沙漠油料作物好好芭的物候学研究

好好芭（jojoba）是当今世界具有重要经济价值的特殊油料作物，栽培仅有20多年的历史．由于好好芭物候学研究的欠缺，在我国大面积引种好好芭存在许多尚需解决的问题．1997—2001年间，在北京和河南南阳地区试种好好芭，在北京地区利用温室种植好好芭获得成功，2000年开始座果．本研究调查了这两地水质、土壤、气候等物候条件，比较了两地的差异．结果表明：温度是好好芭引种的决定因素，并初步明确了好好芭生长的气候条件，为今后引种奠定了良好基础．     

特种油料作物"沙漠克星"——好好芭生物学及其研究进展概述

对近年崛起的被誉为"沙漠克星"的特种油料作物好好芭(Simmondsia chinensis (Link) Schneider)的生物学特性、引种栽培、微繁殖技术及抗寒驯化研究进行了系统介绍;综述了好好芭油合成相关基因、抗盐、抗旱等资源基因的最新研究进展,并对好好芭的研究前景进行了展望.     

好好芭叶片RNA提取方法和cDNA干旱消减文库的构建

介绍了从好好芭成熟叶片中提取总RNA的方法;用含有SDS的裂解液裂解细胞,用DNase除去可能出现的DNA干扰,再经植物提取试剂盒纯化后,获得了高质量的完整RNA,可用于基因编码序列的克隆和基因表达的mRNA分析等精细研究,并进一步构建了好好芭干旱消减文库.     

"好好芭"种子实生苗茎节无性系建立及其遗传差异

采用“好好芭”种子萌发的无菌苗茎节作为外植体,利用体外微繁殖技术,通过茎节培养获得大量体细胞无性系克隆,研究了植物生长调节物质脱落酸、矮壮素对试管苗生长繁殖的影响,随机选择一些克隆进行表型的遗传差异分析,结果表明克隆间的遗传差异是显著的,因此,利用种子实生苗的遗传变异广泛性进行种子实生苗茎节克隆的筛选和培育新品种,是完全可行的。     

小麦果聚糖合成酶基因6-SFT克隆和功能验证

许多研究表明植物中果聚糖累积有助于提高植物耐逆境胁迫能力,但对小麦中果聚糖合成相关基因及其与抗逆性关系还缺乏研究。本研究拟从普通六倍体小麦中克隆出果聚糖合成酶基因6-SFT并进行生物学特性及功能研究,以期解析小麦通过累积果聚糖途径提高自身抗逆境胁迫能力的分子机理,为转6-SFT基因提高小麦抗逆境胁迫能力提供理论依据。利用touch-down PCR技术从小麦品种杨麦6（Triticum aestivum cv. Yangmai 6）基因组及cDNA中分离出编码6-SFT基因的全长序列,运用生物学信息技术对其编码蛋白的相关理化及生物学功能进行了预测,同时利用农杆菌介导法将所克隆的小麦6-SFT基因转入了烟草,对转基因烟草植株进行了抗旱、抗盐和抗低温鉴定。结果表明,小麦中6-SFT基因的gDNA和cDNA长度分别为3134bp和1851bp,序列结构分析表明该基因含有4个外显子、3个内含子,编码产物为约68kD的蛋白质,其等电点（pI）约为5.25,具有液泡定位信号;转小麦6-SFT基因烟草植株表现出较强的抗旱、抗盐和抗低温能力。本研究为利用基因工程技术改良小麦抗逆性提供重要理论依据。     

拟南芥逆境诱导型启动子rd29A的克隆及活性检测

以拟南芥基因组DNA为模板,通过特异PCR扩增,克隆逆境诱导表达启动子rd29A。序列分析表明,该启动子与NCBI上已报道rd29A的启动子有9964%的同源性,其序列含有干旱诱导表达元件DRE和ABA作用元件ABRE等顺式作用元件。用rd29A启动子替换pBI121载体上的35S启动子构建新的载体pBrd-GUS,并以农杆菌介导法将其转入烟草。转基因烟草的GUS组织化学染色及PCR分析结果表明,在低温、干旱及高盐等胁迫诱导下,rd29A启动子可增强GUS基因表达,因此,rd29A启动子可应用于植物抗逆基因工程研究。     

