构建高产谷胱甘肽啤酒酵母基因工程菌提高啤酒抗老化能力的研究

根据同源重组的原理,将来源于啤酒酵母工业菌株G03的γ-谷氨酰半胱氨酸合成酶基因(GSH1)和筛选标记Kan取代质粒pRJ-5中18S rDNA内部约340bp的DNA片段,构建重组质粒pRKG。以pRKG为模版,PCR得到以18S rDNA为整合位点包含GSH1和Kan的基因片段18S rDNA::(Kan-GSH1)。用此片段转化啤酒酵母工业菌株G03,通过G418抗性筛选得到啤酒酵母工程菌。实验室小试表明,工程菌的谷胱甘肽含量比受体菌株提高16.6%,啤酒的抗老化能力得到了显著提高,而常规指标没有发生显著变化。连续传代5次后胞内GSH含量基本不变遗传稳定性良好。由于表达γ-谷氨酰半胱氨酸合成酶的基因来源于受体菌株自身,是通过自克隆技术改造工业啤酒酵母的一次有益的尝试。     

低乙醇脱氢酶Ⅱ活性的抗老化啤酒酵母工程菌的构建

采用自克隆技术,破坏啤酒酵母工业菌株YSF31的ADH2基因,在ADH2基因位点插入来源于YSF31的编码γ-谷氨酰半胱氨酸合成酶的GSH1基因和铜抗性筛选标记CUP1基因.通过铜抗性筛选转化子,经PCR和乙醇脱氢酶Ⅱ(ADH Ⅱ)活性测定验证,获得了1株啤酒酵母工程菌.10°P麦芽汁发酵实验显示,自克隆菌株的乙醇脱氢酶Ⅱ活性是受体菌的65%,谷胱甘肽含量比受体菌YSF31的高34%.其他发酵指标并没有发生明显改变.由于DNA操作过程中没有外源基因介入,因此啤酒酵母工程菌为生物安全的自克隆菌株,具有重要的应用价值.     

低双乙酰抗老化啤酒酵母工程菌的构建

用来源于啤酒酵母的γ-谷氨酰半胱氨酸合成酶基因（GSH1）和铜抗性基因（CUP1）取代质粒pLZ-2中α-乙酰乳酸合成酶基因（ILV2）内部约2.3kb的DNA片段,构建成重组质粒pICG。限制酶酶切质粒pICG后获得在基因GSH1和CUP1两端含有ILV2序列的6.0kb的DNA片段。用此片段转化啤酒酵母YSF31,得到铜抗性高的转化子。并通过PCR和α-乙酰乳酸合成酶（AHAS）活性测定筛选到酵母工程菌。小试实验结果表明酵母工程菌谷胱甘肽含量比受体高34％,而双乙酰含量是受体的75％。其他发酵指标并没有发生改变。中试实验表明酵母工程菌发酵周期缩短3d,而且成品啤酒的保鲜时间延长50％。由于DNA操作过程中没有外源基因介入,因此啤酒酵母工程菌为生物安全的自克隆菌株,具有重要的实际应用价值。     

敲除啤酒酵母 fks1 基因对啤酒酵母自溶性能的影响

以啤酒酵母G-03为模板,扩增得到铜抗性基因(cup 1)和β-葡聚糖合成酶基因(fks 1)。将fks 1连接pMD-18T Vector得到重组质粒pTK,重组质粒pTK和cup1经Bgl Ⅱ、Sal Ⅰ酶切后连接得到重组质粒pKC。Bam HI酶切重组质粒pKC得到以fks 1为整合位点包含cup1的基因片段fks 1::cup1。用此片段转化啤酒酵母工业菌株G-03,通过硫酸铜抗性筛选得到一株啤酒酵母工程菌G-03/C。工程菌连续传代10次后依然能在筛选平板上生长,遗传稳定性良好。主酵结束G-03/C和G-03的辛酸和癸酸含量基本相同。25℃诱导自溶20 d,G-03/C的辛酸、癸酸分别下降57.3%、81.8%,自溶性能减弱。G-03/C的死亡率、双乙酰、浊度及TBA均较原菌有所下降。G-03/C与G-03酿制成品啤酒的常规指标没有较大差别,品评结果表明,G-03/C风味更优。     

木酮糖激酶基因整合表达载体构建及在酿酒酵母中的过表达

【目的】以载体p406ADH1为构建骨架,构建一个酿酒酵母(Saccharomyces cerevisiae)工业菌株的整合表达载体。【方法】通过酶切连接的方式,将4个元件片段:作为筛选标记的G418抗性基因KanR,用于基因表达的ADH1终止子片段,酿酒酵母W5自身木酮糖激酶基因,18S rDNA介导的同源整合区,插入到骨架质粒p406ADH1中,得到多拷贝整合表达载体pCXS-RKTr。将该载体线性转化酿酒酵母后,对转化子中木酮糖激酶酶活进行测定,检测其表达情况。【结果】重组质粒在酿酒酵母体内实现了木酮糖激酶的高水平稳定表达,其酶活力是初始菌株的2.87倍。【结论】本实验构建了一个酿酒酵母工业菌株整合表达载体,并用此载体过表达了其自身的木酮糖激酶基因。该重组质粒载体的构建可以有效解决酿酒酵母中自身木酮糖激酶酶活较低的情况,这为利用木糖高产乙醇酿酒酵母基因工程菌株的构建和其它酵母重组质粒载体的构建奠定基础。     

