一株新的豆豉纤溶酶产生菌的研究

从全国各地采集豆豉样品,经富集培养并利用纤维蛋白平板法获得一株形态与现存产纤溶酶微生物差异较大的菌株HS9。通过传统方法、化学方法以及16SrRNA序列分析对HS9进行分类鉴定,属于Pseudomonas aeruqinosa,是未见报道的产豆豉纤溶酶菌株。发酵培养HS9获得粗酶,经20%~70%硫酸铵梯度盐析、SephadexG-75凝胶过滤以及CM-Sepharose Fast Flow阳离子交换层析分离纯化后,得到了电泳纯酶。通过SDS-PAGE了解该酶分子量约为34kD,pH8.0~8.5时酶活性最高,最适作用温度48℃,作用方式为直接水解纤维蛋白,胃蛋白酶抑制剂在工作浓度1μmol/L时能完全抑制其活性,推测该酶为天冬氨酸蛋白酶,是一种新型的豆豉纤溶酶。     

富营养化浅水湖泊蓝藻的种群结构和多样性研究

比较研究不同富营养化浅水湖泊中蓝藻种群结构和多样性,对于认识蓝藻水华的区域分布特征具有重要的生态和环境意义。于2009年7月在富营养化严重的滇池、巢湖和太湖各选取4个采样点,采用16S-23SrRNA基因间隔区（Internaltranscribedspacer,ITS）序列作为生物标记,通过末端限制性片段长度多态性技术（Terminal restriction fragment length polymorphism,T-RFLP）分析蓝藻种群结构和多样性。实验共得到36个不同的末端限制性酶切片段（Terminal restriction fragment,T-RF）,反映3个湖泊中蓝藻种群具有丰富的基因多样性。T-RFLP聚类分析结果表明,在所研究的湖泊中蓝藻种群结构存在空间差异性,其中滇池和巢湖间蓝藻种群结构更为相似。环境因子与蓝藻种群的多元统计分析表明,正磷酸盐（POa-P）和pH与附着细菌样品蓝藻种群结构动态变化具有显著的相关性（P〈0．05）,电导率（Conductivity）和总有机碳（TOC）与浮游细菌样品蓝藻种群结构动态变化具有显著的相关性（P〈0．05）。因此,不同湖泊蓝藻水华的基因型和驱动因子存在差异。     

鸭脂肪型脂肪酸结合蛋白基因（A-FABP）内含子1序列多态性分析

根据鸭A-FABPmRNA序列和鸡A-FABP基因组序列设计1对引物扩增出鸭脂肪型脂肪酸结合蛋白基因(A-FABP)内含子1序列(GenBank登录号:FJ536257),并根据扩增产物设计4对引物,利用PCR-SSCP对鸭A-FABP基因部分序列进行SNP检测,且对不同鸭群体进行群体遗传学分析.结果表明:克隆序列包含鸭A-FABP基因外显子1、2部分序列和完整的内含子1序列,与鸡A-FABP基因内含子1同源性为75.1%;经SSCP检测,发现引物P1扩增片段有3处碱基突变,引物P3扩增片段有6处碱基突变,这些突变分别产生了3种和8种基因型.在P1位点樱桃谷鸭和苏牧麻鸭偏离了Hardy-Weinberg平衡(P0.05),在P3位点3个群体均符合Hardy-Weinberg平衡(P＞0.05).同时,这些基因型在不同鸭群体中分布存在显著或极显著差异(P<0.05或P<0.01).群体遗传学分析结果显示A-FABP基因在不同鸭群体中具有丰富的单核苷酸多态性,可以进一步作为候选基因来分析其与脂肪性状相关性.     

