天麻抗真菌蛋白基因的克隆及其启动子活性

通过构建和筛选天麻(Gastrodia elata Bl.)基因组文库,克隆了一个天麻抗真菌蛋白基因组DNA.该基因组DNA含有一个516碱基组成的编码区,没有内含子结构.其启动子区含有保守的TATA盒及CAAT盒.为研究启动子活性,构建了-1 157 bp启动子区与GUS基因的融合表达载体.并将其用农杆菌(Agrobacterium tumefaciens)介导的遗传转化方法导入烟草(Nicotiana tabacum)中,获得了稳定转化的烟草.利用荧光检测及组织化学染色法对GUS表达进行了分析.结果表明,该启动子能够启动GUS基因在转基因烟草中组织特异性地表达.GUS基因在根中的表达水平最高,茎中次之,叶中只有低水平表达.而且该启动子具有诱导表达活性,可被真菌及水杨酸、茉莉酸强烈诱导表达.     

小桐子低温诱导型启动子JcDnaJ20p的克隆及烟草转化功能鉴定

低温转录组数据显示,DnaJ20是小桐子低温诱导基因。为鉴定该基因启动子的低温诱导活性,基于PCR技术从小桐子叶片基因组DNA中克隆到DnaJ20基因(JcDna20)启动子序列,命名为JcDnaJ20p,利用重组技术构建了JcDnaJ20p启动子驱动GUS标记基因的植物双元表达载体pCambia1381Z-JcDnaJ20p-GUS,并通过农杆菌介导转化烟草进行功能解析。结果表明,克隆的小桐子DnaJ20基因启动子序列长度2023 bp,序列分析显示该启动子中具有真核生物典型的核心启动子区元件TATAbox和CAAT-box,另外,还鉴定到激素如脱落酸、赤霉素响应元件与植物抗逆性相关如低温、干旱胁迫响应元件。以转化空质粒pCambia1381Z-GUS与35S启动子驱动pCambia1381Z-35S-GUS的烟草为对照,对转化pCambia1381Z-JcDnaJ20p-GUS的烟草叶片分别进行15、4℃低温处理24 h,通过GUS组织化学染色表明,JcDnaJ20p在低温处理下能够提高GUS基因的表达量,具有低温诱导启动子活性。     

拟南芥冷诱导型启动子CBF 3的克隆及活性检测

目的：构建冷诱导型启动子CBF3基因的植物表达载体,并将其转入烟草。方法：以拟南芥基因组DNA为模板,通过特异PCR扩增,克隆冷诱导表达启动子CBF3（C-repeat binding factor）。用CBF3启动子替换pBI121载体上的35S启动子构建新的载体pBC-GUS,通过农杆菌介导的叶盘法转化烟草。结果：获得了转基因烟草,转基因烟草的GUS组织化学染色及PCR分析结果表明,在低温诱导下,CBF3启动子可增强GUS基因表达。结论：CBF3启动子可应用于植物抗冷基因工程研究。     

水曲柳节律基因LHY启动子的克隆和功能分析

以水曲柳基因组DNA为模板,用Site Finding-PCR法扩增得到节律基因LHY（late elongated hypocotyl）启动子序列,长度为1 360 bp。PLACE启动子预测工具分析表明,序列中含有转录必备的TATA box、CAAT box以及一些非生物胁迫和激素响应元件等。构建植物GFP瞬时表达载体p PXGFP-P-LHY,农杆菌介导转化烟草叶片和白桦悬浮细胞,GFP检测结果表明,LHY启动子能够启动GFP基因在烟草和白桦细胞中表达,且对非生物胁迫（低温、高温、盐）产生响应;构建植物GUS报告基因整合表达载体p PCXGUS-P-LHY,农杆菌介导法瞬时转化烟草,GUS染色结果表明,LHY启动子的活性具有不同程度的时空特性。     

玉米淀粉分支酶基因启动子的克隆与功能分析

以玉米品种“吉糯1号”的基因组DNA为模板,通过PCR扩增得到玉米淀粉分支酶基因的启动子序列,克隆到pMD18-TVector上,经测序,该启动子大小为934bp。与已报道的序列比较仅有14个核苷酸发生改变,同源性为98.5%。用该启动子取代植物表达载体pBI121的35S启动子,与GUS基因编码区连接,构建成融合质粒pSBE-GUS。经农杆菌介导法转化烟草,获得了转基因植株。GUS活性检测结果表明,由该启动子序列引导的GUS基因能在种子中表达,而在其他组织中表达微弱或未表达,证实该启动子具有种子特异性表达的功能。     

竹节花黄斑驳病毒启动子的缺失分析及功能

竹节花黄斑驳病毒(CoYMV)是侵染单子叶植物竹节花的一 种双链环状DNA病毒,它的启动子可介导外源基因在烟草韧皮部特异表达。为了研究其组织 特异性表达的最佳启动子区域,对CoYMV启动子进行了5′端五种不同长度的缺失分析,用不同长度的启动子片段与GUS基因及NOS3′端转录中止序列构建了全长启动子及5 个缺失启动子序列的六个嵌合GUS基因植物表达载体。利用农杆菌将上述嵌合基因转化烟草 外植体后,每种表达载体都获得了一批转基因烟草植株。转化再生烟草植株的PCR分析、GUS 酶活测定及GUS组织染色的结果表明六种类型的嵌合基因已整合到烟草染色体中,并有五种 表达出GUS活性。缺失到870bp的启动子比全长启动子(1040bp)的活性约高78%,870bp比585bp启动子介导的GUS活性略高但差别不明显,缺失到447和232时GUS活性有明显下 降,但仍具有韧皮部特异表达的特性。当缺失到TATA box附近的44bp时启动子丧失组织特 异性,GUS活性也降低到测不出来的水平。以上结果表明CoYMV启动子从转录起始位点上游 870bp～230bp及232bp下游区分别与启动子的活性和韧皮部组织特异性密切相关,870bp上游可能存在一个负调控序列,所以该启动子的活性和组织特异性的最佳调控区应在87 0bp或585bp的下游区。CoYMV启动子与35S启动子驱动GUS基因在烟草中表达的活性相比, 前者为后者的70%左右,考虑到前者仅在韧皮部细胞表达而后者为组成型表达,所以CoYMV启 动子在韧皮部的活性可能与35S启动子相当或更高。CoYMV启动子在其它转基因植物中驱动外 源基因表达的特点正在研究中。     

