过表达本氏烟基因NbPAP2对转基因植物生长发育的影响

为探究紫色酸性磷酸酶基因在本氏烟草中所发挥的生物学功能,该研究利用生物信息学软件发现本氏烟NbPAP2与拟南芥AtPAP2的氨基酸同源性达到64.2%,其具有特征性的信号肽和靶向叶绿体的C端疏水基序以及双核金属离子中心. qRT-PCR结果显示NbPAP2在植物中组成性表达,花瓣中表达量最高,测定叶片气孔导度平均提高30.5%、净光合速率平均提高23%,然而蒽酮比色法测定糖平均含量下降21%.因此,NbPAP2通过介导碳代谢促进本氏烟草花期提前、生物量提高、种子体积增大等.     

利用TRV过表达和沉默系统验证植物油脂合成相关基因的功能

为探究烟草脆裂病毒过表达和沉默系统进行油脂相关基因研究的可能性,该研究克隆了拟南芥的WRI1和FAD2基因,分别构建了TRV过表达和沉默载体. 利用瞬时侵染技术侵染本氏烟草,取材料进行RT-PCR和脂肪酸含量检测. 结果显示在过表达WRI1基因的本氏烟草中,侵染叶和非侵染叶的WRI1基因及其相关参与脂肪酸合成基因ACP1、KAS1和BCCP2等都有明显上调,且脂肪酸含量检测结果显示分别增加16%和28%. 在沉默FDA2基因的本氏烟草植株中,发现多不饱和脂肪酸含量分别减少约25%和24%. 因此,利用TRV系统对油脂相关基因进行研究是可能的.     

Plant polar growth in tobacco disturbed by y-tubulin gene silencing

To further understand the functions of y-tubulin in plant cells, we conducted a study in which the y-tubulin gene was down-regulated in tobacco plants (obtained by the Agrobacterium-mediated method). This involved transforming the target fragments, in which the sense and antisense partial y-tubulin cDNA fragments were ligated together, into Nicotiana tabacum var. Samsun NN. The y-tubulin down-regulated transformants developed multiple meristems or branches with trumpet-shaped leaves; their root generation also appeared abnormal, with the taproots undeveloped, whereas lateral roots were developed. In addition, the content of indole-3-acetic acid (IAA) and expression of polarity transportation vector PGPI were aberrant. These results suggest that y-tubulin gene silencing disturbed the polar growth of tobacco plants, and that this phenomenon was probably correlated with the IAA content and the polar transpor-tation process.     

砷胁迫对烟草生理特性的影响

为探明砷胁迫对烟草生理特性的影响,选用本生烟草(Nicotiana benthamiana)为材料,采用盆栽试验,挑选了生长4周后健壮的烟草苗进行砷(As2O3)胁迫处理(50μg·L-1),并于处理后0、24、48、72和96h分别取样并进行相关生理指标(叶绿素含量、丙二醛含量、超氧化物歧化酶、过氧化物酶和过氧化氢酶活性)的测定.结果显示:随着砷处理时间的延长,本生烟草叶片叶绿素含量、超氧化物歧化酶和过氧化物酶活性呈先升后降趋势,过氧化氢酶活性呈下降趋势,丙二醛含量呈上升趋势.试验数据表明:短时间的砷胁迫烟草会出现自我保护现象,但胁迫超出一定时间(24h)后对烟草造成一定的伤害.     

转BnTR1基因的烟草对多种逆境胁迫的抗性研究

将BnTR1基因转化到了本生烟中,发现与非转基因对照相比,转基因烟草对甘露醇和PEG模拟干旱表现出较强的抗性,尤其是PEG胁迫时,三个转基因株系在生物量的积累和整体长势方面都优于对照.干旱处理20d后复水并培养14d后,对照烟草全部枯死,但是所有转基因烟草都恢复生长直至开花结果.当把烟草叶片圆片浸泡在系列盐水中,盐浓度超过600mM时,对照烟草圆片受损白化,而转基因烟草圆片能保持较大面积的绿色。热激蛋白基因HSP90和HSP70在所有烟草中的表达量都有明显提高,其表达量的增加在对照和转基因株系中并无差异,但是在正常条件下不表达的HSF30和sHSP17.6基因在热激处理后都表达了,而且HSF30的表达量增加了50%,sHSP17.6的表达量增加了30%.     

本生烟组织培养及遗传转化体系的建立

本文研究了通过农杆菌侵染获得本生烟(Nicotiana benthamiana)转基因植株的方法。将本生烟中上部叶片消毒后切成5mm×5mm左右的小块作为外植体,在含有6-BA(3mg/L)和NAA(0.2mg/L)的MS培养基中培养2~3d,用含有表达载体的农杆菌侵染后共培养3~4d,转入含有6-BA(3mg/L)、NAA(0.2mg/L)和卡那霉素(100mg/L)的MS培养基中培养2~3周。将外植体边缘产生的愈伤组织和芽点转移到只含有卡那霉素(100mg/L)的MS培养基中继续培养,2周后分化出大量不定芽。继续培养2周,当芽长1~3cm时,将芽转移到含IBA(0.1mg/L)的MS培养基中分化生根,得到再生植株。当再生植株高8~10cm、根长4~5cm以上时,移栽到经过灭菌的营养土中。通过实时荧光PCR扩增,从获得的植株中检测35S启动子和NOS终止子外源基因片段,判定所得的植株的确为转基因植株。最后对本生烟组织培养中应该注意的问题进行了讨论。     