绵羊骨形态发生蛋白受体IB(BMPR-IB)基因部分3'非翻译区的多态性分析

根据绵羊骨形态发生蛋白受体IB(bone morphogenetic protein receptor IB,BMPR-IB)基因部分调控区的mRNA序列设计3对引物,采用PCR-SSCP技术检测BMPR-IB基因部分3'非翻译区序列在绵羊(Ovis aries)高繁殖力品种(小尾寒羊和湖羊)和低繁殖力品种(特克塞尔和中国美利奴绵羊)中的单核苷酸多态性,同时研究该基因对小尾寒羊和湖羊高繁殖力的影响.结果发现,引物P1扩增片段存在多态性,其余2对引物的扩增片段均不存在多态性.对于引物P1,只在湖羊中发现AA和BB两种基因型,其余3个品种均为AA型;测序表明BB型与AA型相比有2个碱基突变(121T→C和195T→C);湖羊AA和BB基因型频率分别为0.575和0.425,湖羊AA和BB基因型之间产羔数的最小二乘均值差异不显著(P>0.05).研究结果初步表明,检测的BMPR-IB基因位点对小尾寒羊和湖羊高繁殖力都没有显著影响.     

绵羊骨形态发生蛋白受体IB（BMPR-IB）基因部分3′非翻译区的多态性分析

根据绵羊骨形态发生蛋白受体IB（bone morphogenetic protein receptor IB,BMPR-IB）基因部分调控区的mRNA序列设计3对引物,采用PCR-SSCP技术检测BMPR-IB基因部分3′非翻译区序列在绵羊（Ovis aries）高繁殖力品种（小尾寒羊和湖羊）和低繁殖力品种（特克塞尔和中国美利奴绵羊）中的单核苷酸多态性,同时研究该基因对小尾寒羊和湖羊高繁殖力的影响。结果发现,引物P1扩增片段存在多态性,其余2对引物的扩增片段均不存在多态性。对于引物P1,只在湖羊中发现AA和BB两种基因型,其余3个品种均为AA型;测序表明BB型与AA型相比有2个碱基突变（121T→C和195T→C）;湖羊AA和BB基因型频率分别为0.575和0.425,湖羊AA和BB基因型之间产羔数的最小二乘均值差异不显著（P〉0.05）。研究结果初步表明,检测的BMPR-IB基因位点对小尾寒羊和湖羊高繁殖力都没有显著影响。     

来源于水牛瘤胃未培养微生物的内切葡聚糖酶的碱性羧基末端结构域的功能鉴定

本碱性羧基末端结构域（basic C-terminal domain,BTD）是瘤胃细菌的碳水化合物活性酶（carbohydrateactive enzymes）中未知功能的一个结构域。BTD都位于酶或蛋白质的羧基最末端,大小为30～80个氨基酸（aa）,含有较多的碱性氨基酸,一般该结构域等电点都大于10。本工作构建了来源于水牛瘤胃未培养微生物的内切葡聚糖酶C5614-1的BTD缺失酶C5614-1RBTD57（缺失C5614-1羧基末端的57个aa）和C5614-1RBTD40（缺失C5614-1羧基末端的40个aa）以及C67-1的BTD缺失酶C67-1ΔBTD（缺失C67-1羧基末端的42个aa）,酶学特性分析发现BTD缺失酶与野生酶对pH和温度的稳定性是相似的,说明BTD结构域对酶的酶学特性方面贡献不大。尽管缺失了C5614-1的羧基末端57个氨基酸后,缺失酶C5614-1RBTD57对温度和pH的稳定性明显降低,但同时缺失酶对Avicel的结合能力也显著下降,说明该缺失酶是因为该酶的碳水化合物结合组件（carbohydrate binding module,CBM）的部分缺失而导致了酶的稳定性的改变。本文还对BTD可能的功能做了预测。     

