玉米Ubi-1启动子驱动挪威鼠Hspa4基因植物表达载体的构建

根据Genbank中挪威鼠（Rattus norvegicus）Hspa4基因序列(登录号：NM 153629)和玉米Ubi-1启动子序列(登录号：DQ141598 )设计引物,采用RT-PCR和PCR技术分别进行克隆。测序及同源性分析结果表明：Hspa4基因片段含有2530bp,编码840个氨基酸;获得的启动子Ubi-1序列大小为1992bp;克隆的序列与相应原序列同源性分别为99.96％和99.55%。以植物表达载体pCAMBIA1301质粒为基础,成功构建了植物表达载体pCAMBIA1301-Ubi-Hspa4,为利用Hspa4基因改良植物抗逆性打下了基础。     

TLR4基因在小尾寒羊乳房炎乳腺组织中的表达

Toll样受体4(Toll-like receptor 4,TLR4)是一种Ⅰ型跨膜蛋白,广泛表达于哺乳动物细胞表面,在抗感染免疫中发挥重要作用.为探讨TLR4基因在大肠杆菌(Escherichia coli引起的小尾寒羊(Ovis aries)乳房炎的乳腺组织中的表达情况,本实验以大肠杆菌(O113型)人工感染小尾寒羊乳房,建立乳房炎病理模型,采用qRT-PCR检测TLR4基因在正常乳腺组织和感染大肠杆菌后乳腺组织中的相对表达量,用蛋白免疫印迹(Western blot,WB)和免疫组织化学(immunohistochemistry,IHC)SP法研究了正常乳腺组织和感染大肠杆菌后乳腺组织中TLR4蛋白的表达量和组织内分布.结果显示,正常乳腺组织和感染大肠杆菌后乳腺组织中均有TLR4的表达,而感染后48 h时TLR4 mRNA和蛋白相对表达量最高,96 h时TLR4mRNA和蛋白相对表达量显著低于48 h(P＜0.05),正常对照组TLR4的相对表达量最低(P＜0.05).免疫组织化学染色发现,正常乳腺组织和感染大肠杆菌后的乳腺组织中乳腺腺泡上皮均有TLR4阳性表达,与对照组相比,感染后阳性表达明显增强(P＜0.05).本研究结果表明,小尾寒羊感染大肠杆菌后乳腺组织中TLR4的表达明显上调,这为进一步研究TLR4在绵羊乳房炎发病过程中的作用机制提供了基础资料.     

猪伪狂犬病病毒GB株蛋白激酶(PK)基因的克隆及序列分析比较

以伪狂犬病病毒GB株细胞培养物为模板，用PCR方法扩增蛋白激酶(PK)基因，对其进行了克隆、序列测定，并与PRV NIA-3株、Ka株和其他α-疱疹病毒进行了同源性比较。结果显示：所扩增的PK基因片段长1396bp，该片段包含一个开放阅读框(ORF)，长1005bp，编码由334个氨基酸组成的蛋白质。与NIA-3株及Ka株的核苷酸同源性为98．4％和97．5％，基因编码区氨基酸序列的同源性为98．2%和96．4％。具有真核细胞丝氨酸／苏氨酸蛋白激酶氨基酸亚结构域特征序列，而且α-疱疹病毒PK基因具有一些特征性的、共有的氨基酸序列。为今后研究PK基因的功能和构建PK缺失株奠定了基础。     

番鸭呼肠孤病毒ZJ99株σC基因的序列分析及其在大肠杆菌中的表达

参考已发表的番鸭呼肠孤病毒(Muscovy duck reovirus, mDRV)的S4序列，自行设计1对扩增σC基因的引物，对mDRV ZJ99株的RNA抽提物进行RT-PCR扩增，成功地获得了843 bp特异性的扩增片段(GenBank: AY619690)。将PCR产物克隆测序，BLASTn比较结果显示, mDRV ZJ99株σC基因与福建MW9710株对应基因的同源性为98％，与法国89026和89330株对应基因的同源性分别为92％和93%； BLASTp比较结果显示, ZJ99株编码的σC蛋白与MW9710、89026和89330株相应蛋白分别具有94％、89％、 89％同一性(identity)和95％、92％、93％相似性(similarity)。进一步将σC基因亚克隆至原核表达载体pET28a， 转化大肠杆菌(Escherichia coli )BL21(DE3)，IPTG诱导阳性重组子，SDS-PAGE电泳检测发现, σC蛋白在大肠杆菌中以包涵体的形式高效表达，目的蛋白的表达量可达到细菌总蛋白的22.9％。     