敲除和过表达miRNA446以研究其靶基因APP1的表达模式

MicroRNA是一类小的非编码RNA,参与植物生长发育和胁迫应答等多种生理过程。在本课题组前期研究中,通过基因芯片数据发现了一系列在胁迫应答中表达量有变化的microRNA。其中以miRNA446的变化尤为显著。为进一步探求miRNA446在胁迫应答中的变化与功能,本实验通过在水稻中分别敲除和过表达miR446,检测干旱和冷胁迫下,miR446及其靶基因APP1的表达模式。结果表明,过表达株系中,miR446的表达量与野生型的持续升高不同,是先降后升的,而APP1基因由于受到高水平miR446的剪切作用而一直降低。敲除株系中,miR446的表达量被完全抑制,在整个胁迫过程中均保持接近于零的水平,而APP1在胁迫中也仅有微弱的先降后升趋势。     

水稻Rho家族OsRac2启动子的转基因功能鉴定

采用PCR扩增的方法,构建了OsRac2基因转录起始位点上游1.7 kb的启动子5′缺失植物表达载体,转化烟草并筛选阳性转基因植株.以多种激素处理转基因烟草,通过组织化学分析和GUS荧光活性的检测,证实Os-Rac2启动子上存在着一些激素应答元件,该基因的表达可能受茉莉酸的正调控,脱落酸等激素的负调控.     

冷诱导基因的转录因子CBF1转化油菜和烟草及抗寒性鉴定

为研究冷诱导基因的转录因子CBF1(C-repeat binding factor)基因对植物抗寒的作用,利用PCR技术从拟南芥菜(Arabidopsis thaliana)中扩增并克隆了该转录因子,并将其与CaMV 35S启动子融合构建成植物表达载体pBPCBF1.以农杆菌介导的叶盘法,分别转化了油菜和烟草.经PCR程序的DNA分析和Southern杂交对具有卡那霉素抗性的再生植株进行了鉴定,表明CBF1基因已整合进烟草和油菜基因组中.以电解质渗漏法分别检测了转化的油菜和烟草的抗寒性,结果显示转基因油菜的抗寒性较未转基因油菜有明显提高,转基因烟草抗寒性也有一定的提高.因此利用对转录因子的调控来提高植物的抗寒性可能有很好的应用前景.     

新疆小拟南芥ApCBF1基因的克隆及其过量表达转基因的研究

利用同源克隆法,克隆了小拟南芥的CBF1基因的cDNA（FJ491244）,比对分析结果表明,ApCBF1的cDNA序列的A252,在拟南芥的AtCBF1的cDNA是T252,但翻译的氨基酸均是Ala,表明ApCBF1和AtCBF1翻译的氨基酸序列完全一致。构建了过量表达载体：p35S：ApCBF1,通过花滴法转入小拟南芥中。RT-PCR检测结果表明,转基因T0代AtCBF1基因过量表达。     

转蔗糖:蔗糖-1-果糖基转移酶基因提高烟草的耐旱性

蔗糖: 蔗糖-1-果糖基转移酶（sucrose: sucrose 1-fructosyltransferase, 1-SST）以蔗糖为底物催化生成蔗果三糖等低聚合度的果聚糖.将从莴苣中克隆的1-SST基因重组到pCAMBIA1300-als中,构建了在CaMV 35S启动子调控下的植物表达载体,利用农杆菌介导的叶盘转化法将1-SST基因导入烟草中,PCR和Southern杂交检测表明获得了转基因植株,RT-PCR结果表明该基因在烟草中正常表达. 对T₀代转基因烟草进行的耐旱性分析结果表明,干旱胁迫6d的转基因植株丙二醛含量和电解质渗漏率显著低于未转基因对照,叶片相对含水量下降速度也明显比对照慢. 对转基因植株叶片糖分分析表明,转基因烟草植株积累果聚糖,并在干旱胁迫后含量明显增加,而未转基因对照植株不积累果聚糖. 在14%PEG溶液中未转基因烟草种子的萌发率仅为转基因烟草种子的一半;在附加200mmol/L甘露醇的培养基中未转基因烟草种子根的生长明显受到抑制,而转基因烟草根的生长发育正常. 以上研究结果表明,转1-SST基因烟草植株耐旱性的提高可能与该基因的表达有关.     

项目信息门户(PIP)在建设工程 项目管理中的应用分析

从为工程项目组织建立统一信息共享与沟通平台的必要性出发,在明确项目信息门户(PIP)概念和使用意义的基础上,对其功能和实施要素加以分析;重点探讨了项目信息门户与项目管理信息系统的区别与联系,建立了项目信息门户的功能结构,并对项目信息门户在大型建设工程项目中的实施工作进行了分析.     

