转基因tritordeum的遗传分析

从一批使用无启动子“uidA转化策略转化的tritordeum中,分离出标定有花药组织特异性启动子的单株并考察其外源基因的遗传稳定性,为即将开展的启动子分离工作奠定基础;对转基因材料不同生长时期的不同组织进行Gus组织化学检测,分析Gus表达的特异性,应用RT-PCR从转录水平进一步确证启动子的特异性;所筛选出的植株中,Gus的表达特异性和To代的表现完全一样,只有花药原基和花粉粒,整合位点在各代之间传递的频率较低,明显不符合显性：隐性（3：1）规律,但Gus的表达特异性在各代之间传递时有着极强的稳定性;成功地分离出了被标定有花药组织特异性启动子的tritordeum,对其遗传特性作了进一步的分析．     

Maize transformation via pollen tube pathway and the inheritance of transgene in progeny

The herbicide-resistant gene als was introduced into maize inbred line Qi319 through pollen-tube pathway. The inheritance of transgene was studied up to 33 generation. Transformation was performed by applying plasmid DNA carrying solution on the severed styles after self-pollination, or just prior to selfpoll/nation. After herbicide screening, PCR and Southern blot analysis, seventeen plants were confirmed positive in TO generation, and sixteen of them were self-bred to construct family to 33 progeny. Through PCR analysis and herbicide screening in offspring, four lines were confirmed following a 3 : 1 Mendelian segregation ratio. In other lines the numbers of positive plants were lower than expected. Therefore, it is necessary to select large population in progeny to produce stable inherited lines when transgene was delivered by pollen tube method.     

pACT E3质粒的构建及其在转基因烟草中的表达

将1个从棉花中分离的新的启动子ACT E3插入质粒pBI101,ACT E3与GUS基因融合，构建成pACT E3表达载体。通过农杆菌介导转化法将ACT E3/GUS融合基因导入烟草。GUS组织化学染色分析表明，在ACT E3启动子控制下，GUS基因仅在叶片及茎等组织中特异表达。     

Robust and regulatory expression of defensin A gene driven by vitellogenin promoter in transgenic Anopheles stephensi

The use of genetically modified mosquitoes to reduce or replace field populations is a new strategy to control mosquito-borne diseases. The precondition of the implementation of this strategy is the ability to manipulate the genome of mosquitoes and to induce specific expression of the effector molecules driven by a suitable promoter. The objective of this study is to evaluate the expression of defensin A gene of Anopheles sinensis under the control of a vitellogenin promoter in transgenic Anopheles ste- phensi. The regulatory region of Anopheles gambiae vitellogenin was cloned and subcloned into transfer vector pSLFa consisting of an expression cassette with defensin A coding sequence. Then, the expression cassette was transferred into transformation vector pBac[3xP3-DsRedafm] using Asc I di- gestion. The recombinant plasmid DNA of pBac[3xP3DsRed-AgVgT2-DefA] and helper plasmid DNA of phsp-pBac were micro-injected into embryos of An. stephensi. The positive transgenic mosquitoes were screened by observing specific red fluorescence in the eyes of G1 larvae. Southern blot analysis showed that a single-copy transgene integrated into the genome of An. stephensi. RT-PCR analysis showed that the defensin A gene expressed specifically in fat bodies of female mosquitoes after a blood meal. Interestingly, the mRNA of defensin A is more stable compared with that of the endogenous vitellogenin gene. After multiple blood meals, the expression of defensin A appeared as a reducible and non-cycling type, a crucial feature for its anti-pathogen effect. From data above, we concluded that the regulatory function of the Vg promoter and the expression of defensin A gene were relatively con- served in different species of anopheles mosquitoes. These molecules could be used as candidates in the development of genetically modified mosquitoes.     

一个假单胞菌MY1402基因启动子的分离与鉴定

利用构建的启动子探针载体pUE,以大肠杆菌(Escherichia coli)DH5α作为宿主菌,从耐低温假单胞菌(Pseudomonas sp.)菌株MY1402分离基因启动子片段,获得7个具有不同大小的启动子功能片段的重组子,命名为pUE1～pUE7.将其中具有p1片段的重组子PUE1导入假单胞菌MY1402中进行卡那霉素耐受浓度分析,结果显示阳性转化子MY1402/pUE1的最高耐受浓度为250 mg/L,而且在相同抗生素浓度下培养温度从28℃降到15℃并不影响转化子MY1402/pUE1的生长,表明pUE1中的插入片段p1具有低温启动子的活性.将所预测的p1核心序列p1a通过化学合成并连接到探针载体pUE上,构建出重组质粒pUE1a并转化MY4102中进行功能分析.结果显示,28℃条件下p1a片段仍保持相同强度的启动子活性,但15℃条件下活性下降明显,卡那霉素质量浓度高于100 mg/L基本没活性,表明p1a两端序列可能与p1低温转录启动活性有关.     

