/OGT的表达量来评价OGT-siRNA_1～3对OGT基因表达的抑制率．将筛选出的具有最佳沉默效果的OGT-siRNA与质粒pCI/ tau₄₄₁共转染HEK293T细胞,48 h后Western blot检测tau蛋白糖基化与磷酸化修饰的变化．OGT-siRNA₃在100 nmol/L的终浓度时对OGT基因的抑制效率最高．与Mock组相比,对OGT基因mRNA及蛋白质水平的抑制率分别可达80.0%与51.3%左右．OGT基因表达的下调使tau蛋白糖基化水平下降,而磷酸化水平增高,证实tau蛋白的糖基化负调节其磷酸化,葡萄糖摄入减少或代谢降低可能在AD的发生发展中起关键作用．     

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。