Subcellular localization and functional analyses of structural domains of COP1 in transgenic tobacco

Plants have evolved an extremely exquisite light signal regulatory network to adapt to the changing ambient light conditions, in which COP1 plays a critical role of the light signal transduction. Based on the cloned pea COP1 cDNA sequence and its protein structure, four individual gene fragments encoding different structural domains of the COP1 were designed to fuse to the GFP gene. The plant expression vectors containing these fusion genes as well as the COP1GFP fusion gene were constructed and used to transform tobacco by Agribacterium as confirmed by Southern analyses. Antibodies were raised against the recombinant GFP-COP1 overproduced in Escherichia coli. Immunoblotting results demonstrated that all of the fusion genes were constitutively expressed in transgenic tobacco plants. We systematically investigated the different subcellular localization of these fusion proteins and the resulting phenotypic characteristics of these transgenic plants under light and dark conditions. Our data show that (1) the molecular mass of the tobacco endogenous COP1 protein is 76 kD. It is constitutively expressed in all of the tested tissues and the total cellular content of COP1 protein is not noticeably affected by light conditions. (2) The nuclear localization signal of COP1 plays a critical role in regulation of its nuclear-cytoplasmic partitioning. The subcellular localization of the COP1 protein containing nuclear localization signal is regulated by light in the epidermal cells of leaves, but, it is located in nucleus constitutively in root cells. (3) The coiled-coil domain is very critical to the function of COP1 protein, while the zinc binding RING finger domain only plays a supportive role. (4) The WD-40 repeats domain is essential to the COP1 function, but this domain alone does not affect photomorphogenesis. (5) Overexpression of COP1 protein not only inhibits the photomorphogenesis of the stems and leaves of the transgenic tobacco, but also results in the generation of short and clustered roots. In contrast, overexpression of COP1 protein without WD-40 repeats domain promotes the photomorphogenesis process in the stems and leaves and lead to root elongation and lack of lateral roots. The COP1-COP1 interaction happens not only in the nucleus, but also in cytoplasm.     

IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice

Studies in invertebrates have led to the identification of a number of genes that regulate lifespan, some of which encode components of the insulin or insulin-like signalling pathways. Examples include the related tyrosine kinase receptors InR (Drosophila melanogaster) and DAF-2 (Caenorhabditis elegans) that are homologues of the mammalian insulin-like growth factor type 1 receptor (IGF-1R). To investigate whether IGF-1R also controls longevity in mammals, we inactivated the IGF-1R gene in mice (Igf1r). Here, using heterozygous knockout mice because null mutants are not viable, we report that Igf1r(+/-) mice live on average 26% longer than their wild-type littermates (P < 0.02). Female Igf1r(+/-) mice live 33% longer than wild-type females (P < 0.001), whereas the equivalent male mice show an increase in lifespan of 16%, which is not statistically significant. Long-lived Igf1r(+/-) mice do not develop dwarfism, their energy metabolism is normal, and their nutrient uptake, physical activity, fertility and reproduction are unaffected. The Igf1r(+/-) mice display greater resistance to oxidative stress, a known determinant of ageing. These results indicate that the IGF-1 receptor may be a central regulator of mammalian lifespan.     

rAAV1载体介导外源性VEGF-165和Angiopoietin-1基因治疗大鼠脑缺血的疗效评价及其机制研究

为研究经脑室途径联合应用血管内皮生长因子（VEGF）和血管生成素（Angiopoietin-1,Ang-1）,治疗大鼠急性脑梗塞的效果,并探讨治疗的机制。采用脑立体定向输注的方法,将rAAV1-VEGF治疗载体或者rAAV1-VEGF和rAAV1-Ang-1的混合治疗载体,通过侧脑室转染途径对大鼠大脑中动脉阻塞缺血再灌注模型进行基因治疗。观测VEGF和Ang-1蛋白表达、血脑屏障通透性、脑微血管密度等指标,并对大鼠进行神经功能行为评分等。结果显示,联合应用VEGF和Ang-1治疗急性脑梗塞可降低血脑屏障通透性,减轻脑水肿。增加缺血灶周围脑区的微血管密度,改善大鼠的神经功能。由此得出结论：联合应用VEGF和Ang-1基因治疗大鼠急性脑缺血。可保护脑细胞,促进新生血管生成,减轻脑水肿。改善大鼠的神经功能。     