山羊IL-2基因cDNA的克隆、鉴定及序列分析

根据GenBank发表的山羊（Capra hircus)IL-2基因序列，设计了一对引物。以ConA刺激的外周血淋巴细胞为材料用RT－PCR的方法。从总RNA中扩增出山羊IL－2基因。琼脂糖电泳显示扩增片段约为500bp。分离纯化片段，克隆入表达质粒pBV220，经酶切鉴定，测序结果显示，克隆的山羊IL－2基因与GenBank上发表的序列一致。该序列与绵羊（Ovis aries)和牛(Bas taurus)的IL－2基因序列同源性最大，在96％以上，氨基酸和抗原性比较分析认为，山羊IL－2与绵羊及牛的IL－2在这两方面有较大的同源性。     

新疆褐牛Toll样受体基因(TLR4)外显子Ⅲ2021位点突变与奶牛体细胞评分的相关性研究

Toll-样受体(toll like receptors,TLRs)可以通过识别病原微生物启动天然免疫并激活适应性免疫.以中国荷斯坦奶牛和新疆褐牛为研究对象,对TLR4基因外显子3进行了序列分析,并对发现的多态性位点进行PCR-RFLP分析,共发现TLR4 E3+1656、TLR4 E3+2021和TLR4 E3+2414三个多态位点.对TLR4E3+1656和TLR4 E3+2021位点x2检验表明:2个品种的突变均处于Hardy-Weinberg平衡状态(P>0.05).运用最小二乘法分析表明,TLR4E3+1656位点的各基因型个体间的体细胞评分(SCS)差异不显著(P>0.05);而TLR4 E3+2021位点中AA与AB基因型SCS水平差异显著(P<0.05),AA与BB基因型SCS水平差异极显著(P<0.01),但AB与BB基因型SCS水平差异不显著(P>0.05).研究结果提示,TLR4E3+2021位点的AA基因型可能与抗乳房炎的抗性有关,A等位基因是乳房炎抗性的有利基因.采用RT-PCR技术检测了不同基因型的新疆褐牛个体中TLRs信号通路下游的部分基因mRNA的表达水平,结果显示,NF-κB1、NF-κB2和IL-1β基因mRNA表达水平与TLR4 E3+2021位点AB和BB两种基因型差异显著(P<0.05),TLR4、IL-10和IL-8基因mRNA表达水平与TLR4 E3+2021位点AB和BB两种基因型差异不显著(P>0.05).     

番茄ACO基因的克隆及其RNAi对乙烯释放的抑制

筛选番茄(Lycopersicon esculentum)幼苗cDNA文库获得与番茄抗性相关的克隆E11,设计引物,利用RT-PCR方法从受到病原侵染的番茄叶片中获得长1018bp的候选片段,同源性分析发现该片段与其它作物上发表的ACO基因序列高度同源,同源率83%～99%,推断该基因为番茄ACO基因家族的新成员。在此基础上,用BP克隆的方法构建该基因的RNA干涉(RNAi)载体pD311,对番茄进行遗传转化,获得卡那霉素抗性植株27棵,分子检测证实外源片段成功导入番茄基因组中。对获得的转基因植株的乙烯生成量测定结果表明,RNAi结构的导入大大抑制了内源ACO基因的表达,从而导致乙烯的生成大大降低。     