宽体金线蛭不同工艺提取物的抗凝溶栓活性比较研究

宽体金线蛭（Whitmania pigra）具有抗凝溶栓的药理作用,但其药效受提取工艺的影响。为全面评价并筛选出合适的提取工艺,本试验采用脱脂、水提、酸提和酶解等不同工艺制备了14种提取物,并对提取物的抗凝活性、纤溶活性与体外溶栓活性进行分析。结果表明,脱脂胃蛋白酶酶解提取物的抗凝血酶活性显著高于其他提取物,且具有较强的纤溶活性和体外溶栓活性;仿生酶解提取物具有较强的抗凝血酶活性和体外溶栓活性;脱脂仿生酶解提取物具有较强的纤溶活性和体外溶栓活性;脱脂加热的水提提取物具有较强的纤溶活性和体外溶栓活性。脱脂胃蛋白酶酶解提取物中,分子量小于3 kDa的多肽具有较强的抗凝血酶活性和纤溶活性。此外,三个水提提取物、仿生酶解物、脱脂仿生酶解物和粗酶酶解提取物对纤维蛋白原的α肽链均具有溶解作用。综上所述,宽体金线蛭提取物均具有抗凝溶栓的药理作用,酶解提取物优于水提提取物和酸提提取物,脱脂处理有利于活性物质的提取,其中胃蛋白酶酶解提取物效果最佳,并且脱脂胃蛋白酶酶解物的小分子多肽成分具有最佳的抗凝血酶活性和纤溶活性。本试验结果为进一步开发利用宽体金线蛭的药理作用提供了理论依据。     

辣椒泛素结合酶变体类似蛋白基因克隆及其与氮吸收利用的相关性分析

UEVs蛋白是泛素结合酶E2的变体,可通过与E2形成复合物催化底物泛素化。为探究UEVs蛋白在氮吸收利用中的作用,本研究参考辣椒低氮胁迫相关转录组数据,利用反转录PCR方法从辣椒叶片中克隆了泛素结合酶变体类似蛋白基因,命名为CaUev1D-L,对其进行生物信息和系统进化分析,并对转CaUev1D-L基因烟草进行低氮胁迫后的表型和生理特性分析。结果表明,CaUev1D-L开放阅读框长402 bp,编码133个氨基酸,具有E2结合酶UBC结构域,比辣椒、烟草、番茄和马铃薯等茄科作物的Uev1D蛋白氨基酸序列末端少14个氨基酸,与Uev1D蛋白的UBC结构域存在6个氨基酸的差异,与辣椒等作物的Uev1D蛋白不在同一进化分支。将CaUev1D-L转入烟草,在正常氮素水平下,转基因植株的茎叶干重显著小于野生型植株;在低氮水平下,转基因烟草植株的茎叶干重和根干重均显著大于野生型植株。本研究结果为氮吸收利用分子机制研究和相关基因的发掘利用提供了参考。     

影响原料乳品质的纤溶酶活性的因素分析

为了充分了解原料乳中纤溶酶活性,以便从源头上提高原料乳品质,该文针对中国原料乳的生产现状,分析了奶牛品种、胎次、养殖模式及挤奶时间对原料乳中纤溶酶活性的影响,并对纤溶酶的热稳定性进行了研究。结果表明,养殖模式、奶牛品种及胎次对原料乳中纤溶酶活性影响显著(P0.05),牧场养殖、1、4胎和娟珊牛原料乳中纤溶酶活性显著低于养殖小区、2、3胎及荷斯坦牛乳中的纤溶酶活性;挤奶时间对原料乳中纤溶酶活性影响不显著(P0.05);原料乳中纤溶酶活性随热处理温度的升高而逐渐下降,巴氏杀菌(75℃、15 s)仅可使原料乳中纤溶酶活性下降25%;半胱氨酸对乳中纤溶酶活性具有一定的抑制作用。研究结果对根据纤溶酶活性对原料乳进行品质评价及分级具有一定借鉴作用。     

利用生物信息学方法进行基于表达序列标签的玉 米单核苷酸多态性标记的开发

针对简单重复序列（SSR）标记密度不足、单核苷酸多态性（SNP）标记开发一般只基于2种基因型的序列差异,用以检测其他基因型的材料时多态性不高,不能满足玉米基因精细定位需要的现状,本研究从公共数据库下载来自不同遗传背景的玉米表达序列标签（EST）,结合运用各种生物信息学软件,开发基于EST序列的高多态性SNP标记。通过对2018530条EST序列的比对分析,拼接发掘出遍布全基因组的80363个SNP位点。在SNP位点两侧保守序列上设计PCR引物,开发出12388 个SNP标记（www.sicau.edu.cn/web/yms/snp/snp.html）,包含34721个SNP位点。其中,6008个标记只含单一SNP位点,12762个位点的多态性信息含量（PIC）大于04,具有高度多态性。     