拟南芥 AtTR1 在盐胁迫应答中的功能初探

本研究主要探索油菜中E3泛素连接酶BnTR1在拟南芥中的同源基因AtTR1(At3g47550)的功能.通过体外泛素化实验证明AtTR1具有E3连接酶活性.基因表达分析显示该基因受200mmol/L NaCl显著诱导,说明该基因可能在响应盐胁迫中发挥一定的功能.为了更深入的探究该基因在植物耐盐中的作用,构建了植物表达载体pZH01-AtTR1转化突变体.在含有潮霉素的培养基上筛选阳性苗,并利用荧光定量PCR检测表明AtTR1基因已经成功转入突变体中.     

Transgenic tobacco plants expressing synthetic Cry1Ac and Cry1Ie genes are more toxic to cotton bollworm than those containing one gene

Transgenic tobacco plants carrying Cry1Ac, Cry1Ie or both genes were obtained. In the leaves of transgenic plants carrying both genes, the contents of Cry1Ac and Cry1Ie proteins were 0.173% and 0.131% of the total proteins, respectively. Cry1Ac protein content was 0.182% and Cry1Ie protein con- tent was 0.124% of the total proteins in the leaves of transgenic plants containing only one Bt gene. Fresh leaves of transgenic tobacco and wild-type plants were used for the insect bioassay against wild-type and Cry1Ac-resistant cotton bollworm (Helicoverpa armigera). The bioassay results showed that transgenic plants carrying both genes were significantly more toxic to wild-type and Cry1Ac-resistant cotton bollworm than those carrying Cry1Ac or Cry1Ie alone. This study indicates that the higher toxicity of transgenic tobacco plants carrying both genes is caused by the cooperative function of both Bt proteins, thus providing a potential way to delay the development of insect resis- tance to transgenic crops.     

SRO1 regulates heavy metal mercury stress response in Arabidopsis thaliana

Heavy metals in the environment are harmful limiting factors for the normal growth and development of plants. Here, we isolated and identified an Arabidopsis thaliana T-DNA insertion mutant, named srol-1, which showed a hyper-sensitive response to HgCl2. The SRO1 protein contains a WWE domain that mediates proteinprotein interactions. Under HgCl2 treatment, when compared with the wild-type plants, the growth of srol-1 was repressed dramatically and the number of true leaves was reduced and etiolated. The electrolyte leakage rates showed that cell membrane integrity in srol-1 was damaged more severely than in the wild type. DAB （3,5-diaminobenzidine） staining and confocal microscopy showed that Hg2＋ stress induced more hydrogen peroxide accumulation in srol-1 than in the wild type. The qRT-PCR results indicated that the expression of some abiotic stress-induced genes, such as L-ascorbate peroxidase （APX1）, was reduced under oxidative or Hg2＋ stress. Transgenic plants containing a GFP：：SRO1 fusion protein showed that SRO1 was localized in the nucleus of the cells. SRO1 was shown to be expressed in various tissues, and was most highly expressed in the vigorous tissues. Our results suggest thatSRO1 may play an important role in the stress response of A. thaliana to heavy metals.     

Molecular dynamics simulations exploring drug resistance in HIV-1 proteases

Although HIV-1 subtype B still dominates the epidemic AIDS in developed countries, an increasing number of people in developing countries are suffering from an epidemic of non-subtype B viruses. What is worse, the efficacy of the combinational use of an-tiretroviral drugs is gradually compromised by the rapid development of drug resistance. To gain an insight into drug resistance, 10-ns MD simulations were simultaneously conducted on the complexes of the TL-3 inhibitor with 4 different proteases (B_wt, B_mut, F_wt and F_mut), among which the complex of the B_wt protease with the TL-3 inhibitor was treated as the control group. Detailed analyses of MD data indicated that the drug resistance of B_mut against TL-3 mainly derived from loss of an important hydrogen bond and that of F_wt was caused by the decrease of hydrophobic interactions in S1/S1’ pocket, while both of the two reasons mentioned above were the cause of the F_mut protease’s resistance. These results are in good agreement with the previous experiments, revealing a possible mechanism of drug resistance for the aforementioned protease subtypes against the TL-3 inhibitor. Additionally, another indication was obtained that the mutations of M36I, V82A and L90M may induce structural transforms so as to alter the inhibitor’s binding mode.