一种适用于产朊假丝酵母的基因敲除系统及其在gsh1基因敲除中的应用

报道一种适用于产朊假丝酵母Candida utilis的基因敲除系统,利用该敲除系统获得gsh1基因敲除杂合突变株。根据不同种属酵母菌γ-谷氨酰半胱氨酸合成酶(γ-GCS)蛋白质的保守序列,克隆C.utilis SZU 07-01的gsh1基因;以商品化质粒pPICZalpha A为基础,构建gsh1基因的敲除载体pPICZalpha A-kan 3,其中,kan基因的启动子TEF被替换为来自于C.utilis SZU 07-01的GAP启动子(pGAP:kan)。质粒电转化C.utilis,获得gsh1基因敲除杂合突变株C.utilis GSH-6。结合发酵培养得到的数据进行分析,突变株的γ-GCS酶活比出发菌株降低17.5%,GSH合成量降低61%,细胞干重降低18.5%。所构建敲除组件pGAP:kan的成功应用为从分子水平研究C.utilis中谷胱甘肽(GSH)的生理功能提供了一种新借鉴。     

黑曲霉木聚糖酶在工业酒精酵母中的分泌表达

用重叠延伸PCR方法从黑曲霉 (Aspergillusniger)UV 11的基因组DNA中克隆出木聚糖酶的cDNA基因 ,构建了由酵母乙醇脱氢酶 (ADH1)启动子和终止子引导表达、木聚糖酶自身信号肽引导分泌、rDNA序列介导的酵母整合型分泌表达质粒pAX2。用pAX2与酵母YEp型G4 18抗性质粒共转化野生型工业酒精酵母S .cerevisiae 2 346 ,获得了整合型分泌表达木聚糖酶的酵母重组菌株XY2。发酵分析表明该工程菌能够明显提高酒精生产率     

鸡γ-干扰素基因重组质粒在工程菌株中的稳定性研究

本文通过传代的方法研究了鸡γ-干扰素基因的重组质粒pET-ChIFN-γ在工程菌CH1中的遗传稳定性.结果表明重纽质粒在没有筛选压力下存在质粒不稳定性,100代时质粒稳定性只有69%.适当的筛选压力可以显著降低质粒的遗传不稳定性,100代时稳定性提高到95%.各代重组菌株在生长性能、质粒酶切图谱、蛋白表达等方面均保持一致,未见明显差异.生物活性检测原代和100代均达到1.0×105 IU/mL以上.以上结果表明重组质粒pET-ChIFN-γ在工程菌株CH1中具有良好的遗传稳定性,其质粒遗传不稳定性可由适当的筛选压力来控制.     

红霉素对支气管上皮细胞谷氨酰半胱氨酸合成酶（γ-GCS）的影响

目的：探讨红霉素对支气管上皮细胞16-HBE谷氨酰半胱氨酸合成酶（γ-GCS）和谷胱甘肽（GSH）的影响。方法：应用红霉素5μg／ml分别孵育细胞16-HBE细胞2h,8h,16h,24h。分别应用显色法,Westemblot和荧光定量PCR方法检测细胞内GSH,γ-GCS重链蛋白和mRNA的表达。结果：经红霉素孵育后,16、24h后,细胞内GSH、γ-GCS重链蛋白和mRNA的表达较对照组增加。结论：红霉素对支气管上皮细胞谷氨酰半胱氨酸合成酶（γ-GCS）和GSH的合成有上调作用。     

枯草芽孢杆菌α-乙酰乳酸脱羧酶基因的克隆及表达*

利用鸟枪法构建了供体菌的基因组文库,通过VP反应显色法进行筛选,从约7000个转化子中选出携带ALDC基因的重组质粒（pBG4～pBG5）．绘制了pBG4插入片段的限制酶图谱．Southern杂交证明该外源片段来源于供体菌。酶活性测定结果表明ALDC基因在大肠杆菌中获得了表达,为构建带有ALDC的啤酒酵母工程菌奠定了基础．     

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.     

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[Panisea(Lindl.)Steud.]中国分布新记录种——林芝曲唇兰(P.panchaseensis Subedi)。此二新记录种均生长在海拔约2000 m的常绿阔叶林中的树干或岩壁上。此外,还提供了二新记录种的形态特征描述和彩色图片等信息,并附有国产曲唇兰属的分种检索表。该研究结果扩充了我国兰科植物的记录,为我国兰科植物多样性和保护研究提供了新资料。     

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,而白及的球茎大小与红光的占比呈正相关;在红蓝组光合中增加25%的绿光(2R1B1G),可显著提高白及叶片的叶绿素含量和净光合速率,促进组培苗根系和叶片的生长。综上结果表明,2R1B1G处理下的白及组培苗株高、茎粗、叶绿素含量、净光合速率和根系发育均表现出最佳优势,2R1B1G处理的LED光谱可推荐作为白及组培育苗的光质配方。     

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。