Comparing four mitochondrial genes in earthworms – Implications for identification,phylogenetics, and discovery of cryptic species

Earthworms play a key role in soil ecology as they can reach high densities, are well known as soil engineers, and occupy a central position in soil food-webs. The identification of earthworms, however, is notoriously difficult and morphologically only possible for well-preserved adult specimens. Molecular markers could facilitate earthworm identification and would be a huge advantage in studies where it is important to species-specifically identify juveniles or where badly preserved specimens and remains of earthworms need to be identified. The aim of this study was to compare four mitochondrial genes (12S rRNA, 16S rRNA, COI, and COII) with respect to their value for identifying earthworms, calculating earthworm phylogenies, and discovering cryptic species. Our results indicate that all four genes are suitable for species identification. However, the genetic distances were approximately twice as high for the protein coding genes than for RNA coding ones. High genetic distances and deep genetic lineages, e.g. for Octolasion lacteum, Lumbricus rubellus and Aporrectodea rosea, indicate the possible presence of cryptic species and hamper molecular identification. The Bayesian analysis based on concatenated sequence data resulted in a phylogenetic tree with high posterior probabilities. The con-specific relationship of Aporrectodea spp. and Allolobophora spp. was not confirmed, underlining the ongoing discussion about the revision of these two genera. In conclusion, our findings suggest using 12S and 16S rRNA sequences as molecular markers for species identification whereas the COI gene is better suited to address genetic lineages and to explore possible cryptic species. Taxonomy and the identification of species are essential for most ecological studies. This study provides the needed molecular sequence information to develop molecular tools that can overcome many hurdles in studies on earthworms, their relationship and their ecology.     

Vermipharmaceuticals and active proteins isolated from earthworms

The earthworm is one of the typical saprophagous organisms that has been successfully used to convert organic waste into biomass. Indeed, vermicomposting occurs throughout the world. More recently, research on pharmaceuticals derived from earthworms, known as green biomedicine, has been increasing. As a result, earthworms have become an international medicine, even though their original utilization in traditional medicine has been known for thousands of years. With the development of biomedicine, scientists have rediscovered the medicinal value of earthworms related to many chemical components, including (1) earthworm proteases (lumbrokinase, collagenase, superoxide dismutase, cholinesterase, catalases, glycosidases); (2) metal-binding protein (metallothionein, calmodulin-binding protein); (3) other active proteins including those with proliferative improving activity like lysenin, eiseniapore, antitumor proteins, and glycoprotein; (4) active peptides (gut mobility regulation peptide, antibacterial peptide); (5) earthworm metabolites (carbamidine, lumbrinin, lumbrofobrin, terrestrolumbrolysin); (6) special organic acids (succinic acid, lauric acid, and unsaturated fatty acid) and (7) other components such as purin, vitamin B, tyrosine and Se. In this paper, we mainly describe earthworm fibrinolytic enzymes and antibacterial peptides in particular.     

Isolation of polymorphic microsatellite markers in Aporrectodea icterica (Savigny 1826)

Earthworms play a major role in soil dynamics acting as modifiers of properties and soil characteristics. Although population genetics is a promising approach to get a better understanding of their ecology, the use of molecular tools in earthworm studies is still scarce. Here, we developed and analyzed seven microsatellite loci for Aporrectodea icterica, a common endogeic species of most temperate natural and agricultural soils, with the aim to investigate its dispersal capacity in further researches. Although sequences of a fragment of the mitochondrial gene cytochrome oxidase subunit I (COI) showed the existence of two mitochondrial lineages within the species, with a mean divergence between them of 10%, microsatellite data proved that these two COI lineages are interbreeding and form part of a single species.     