香蕉束顶病毒DNA组分2、3的启动子区的组织特异性分析

香蕉束顶病毒（BBTV）基因组至少由6个大小约为1.0-1.1kb的单链环状DNA组分所组成,每一个DNA组分包含编码区与非编码区。本文在前人的研究基础上进一步了解BBTV DNA组分启动子的功能。首先根据BBTV 海南分离物的全序列,通过常规PCR扩增出长为540bp的 BBTV DNA3组分启动子序列BV3.1,同时通过重叠PCR扩增出646bp的DNA2与DNA3组分非编码区拼接的重组启动子序列BV23,分别替代pBI121 35S启动子序列与gus基因进行融合,构建植物表达载体pBIBV3.1、pBIBV23。农杆菌介导转化获得的pBIBV3.1转基因烟草经GUS化学组织染色后,在其叶片的叶脉处检测到微弱的GUS活性,证实了DNA3组分的韧皮部特异表达活性;而pBIBV23转基因烟草,其叶片经GUS组织化学染色后,在叶肉、叶缘及一些叶脉上检测到弱GUS活性,这表明由BV23驱动的gus基因在烟草中类似于组成型表达,则DNA2组分转录方式可能有异于DNA3组分。     

棉花曲叶病毒大基因间区启动子在宿主植物中的表达活性

棉花曲叶病毒(CLCuV)是一种单链DNA病毒,属于双生病毒亚组Ⅲ.为了研究其基因组大基因间区(LIR)的功能,以CLCuV侵染的烟草叶片组织总DNA为模板,通过聚合酶链反应扩增LIR并插入克隆载体.将LIR分别以互补链及病毒链基因转录方向与gus报告基因和nos终止子融合,构建了植物表达载体.通过农杆菌介导的方法转化烟草并测定转化植株的GUS活性,实验结果表明,LIR在互补链基因方向具有强启动子活性,GUS平均活性是CaMV 35S启动子的5～6倍,单株最高的GUS活性达到CaMV 35S启动子的10倍;组织化学定位证实其在叶肉及维管组织均有活性.LIR在病毒链基因方向的启动子活性较低.报道了CLCuV来源的分离的LIR在互补链基因方向可作为一种新型的强启动子元件应用于植物遗传操作.     

杜氏盐藻硝酸盐还原酶基因5′上游序列的克隆与功能分析

目的:克隆杜氏盐藻硝酸盐还原酶（NR）基因5′上游序列序列,并对其功能进行分析。方法: 利用BamHI、EcoRI、HindIII、PstI、SalI、Xbal 6种限制性内切酶分别酶切盐藻基因组DNA,并与接头连接,构建成盐藻基因组步行文库。采用 LA-PCR方法,从上述盐藻步行基因组文库中扩增NR基因5′上游序列序列,测序并进行分析。为检测其表达特性,构建了该片段与GUS 嵌合基因的表达载体pNR-GUS, 通过电击法将所构建的重组表达载体转化盐藻,组织化学染色法观察GUS的表达。结果: 从盐藻基因组步行文库中扩增出约1200bp特异片段,序列分析表明5′上游序列含有启动子的特征性序列。GUS瞬时表达染色结果显示,该DNA 片段具有硝酸盐诱导和铵抑制的启动子活性。结论：所克隆的盐藻的5′上游序列可能是一种具有"开关"活性的可控性启动子。     

大豆转录因子GmERF5启动子的克隆及活性分析

乙烯响应因子(Ethylene-responsive factors,ERFs)广泛参与植物对生物和非生物胁迫的应答。从大豆吉林32的基因组DNA中分离到GmERF5的启动子片段,全长1 924 bp。该区段含有9种与逆境相关的顺式作用元件,即G-box、GT-1、LTRE-1、W-box、TL1、DPBF、MYB、MYC和BIHD10S。将该启动子构建到植物表达载体pCAMBIA1301上与葡萄糖苷酸酶(GUS)基因融合,注射法转化烟草叶片,并进行干旱、高盐和低温处理,通过GUS组织化学染色和GUS荧光值的测定分析启动子的启动活性。结果表明,在未处理条件下,该启动子能驱动下游GUS基因的表达。干旱和低温处理10 h能明显增强GmERF5P的启动活性。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

医疗机构合理用药评价模型的构建研究

目的 针对医疗机构的合理用药水平进行评价研究。方法 根据医疗机构合理用药的具体要求,构建医疗机构合理用药评价指标体系,采用基于模糊群决策的方法和多指标评价分析法构建医疗机构合理用药评价模型。结果 构建了基于模糊群决策的医疗机构合理用药评价模型,并通过实例分析证明了评价模型的可行性。结论 建立的基于模糊群决策的医疗机构合理用药评价模型能够对医疗机构的合理用药水平进行科学评价,为提高医疗机构合理用药水平奠定基础。     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.