慈姑雌花在自然居群中的受粉过程--花粉粒落置、萌芽及花粉管生长的荧光显微观察

用荧光显微术观察了慈姑（Ｓａｇｉｔｔａｒｉａ ｔｒｉｆｏｌｉａ Ｌｉｎｎ）不同时期的雌花柱头上花粉粒的落置、萌发及花粉管的生长过程与途径。结果表明：该种雌花开花２ｈ 柱头受粉率为０,开花４ｈ 和８ｈ受粉率分别为５３８％ 和９８３％ ,柱头上花粉萌发率达１００％ ;花粉管沿雌蕊之向心一侧的组织中穿行,至子房基部后部分花粉管转向胚珠,由珠孔进入珠心。本研究发现该种花粉管常穿过子房基部至花托组织,并可进入其他雌蕊中,作者认为这种行为可能对保证雌花的结实率有一定意义     

人IDE基因启动子生物信息学分析

对人IDE基因启动子进行生物信息学分析以获得人IDE基因启动子、CpG岛及转录因子结合位点特征.从UCSC基因组数据库成功获得人IDE基因5’调控区2 000 bp序列.Promoter 2.0、FPROM、NNPP预测人IDE基因分别有3个、2个、6个启动子.Relative profile score threshold选择80%、85%、90%、95%、100%时,JASPAR预测该序列存在5170、1771、454、87和5个可能的转录因子结合位点.Relative profile score threshold选择80%,搜索到6个潜在的TCF7L2转录因子结合位点.采用进化足迹法,LAGAN预测方法获得位于人和小鼠同源IDE基因启动子保守区域相同位置的转录因子结合位点为14个,包含转录因子SPI-1、cap、c-FOS、FREAC-3、c-ETS、Cdxa、HSF2等.发现一个CpG岛,位于1 303～1 705 bp 之间,大小为403 bp.人IDE基因启动子、CpG岛及转录因子结合位点的生物学信息学分析,为下一步基因表达调控实验奠定了基础.     

超声波在花粉破壁技术中的应用

花粉是种子植物的雄性生殖细胞.直径一般为30～40μm,最大也不过100～200μm.其外壁是由化学性非常稳定的多糖等物质组成,非常坚韧,能耐高压与酸碱.若将花粉直接添加于食品中,则几乎不被消化而排出体外,因此需要解决生物利用率低这一问题.花粉破壁技术有发酵法、冷冻法、磨碎法和超声法等,文献中仅仅提及超声法,但未做进一步报告.本文给出我们从1987年5月开始,用超声波技术进行花粉破壁的一些实验,以及部分测试结果.深入的研究工作仍在继续进行.     

基于PWM扫描算法的启动子区域统计分析

为了寻找启动子区域上转录因子结合位点的分布规律,进而研究这种规律与真核基因表达调控机制之间的关系,该文从已有数据出发,运用位置权重矩阵(PWM)扫描算法对启动子区域上4种与肝脏特异表达相关的转录因子结合位点分布情况进行了初步研究,并提出了一种新的序列评分方法。通过该方法提取的统计特征,肝脏特异基因的鉴别准确率可以达93.33%。实验结果表明:肝脏特异基因的启动子区域上结合位点的分布情况与其他基因相比存在显著差异。新的序列评分方法可以更好地反映这种差异性,实现肝脏特异基因的精确鉴别。     

松树根特异性启动子驱动的转CMO/BADH基因水稻的耐盐性研究

运用松树根特异性启动子PmPgPR10驱动把CMO/BADH双价基因转入水稻.对转基因植株根和叶的CMO酶、BADH酶活性及其它生理生化指标进行了测定.结果表明:CMO/BADH双价基因能在根部特异性表达.文章还对转基因提高植株耐盐性的机理进行了探讨.     

Immunochemical identification of gelsolin in maize pollen

Gelsolin is a representative of a type of actin-binding proteins (ABPs) universally found in eukaryotes. It plays role in nucleation, capping and severing of actin filamentsin vitro. In our experiment, gelsolin was purified from pig plasma and the polyclonal antibodies against it were prepared. The crude extracts of maize pollen were immunodetected by Western-blotting with polyclonal antibody and monoclonal antibody respectively. The immunodetection results show that gelsolin exists in maize pollen and its molecular weight is about 91 ku, similar to that of gelsolin found in animal tissues.     

Membrane skeleton spectrin in pollen and pollen tube

Immuno-western blots, immunofluorescence and immuno-gold labeling methods were used to study the existence and distribution of a homologue of the membrane skeleton spectrin in the pollen and pollen tube ofLilium davidii Duch. A spectrin homologue was found to exist in the pollen and pollen tube, distributed on membranes of secretory vesicles near Golgi apparatus.     