BmNPV几丁质酶基因的表达及其产物对Bt杀虫剂的增效作用

用PCR方法分别扩增出BmNPV几丁质酶基因编码区全片段和去信号肽片段。将全片段克隆入原核表达载体pET28a，在大肠杆菌(Escherichia coli)BL21(DE3)进行融合表达。将去信号肽片段克隆入pFastBacb转移载体，重组到杆状病毒Bac-to-Bac表达系统，在粉纹夜蛾(Trichoplusia ni)细胞系(Tn-5B1-4)中进行融合表达。SDS-PAGE分析和薄层扫描显示，2片段均得到高效表达，表达产物分别占细胞总蛋白的22％和12％。将以上2种表达产物加入Bt菌液中喂食2龄家蚕(Bombyx mori)，观察并记录不同处理下家蚕的死亡时间及体重。结果表明，2种表达产物对Bt杀虫剂均有明显的增效作用。取食添加以上2种表达产物的家蚕与对照处理相比，LT50分别提前24．5和24．6h，体重增量约是对照组的1／4。     

长穗偃麦草HKT1;4基因片段的克隆及序列分析

根据已报道的其他植物高亲和钾离子转运蛋白（HKT1;4）基因的保守序列设计一对简并性引物,以长穗偃麦草幼根总RNA为模板,采用RT-PCR方法克隆出长穗偃麦草HKT1;4,基因命名为EeHKT1;4。结果表明：该基因片段长度为614 bp,编码204个氨基酸,所得序列与其他植物氨基酸序列同源性均在80%以上,特别是和一粒小麦TmHKT1;4氨基酸同源性高达92%。这为长穗偃麦草HKT1;4全长基因的克隆及其耐盐分子机制的研究奠定基础。     

小金海棠MxYSL1基因的克隆与表达分析

小金海棠属于铁高效基因型。为了研究小金海棠的抗缺铁分子机理,根据苹果的EST库得到一个791 bp的YSL(yellow stripe 1-like protein)基因片段序列,设计特异引物,在苹果铁高效基因型小金海棠（Malus xiaojinenesis）中克隆到此基因片段。3'-RACE得到YSL基因的3'端序列,拼接后得到1 114 bp的3'端基因序列。预测该基因片段编码一个含7个跨膜区的蛋白多肽,与拟南芥AtYSL1同源性达74%,命名为MxYSL1。RT-PCR及Northern blot分析表明,该基因在小金海棠各个检测部位都有表达。在根、茎与成熟叶中,该基因在低铁(EDTA-NaFe,4 μmol/L)时减弱表达,在过量铁(EDTA-NaFe,320 μmol/L)供应时增强表达。MxYSL1基因在新叶中的表达趋势与以上器官中的表达趋势正好相反。     

Mobilization of X‐ray amorphous and crystalline aluminum and iron phosphates by common soil extraction procedures

Soil extraction with 0.1 M NaOH completely dissolves X‐ray amorphous as well as crystalline AlPO₄ and FePO₄. Extraction with acidic NH₄ oxalate completely mobilizes X‐ray amorphous AlPO₄ and FePO₄ in addition to phosphate adsorbed to poorly‐crystallized Al and Fe minerals, but only a minor portion of crystalline AlPO₄ and FePO₄. Extraction with dithionite‐citrate‐bicarbonate mobilizes X‐ray amorphous AlPO₄ and FePO₄ as well as crystalline FePO₄, but only a minor portion of crystalline AlPO₄.     

Temporal and spatial variability of the CH4 dynamics of landfill cover soils

Landfills are regarded as important sources of the atmospheric methane (CH₄), one of the major greenhouse gases. In this study we investigated the CH₄ dynamics of landfill cover soils in a long‐term field experiment. The CH₄ emission rates were low, mostly ranging from —100 to 100 μmol m^—2 h^—1, with prevailing negative values. Higher values of up to 130,000 μmol m^—2 h^—1, obtained concurrently, were due to mice burrows, connecting the reduced soil sections with the aerated ones. Thus, the appearance of spatial dissimilarity was the most important factor influencing temporal variability. Reducing the soil cover from 120 cm to at least 60 cm caused a tendency of increased CH₄ emission. The oxidation rates were also low and differed with low temporal variability from 1.0—11.9 nmol g^—1 h^—1 in 0—10 cm soil depth and 0—5.3 nmol g^—1 h^—1 in 40—50 cm, respectively. Highest rates were obtained at 25—30 % soil water content. A mapping of CH₄ concentrations over the whole landfill showed a large spatial variation with values of 3.1—343 nmol g^—1. Subsequent CH₄ emission rates were between —0.2 and 120,000 mmol m^—2 d^—1 and showed a positive correlation to the CH₄ concentrations (r = 0.993, P < 0.05). Thus, by a large scale mapping of CH₄ concentrations a low‐cost procedure is proposed to identify the hot spots of CH₄ release which should be treated with additional thick and well aerated cover soil materials.     