Distribution of a kinesinrelated protein on Golgi apparatus of tobacco pollen tubes

The conventional kinesin and kinesin-related proteins (KRPs) constitute a large family of microtubule-based molecular motors. Members of this family bind to micro-tubules in an ATP-dependent manner. They all have microtubule-activated ATPaes activities, and play essential roles in the transport of various vesicles and organelles in eukaryotic cells[1―3]. The first plant kinesin-like protein was detected in tobacco pollen tube with an antibody against animal kinesin[4]. Recently, a number …     

菊芋低聚果糖促进双歧杆菌生长的研究

研究一种自制菊芋低聚果糖（FOS）在体内外对双歧杆菌的促生长效果,体外试验表明,所实验的5种218株双歧杆菌菌株均利用FOS,在FOS浓度为0．5％时,青春双歧杆菌和婴儿双歧杆菌的生长量分别比对照增加6．5倍和5．4倍,培养液中流加0．2％的FOS也能显著促进双歧杆菌的生长;竞争实验显示双歧杆菌的增殖对大肠杆菌和艰难梭菌的生长有一定的抑制作用,体内试验表明,FOS能显著提高受试动物粪合便中的双歧杆菌菌数。     

Apoptosis in suspension culture of tobacco cells induced by heat shock

The induction of apoptosis in suspension culture of tobacco cells by heat shock is reported for the first time. Heat treatment (48℃ for 4 h) of tobacco cells led to the appearance of typical hallmarks of apoptosis. It was demonstrated by DNA laddering analysis that the cells treated with heat shock at 48℃ for 4 h had a serious degradation of nuclear DNA into multi-nu-cleosomal sizes, suggesting that heat shock activated endogenous nuclease which led to DNA cleavage at the linkage sites between the nucleosomes, but ladders were very faint for DNA from 2 and 9 h heat-treated cells. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) detection also showed that most of these treated cells (48℃ for 4 h) displayed positive reactions, indicating a serious DNA 3'-OH cleavage in their nuclei. Moreover, some other cytological changes in apoptotic cells, such as cell shrinkage, chromatin aggregation, nucleus collapse, have also been observed by 4', 6'-diamidino-2-phenyl-indole (DAPI) staining.     

磷硫代siRNA的抗肿瘤基因沉默活性研究

由于目前尚无高效合成立体特异性磷硫代siRNAs(PS-siRNAs)的化学体系,本研究针对潜在的癌症治疗靶点PLK1,用a-磷硫代三磷酸腺苷(ATPaS)、a-磷硫代三磷酸胞苷(CTPaS)和a-磷硫代三磷酸尿苷(UTPaS)通过T7RNA聚合酶转录合成了部分磷硫代修饰的Rp-磷硫代siRNAs(Rp-PS-siRNAs),探究了nat-siRNAs和PS-siRNAs血清稳定性和基因沉默活性的差异性。发现酶促合成的磷硫代siRNA几乎不影响siRNA的基因沉默效率,但却显著提高siRNA的血清稳定性。因此,酶促转录合成的Rp-PS-siRNA可望作为siRNA的修饰形式,以延长siRNA的生物活性半衰期,使siRNA广泛应用于生物医学临床研究领域。     

Coat protein gene and 3′ non-coding region of tobacco mosaic virus and tomato mosaic virus are associated with viral pathogenesis in Nicotiana tabacum

The camellia isolate of tomato mosaic virus (ToMV-TL) can induce local necrotic lesions on the inoculated leaves in Nicotiana tabacum, whereas the broad bean isolate of tobacco mosaic virus (TMV-B) produces the mosaic symptom on systemic leaves. To examine viral determinant for differential infection phenotype in N. tabacum, the coat protein gene and the 3′ non-coding region of TMV was replaced with that of ToMV, the chimeric virus induced similar local necrotic lesions to that induced by ToMV. The results indicate that the coat protein gene and the 3′ non-coding region of TMV and ToMV influence the virus-induced pathogenesis in N. tabacum.     

In situ localization of calmodulin mRNA in the developing tobacco anthers

The temporal and spatial expression patterns of calmodulin mRNA in the developing tobacco anthers were investigated by in situ RNA hybridization, using digoxigening-labeled anti-RNA probe. Calmodulin mRNA was distributed in various developmental stages of tobacco anthers, but the expression level had temporal and spatial differences distinctly. During early stage of anther development, the expression level of calmodulin mRNA was significantly high, mainly distributed in epidermis, tapetum and transfusion parenchyma cells and so on. Especially, more mRNA was accumulated in the nuclei and chromosomes of microspore mother cells prior to and during meiosis. With the development of anther, mRNA was decreased gradually in the anther wall and pollen. By mature pollen stage, only a stronger positive reaction still existed in the epidermis of anther wall and transfusion parenchyma cells. The results suggest that the temporal and spatial expression of calmodulin mRNA is closely correlated with cell division, pollen development and substance transport.     