植物乳杆菌C88胞外多糖生物合成基因的克隆及序列比对

乳酸菌胞外多糖能显著改善发酵乳制品及食品的流变学和质构特性。为进一步了解乳酸菌胞外多糖的生物合成途径及调控机制,本研究对参与植物乳杆菌C88胞外多糖生物合成基因簇的部分序列进行了克隆和鉴定。根据GenBank中己报道植物乳杆菌基因序列的保守区域设计特异性引物,扩增出植物乳杆菌C88生物合成蛋白基因（cps4A）序列,并通过染色体步移方法克隆了植物乳杆菌C88参与胞外多糖合成基因簇的部分序列（4．9kb）。利用生物信息学方法预测基因簇中6个阅读框的结构和功能,结果表明该序列与己报道的乳酸杆菌胞外多糖生物合成基因具有高度的同源性（〉96％）;对各阅读框功能预测分析发现,这6个基因主要编码参与胞外多糖合成中的多糖合成蛋白、糖链长度检测蛋白、UDP-葡萄糖-4-异构酶和糖基转移酶。本研究将为利用基因工程方法调控多糖的合成和产量提供理论依据。     

7种植物ALAD基因的生物信息学分析

5-氨基乙酰丙酸脱水酶（8-aminoaevulinicaciddehydratase，ALAD）是生物体所有四吡咯化合物生物合成所必需的酶。目前，GenBank共记载了39种绿色植物的ALAD基因。本文采用生物信息学方法对其中常用的模式植物拟南芥、玉米、小麦、大豆、苜蓿以及葡萄、菠菜等植物的5-氨基乙酰丙酸脱水酶基因的核苷酸及其编码的蛋白氨基酸序列、组成成分、导肽、信号肽、跨膜结构域、疏水性／亲水性、蛋白质二级结构、三级结构及功能域等进行预测和分析，并构建了5-氨基乙酰丙酸脱水酶蛋白家族的系统进化树。结果表明，这几种植物的开放阅读框都在1290bp左右，分子量为47kD左右，等电点（pD值为5．5～7．0之间，ALAD蛋白呈中性至微酸性。含量最丰富的氨基酸为Ala、Leu、Val、Arg、Ser、Gly、Pro和Asp。研究还发现这些植物5-氨基乙酰丙酸脱水酶肽链表现出明显的疏水区和亲水区，不存在信号肽，有叶绿体转运肽；可能存在跨膜结构域。蛋白质二级结构中最主要的结构元件是无规则卷曲和α-螺旋，含有5-氨基乙酰丙酸脱水酶的活性结构域、ALAD-PGBS．aspartate-rich保守结构域、舍夫碱残基结构域和一个镁离子结合位点结构域。核苷酸同源性比对结果显示，拟南芥5-氨基乙酰丙酸脱水酶基因与其它植物的同源性较高；进化分析结果表明这些植物5-氨基乙酰丙酸脱水酶基因被分为六个大类。本工作可为今后深入研究植物5一氨基乙酰丙酸脱水酶的结构特征和功能提供一定的依据。     

The contribution of endogenous cellulase to the cellulose digestion in the gut of earthworm (Pheretima hilgendorfi: Megascolecidae)

Cellulase activity has been detected in the digestive tract of earthworms. However, it has not been well clarified whether the origin of those cellulases are the earthworm themselves or the symbionts. In our study, zymogram analysis suggests that one cellulase (endo-β-1,4-glucanase, EC3.2.1.4) mainly works to digest cellulose in Pheretima hilgendorfi. To identify the cellulase in P. hilgendorfi, we carried out cDNA cloning of the cellulase gene from the digestive tract. A novel cellulase gene was identified from the gut of earthworm. The cDNA encoding cellulase of P. hilgendorfi (phhEG) is 1606 bp with an open reading frame encoding a protein of 449 amino acid residues. The deduced amino acid sequence of P. hilgendorfi cellulase showed higher homology to invertebrate cellulases than bacterium cellulases belonging to the glycosyl hydrolase family (GHF) 9. The phhEG gene was detected in intestinal epithelium cell of midforegut using Northern blot and in situ hybridization. Similarly, specific cellulase activity against carboxymethyl cellulose (CMC) was significantly higher in midforegut tissue. Recombinant phhEG produced by wheat germ cell-free protein synthesis system had a cellulase activity which degrade CMC. In zymogram analysis, the molecular size of cellulase was detected as a single band of 51 kDa from the whole gut contents extracts of P. hilgendorfi, and was very similar to the predicted molecular size of the mature phhEG protein. These results strongly suggested that the earthworm has the capacity to produce the endogenous and functional cellulase around the midforegut, and use this cellulase for their cellulose digestion with the support of intestinal caecum.     