气传花粉监测数据研究进展

 综述了基于气传花粉监测数据的研究进展,结果发现,利用花粉观测数据可获取某地的花粉概况,进而绘制具有临床价值的花粉日历。但因其不包含病例信息,故需结合过敏人群特征修正影响浓度阈值。此外,通过分析暴露于不同环境下的患病风险,证实了防治花粉症十分依赖于洁净的空气。局地观测环境及采样器的安放位置对监测结果有极大影响,但整体而言,北半球大多数区域花粉季延长、花粉浓度增加,并可归因于气候变暖所致。为预测花粉关键要素在未来的变化,4大类模型被广泛应用,并取得较好的预测结果。但对于预测效果较差的部分（花粉浓度极值、复杂地形等）,最优解决方法则是结合高分辨率的花粉监测数据进行订正。但是,由于缺乏低成本的自动监测设备,当前花粉监测数据的分辨率仍然较低,由此带来了一系列的数据和技术壁垒。建议该领域应将开发低成本的自动监测设备作为近期发展的重点,并以此建立标准化的观测体系。     

Effect of 5'-deletion of Arabi-dopsis profilin2 promoter on its vascular specific expression

Based on the published sequence of profilin2 promoter of Arabidopsis thaliana, a full-length promoter (1667 bp) was amplified by PCR. The 5' -end deletion fragments with length of 1380, 1153, 969 and 597 bp were then fused with gus (uidA.) gene respectively. Constructed plant expression vectors were individually transferred into Kalan-choe laciniata and transgenic plants regenerated. GUS his-tochemical assay confirmed that the full-length promoter Pfn1.7 was vascular-specific. Deletion assays showed that profilin2 promoter could be divided into three parts. Deletion of fragment 1 ( -1667--1380 bp) resulted in constitutive expression, suggesting that element(s) responsible for vascular-specific expression might exist in this region. Fragment 2 located at -1153 - -597 bp strongly inhibited gus gene expression. Fragment 3 ( -597 - -1 bp) is considered as a basic domain of profilin2.     

南京椴花粉贮藏寿命的研究

采用TTC法对不同贮藏条件下的南京椴花粉生活力进行测定。结果表明：南京椴花粉的寿命相对较短,临界含水量为8.99%。在此含水量下,贮藏温度为-25℃时,经过8 d贮藏,花粉生活力从75%下降到56%,经过180 d贮藏,花粉生活力仅有25%。此外,新采集南京椴花粉的含水量为9.55%,可以不经干燥,直接在-18℃下保存。     

SCFP,a novel fiber-specific promoter in cotton

To investigate the expression pattern of GhSCFP which was isolated from cotton fiber cDNA library, a 1006 bp upstream fragment of the gene was cloned by chromosome walking and fused to GUSand GFP respectively. Histochemical GUS and GFP fluorescence analysis revealed that the expression of the report genes driven by the promoter sequence was detectable only in outer layer cells during the seed development in the transgentic tobaccos. In transgenic cotton, strong GUS activity was observed in spherical protrusions on 0 dpa （days post anthesis） ovule surface, and in the 2-36 dpa fiber cells, while no GUS signals were detected in the root, leaves, stem, corolla, anther and stigma. Our data demonstrated that GhSCFP upstream sequence is a cotton fiber-specific promoter and this promoter will be useful in the molecular research on fiber cell development and in cotton fiber improvements by genetic modification.     

Coat protein promoter from cotton leaf curl virus is not a tissue-specifically expressed promoter

Geminivirus is a kind of single-stranded DNA virus. Experimental results from tomato golden mosaic virus (TGMV) showed that expression pattern of coat protein gene (cp) promoter was phloem specifically expressed. In this note, the studies oncp promoter of cotton leaf curl virus (CLCuV) which is found and identified recently suggest that the promoter is not phloem specifically expressed. The expressing activity ofgus gene driven by the promoter exists not only in phloem but also in mesophyll tissues and root tip meristem. Transient expression suggests thatcp promoter transactivated by AC2 shows expressing activity in mesophyll and vascular tissue of leaf vein.     

Identification of seed-specific promoter nap300 and its comparison with 7S promoter

By fusing seed-specific promoter nap300 with β-glucuronidase gene, it was found that this about 300bp DNA fragment was sufficient to direct seed-specific gene expression. The substitution mutation in both distB and proxB elements had a little effect on the expression efficiency and almost no effect on the organ-specific expression pattern. In the experiment designed to compare nap300 with 7S promoter, the result showed that tissue specificity for nap300 was higher than that for 7S, and its expression level was lower than 7S's. There was no big difference in their expression pattern, and the maximal activity stage for the two promoters was identical, which indicated they could be used simultaneously for expressing different foreign genes in seeds.     

Functional analysis of cis-acting sequences regulating root-specific expression in transgenic tobacco

Two different length fragments, RSF1 and RSF2 which contained the cis-acting sequences of root-spe- cific gene TobRB7, were isolated from tobacco genome. The abilities of these fragments to direct root-specific expression were studied by fusing them to the β-glucuronidase (GUS) report gene with different directions. After the recombined vectors were transformed into tobacco, the expression pattern was performed by histochemical staining and the quantitative analysis of GUS activity. The data suggested that the cis-acting element of TobRB7 gene direct GUS expression not only as root-specific but also as bidirectional. In our studies, the short fragment, RSF2, performed stronger activity than RSF1 with any direction. The stronger activity of GUS expression was determined by reverse inserting of RSF1 or RSF2 than positive inserting.