小鼠生殖细胞特异基因Oct4启动子片段克隆及报告载体构建

本研究通过PCR方法从小鼠(Mus musculus)基因组中扩增出Oct4启动子的CR4区和CR1区,将其克隆到真核报告载体pEGFP-1,构建了携带Dct4启动子片段的小鼠生殖细胞特异性报告载体CR1,4-pEGFP-1.用该载体转染多种细胞,在荧光显微镜下观察绿色荧光蛋白(EGFP)在细胞中的表达情况,同时以半定量RT-PCR检测转染细胞中Oct4及其它生殖细胞特异性基因的转录情况.结果显示,GFP在小鼠胚胎干细胞、小鼠胚胎成纤维细胞(MEF)和C2C12细胞中均不表达,而在P19细胞和睾丸细胞中表达.RT-PCR结果显示,Oct4在P19、睾丸细胞和生殖干细胞中特异性转录.这表明所构建的绿色荧光蛋白报告载体只在生殖细胞和多能生殖干细胞中内表达,该质粒可作为示踪生殖细胞的工具,同时也可以作为生殖干细胞多能性状态的一个监测标记.     

麦季稻秆施用对后续稻季CH_4产生氧化及排放的影响

通过室内培养和田间试验研究了2007年麦季稻秆施用对2008年稻季CH4产生潜力、氧化潜力和排放通量的影响。结果表明,水稻生长前期,稻秆还田处理的CH4氧化潜力仅在水稻移栽后19 d时表现为显著高于稻秆不还田处理,而两处理的CH4产生潜力与排放通量均无显著差异;水稻生长中期,两处理的CH4氧化潜力无显著差异,稻秆还田处理的CH4产生潜力及排放通量均显著高于稻秆不还田处理,其中稻秆还田处理的CH4产生潜力是稻秆不还田处理的2~4倍;水稻生长后期,两处理的CH4产生潜力、氧化潜力和排放通量均无显著差异。CH4排放主要发生在水稻生长中期。水稻全生育期内,稻秆还田处理的CH4排放总量为稻秆不还田处理的1.4倍。     

尿素施用对稻田土壤甲烷产生、氧化及排放的影响

通过田间和培养试验研究了不施尿素(N0kg/hm2)和施用尿素(N300kg/hm2)对水稻生长期稻田土壤CH4产生潜力、氧化潜力和排放通量的影响。结果表明,水稻全生育期内,施用尿素降低土壤CH4产生潜力,并推迟其达到最大值的时间;尿素作为基肥和穗肥施用抑制土壤CH4氧化,而作为分蘖肥施用短暂地促进然后抑制土壤CH4氧化;施用尿素降低稻田CH4排放量。施用尿素通过同时影响CH4产生潜力和氧化潜力来影响稻田CH4排放。     

K+高亲和转运系统吸收动力学特征及其受NH4+影响的研究

为探讨钾的高亲和转运系统是否受到铵离子的影响,采用溶液培养方法研究了水稻、大豆两种作物苗期(16d)的K+高亲和转运系统吸收动力学特征及其受吸收液中NH4+的影响。结果表明,NH4+对K+吸收的Vmax的影响在作物种类间有较大的差异,水稻受影响显著小于大豆。NH4+对供试作物K+吸收的Km值影响均很小,说明NH4+对K+吸收速率的影响主要在于影响了细胞膜上K+载体的数量而非影响了载体吸收位点与K+之间的亲和性。     