Earthworm functional groups are related to denitrifier activity in riparian soils

Riparian buffers, located in the transition zone between terrestrial and aquatic ecosystems, are a hotspot for nitrogen (N) removal through denitrification. Earthworms are abundant in riparian buffers and may enhance denitrification. This study investigated earthworm demographics of three earthworm functional groups (anecic, epigeic, and endogeic) and denitrifier activity in temporarily flooded and non-flooded riparian soils from April to October 2012 in southern Quebec, Canada. Nine earthworm species, mostly endogeic, were found in the temporarily flooded soil, while only six earthworm species were found in the non-flooded soil. On average, there were 11.7 times more earthworms with 12.4 times greater biomass (P<0.05) found in the temporarily flooded soil than in the non-flooded soil. The denitrification enzyme activity (DEA) was of similar magnitude in temporarily flooded and non-flooded soils, with temporal variation associated with rainfall patterns. Endogeic earthworm biomass was positively correlated (P<0.05) with DEA, while epigeic earthworm biomass was positively correlated (P<0.05) with 16S rRNA gene copies and nosZ gene copies from bacteria, indicating an association between earthworm functional groups and denitrifier activity in riparian soils. Stepwise multiple regressions showed that DEA in riparian soils could be predicted using soil moisture, inorganic N concentration, and earthworm functional groups, suggesting that endogeic and epigeic earthworms contributed to denitrifier activity in riparian soils.     

芒果AP1同源基因的克隆及其生物信息学分析

我们采用RT-PCR方法克隆了2个A州同源基因全长cDNA,分别命名为MAPl—1（GenBankac—cession No．FJ529206）和MAPl—2（GenBankaccession No．FJ529207）。MAPl—1编码247个氨基酸,开放阅读框长度为741bp,蛋白质分子量为28．54kD,等电点为8．31;MAPl—2编码248个氨基酸,开放阅读框长度为744bp,蛋白质分子量为28．78kD,等电点为8．70。同源性分析表明,它们的核苷酸序列与其它木本植物A纠同源基因的一致性为72％～81％。实验分析表明,MAPl—1和MAPl—2第1至第61个氨基酸含有一个MADS盒结构域,第88至第178个为K盒结构域;两个基因均定位于细胞核,且功能位点分布存在着不同,推测这两个基因在花器官发育过程中的功能存在差异。蛋白二级结构预测显示,MAPl—1蛋白有12个α-螺旋,4个8折叠区,14个β-转角;而MAP1—2蛋白有11个α-螺旋,5个B折叠区,15个β-转角;其大多数氨基酸具有亲水性。本研究有助于进一步了解芒果的开花分子机理及成花的生物学发育阶段。     

氯丹和灭蚁灵污染场地土壤对蚯蚓的毒性效应研究

以赤子爱胜蚓（Eisenia fetida）为供试生物,氯丹和灭蚁灵污染场地土壤为供试土壤,以蚯蚓体重及体内蛋白质含量、超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性为指标,进行了不同暴露时间（1、3、7、14 d）下场地土壤中氯丹和灭蚁灵污染胁迫对蚯蚓的毒性效应研究。结果表明,随着暴露时间的延长,蚯蚓体重在氯丹和灭蚁灵作用下受到显著抑制,蚯蚓体内蛋白质、SOD和CAT活性对氯丹和灭蚁灵响应不同,其敏感性大小为CAT〉SOD〉蛋白质;在一定暴露时间内,根据暴露-剂量效应关系,表明氯丹浓度为14.13 mg.kg-1、灭蚁灵浓度为4.14 mg.kg-1可能是使蚯蚓CAT活性达到最大值的临界浓度,同时也是SOD活性受到抑制的临界浓度,超过该临界浓度可能对蚯蚓产生生态毒性效应,这为场地风险评价和修复提供了基础数据。