不同无性繁殖复壮措施对桉树生长及抗病性比较研究

以巨尾桉(Eucalyptus grandis×E.urophylla)广林9号幼林为材料,采用组培繁殖和扦插繁殖两种苗木进行桉树生长规律及抗病性衰退的比较研究。组培繁殖设4个处理,分别为第1、8、15、28次继代苗(简称为组培1代、组培8代、组培15代和组培28代);扦插繁殖设3个处理,种植第15代组培苗做采穗圃,分别于第1、2、3年后采穗扦插的扦插苗(简称为1年扦插、2年扦插和3年扦插)。试验结果表明,不同无性繁殖方法的幼林树高、地径生长曲线呈双峰型,峰值分别出现在3月和6月,树高、地径生长量及月增长量变化不同,组培苗在地径生长方面具有较大优势,树高生长方面虽然前期较差,但后期增长快,总体上仍具有优势;同一繁殖方法随着繁殖代数增加,树高、地径生长均呈下降趋势;不同繁殖方法以及不同繁殖代数都会对抗青枯病能力造成影响,组培苗的抗病性优于扦插苗,同一繁殖方法随着繁殖代数的增加抗病性下降,但抗病性衰退速度不同;不同无性繁殖的抗焦枯病能力不同,组培苗随着继代次数的增加而减弱,扦插苗随着繁殖年份的增加而升高;7种无性繁殖复壮措施的抗衰退能力为组培1代最强,组培8代次之,3年扦插最弱。     

Cloning and characterization of a gene encoding cysteine proteases from senescent leaves of Gossypium hirsutum

A gene encoding a cysteine proteinase was isolated from senescent leave of cotton (Gossypium hirsutum) cv liaomian No. 9 by utilizing rapid amplification of cDNA end spolymerase chain reaction (RACE-PCR), and a set of consensus oligonucleotide primers was designed to anneal the conserved sequences of plant cysteine protease genes. The cDNA, which designated Ghcysp gene, contained 1368 bp terminating in a poly(A)^ trail, and included a putative 5‘(98 bp) and a 3‘(235 bp) non-coding region. The opening reading frame (ORF) encodes polypeptide 344 amino acids with the predicted molecular mass of 37.88 kD and theoretical pl of 4.80. A comparison of the deduced amino acid sequence with the sequence in the GenBank database has shown considerable sequence similarity to a novel family of plant cysteine proteases. This putative cotton Ghcysp protein shows from 67% to 82% identity to the other plants. All of them share catalytic triad of residues, which are highly conserved in three regions. Hydropaths analysis of the amino acid sequence shows that the Ghcysp is a potential membrane protein and localizes to the vacuole, which has a transmembrane helix between resides 7-25. A characteristic feature of Ghcysp is the presence of a putative vacuole-targeting signal peptide of 19-amino acid residues at the N-terminal region. The expression of Ghcysp gene was determined using northern blot analysis. The Ghcysp mRNA levels are high in development senescent leaf but below the limit of detection in senescent root, hypocotyl, faded flower, 6 d post anthesis ovule, and young leaf.     

基因沉默抑制子HC-Pro表达载体的构建

RNA沉默广泛存在于植物等大多数真核生物中,能够防御外源基因的入侵和调控基因表达等.然而,基因沉默抑制子HC-Pro蛋白的具体功能的研究并不十分清楚.本文重点介绍了基因沉默抑制子HC-Pro的表达载体pBI121-HC-Pro的构建及HC-Pro基因在烟草(Nicotiana benthamiana)中的稳定表达.并利用半定量RT-PCR技术检测了目的基因HC-Pro的表达,检测结果表明我们获得了HC-Pro基因稳定表达的转基因烟草株系,为进一步深入研究基因沉默抑制子HC-Pro的功能奠定了实验基础.     

磷硫代siRNA的YAP基因沉默活性研究

为了探究RNA聚合酶酶促合成非对映体纯PS-siRNA（pPS-siRNA）的基因沉默活性,本研究针对潜在的癌症治疗靶点Yes相关蛋白（YAP）,通过qPCR、Western blot等方法研究了其对YAP基因的沉默效果和对HeLa细胞的细胞毒性. 结果表明,与未修饰的siRNA（PO-siRNA）相比,pPS-siRNA可以更好地下调YAP基因表达（效率提高约30％）,而没有明显的细胞毒性. 此外,MTT细胞增殖实验与流式细胞术的结果表明,经pPS-siRNA敲降YAP后,HeLa细胞的增殖受到了抑制. 说明pPS-siRNA在基因治疗方面具有优越性以及YAP作为肿瘤治疗靶点的潜力.     

植物根际细菌的促生机制

植物根际促生细菌是指生活在植物根系土壤的一类可促进植物对矿质营养的吸收和利用,并能抑制有害微生物从而促进植物生长的益生菌类。人们对植物根际促生细菌的作用机制进行了广泛深入的研究。对植物根际促生细菌的促生机制进行综述。