Effects of rice‐straw and phosphorus application on production and emission of methane from tropical rice soil

Rice‐straw amendment increased methane production by 3‐fold over that of unamended control. Application of P as single superphosphate at 100 μg (g soil)^–1 inhibited methane (CH₄) production distinctly in flooded alluvial rice soil, in the absence more than in the presence of rice straw. CH₄ emission from rice plants (cv. IR72) from alluvial soil treated with single superphosphate as basal application, in the presence and absence of rice straw, and held under non‐flooded and flooded conditions showed distinct variations. CH₄ emission from non‐flooded soil amended with rice straw was high and almost similar to that of flooded soil without rice‐straw amendment. The cumulative CH₄ efflux was highest (1041 mg pot^–1) in rice‐straw‐amended flooded soil. Appreciable methanogenic reactions in rice‐straw‐amended soils were evident under both flooded and non‐flooded conditions. Rice‐straw application substantially altered the balance between total aerobic and anaerobic microorganisms even in non‐flooded soil. The mitigating effects of single‐superphosphate application or low‐moisture regime on CH₄ production and emission were almost nullified due to enhanced activities of methanogenic archaea in the presence of rice straw.     

茶树控释氮肥的施用效果与合理施用技术研究

采用田间试验方法研究控释氮肥不同施用量和施用次数条件下土壤养分释放动态,以及对茶树养分吸收、叶片叶绿素含量,茶叶产量和品质变化的影响。结果表明,控释氮肥能明显延长氮在土壤中的留存时间,与普通尿素相比,NH4+-N浓度明显提高并能持续较长时间;而NO3--N变化不大,使NO3--N占总无机态氮含量的比例降低。控释氮肥在土壤中的这种动态变化与茶树对氮素的吸收规律基本同步,这不仅明显促进了茶树对氮素的吸收,而且对钾和锌等矿质元素的吸收也有所增强;茶树叶片叶绿素含量提高。与普通尿素相比,控释氮肥可减少30%氮肥用量而不影响茶叶产量和品质;相同的施肥量和施肥次数或施肥次数减少一次,茶叶仍增产6.6%～24.1%,平均12.9%;茶叶品质成分,特别是游离氨基酸含量也有明显提高。     

NO3-和NH4+对水稻和旱稻生长及水分利用的影响

用分根法研究NO3-和NH4 对水稻‘越富’和旱稻‘297’生长和水分利用的影响,结果表明:与水稻相比,旱稻在NO3-中的生长最好。NH4 提高氮素在水稻根中的分配比率,同时也提高了水稻和旱稻根系在NO3-/NH4 处理中NH4 一侧的吸氮速率。旱稻水分利用率高于水稻,且二者的水分利用率都遵循NH4 >NH4NO3≥NO3-的规律。     

秸秆施用对稻田CH4排放、溶解浓度及13C变化特征的影响

CH4 emission and the concentration of dissolved CH4 in soil solution and floodwater in a rice field and their stable carbon isotopic signatures as affected by straw application were investigated in 2009 in a field experiment at Jurong, Jiangsu Province, China. Straw application increased CH4 emission and CH4 concentration in the soil solution and floodwater. A positive seasonal correlation was also observed in the variation between CH4 flux and CH4 concentration in soil solution. The seasonal total CH4 emission (51.6 g CH4 m-2) in Treatment WS (straw applied) was about 168% higher than that in Treatment CK (without straw). The emitted CH4 and CH4 in soil solution were initially relatively enriched, then depleted and finally enriched again in 13C in both treatments, while CH4 in floodwater became isotopically heavier. The carbon isotopic signature of emitted CH4 and CH4 in floodwater averaged around -62‰ and -45‰ for both treatments, respectively, and was not significantly influenced by the application of straw. However, straw application caused the CH4 in soil solution to be significantly depleted in 13C during the middle of the rice season, and the mean δ13C value was lower in WS (-57.4‰) than in CK (-49.9‰). Calculation from the isotopic data showed that straw application increased the fraction of CH4 oxidized, causing no significant difference in the δ13C value of the emitted CH4 between the two treatments.