盐穗木miRNA417的克隆及对种子萌发和幼苗成活率的影响

MicroRNA (miRNA)是植物重要的基因表达调控因子,miR417的表达受盐胁迫的调节,高盐胁迫时,拟南芥miR417的表达能够抑制种子的萌发和幼苗成活。本研究通过分析miRbase数据库中已知植物miRNA417的序列,利用PCR技术成功克隆获得了盐生植物盐穗木的miR417（HcmiR417）的前体序列,将其构建至植物表达载体pCAMBIA1301上,通过花絮浸染法对拟南芥进行遗传转化。结果表明,在150 mmol·L^-1 NaCl的胁迫下,分别过表达HcmiR417和过表达拟南芥miRNA417（AtmiR417）的转基因拟南芥种子的萌发率和幼苗存活率均较野生型低,但两种转基因拟南芥株系之间没有差异。初步验证了盐生植物HcmiR417在种子萌发和幼苗成活率方面也具有负调控作用,盐生植物盐穗木和拟南芥植物miRNA在功能没有显示出差异。     

拟南芥GH3.9基因的过表达及其表型分析

为研究GH3.9基因在植物生长发育过程中的作用,利用RT-PCR成功克隆到GH3.9基因,该基因全长为1 750bp。通过构建pEGAD-GH3.9过表达载体转化拟南芥,获得过表达GH3.9基因纯系转基因株系GH3.9ox-3和GH3.9ox-7。对拟南芥野生型(WT)和转基因株系(GH3.9ox-3和GH3.9ox-7)幼苗用不同光强和光质进行处理,结果显示:在蓝光、红光、远红光等不同光照强度下培养,过表达株系幼苗下胚轴的生长均明显受到抑制,且较野生型明显;采用不同光周期处理拟南芥幼苗,过表达幼苗下胚轴的伸长明显低于野生型;对成年植株表型进行观察,发现过表达株系植株矮小、雄蕊变短、果荚短小。研究表明:GH3.9基因参与了拟南芥生长发育调控,过表达GH3.9基因对拟南芥生长有抑制作用。     

聚乙二醇模拟水分胁迫筛选拟南芥突变体的新方法

在以聚乙二醇（PEG）模拟水分胁迫的条件下,分别在拟南芥种子萌发期和幼苗期以25％和30％为选择浓度,根据一种子是否萌发和幼苗能否存活为指标,筛选拟南芥抗水分胁迫突变体。此法操作简单,方便快捷,特别适合实验室进行大规模筛选。我们用这种方法筛选了T—DNA插入的拟南芥突变体库,得到78株可能是拟南芥抗水分胁迫的突变体。     

玉米ZmCI-1B启动子及其7个5'端缺失体在转基因拟南芥中的活性分析

将ZmCI-1B全长启动子及其7个5'端缺失的启动子片段的表达载体分别转化农杆菌GV3101,经PCR鉴定正确之后,以拟南芥为遗传转化受体,利用花序侵染法将各个表达载体转化拟南芥。用PCR法检测转化后的拟南芥植株,取阳性植株的幼苗或花、角果进行GUS组织化学染色。结果表明,ZmCI-1B启动子在拟南芥中的调控特性与玉米中的不同;ZmCI-1B启动子及其7个5'端缺失启动子转基因拟南芥植株中GUS的染色部位各异,说明了不同长度的启动子功能特性不同,推测其可能与启动子上的顺式作用元件有关。     

拟南芥ABI5基因对BR胁迫响应及其对下胚轴生长的调节作用

植物脱落酸不敏感蛋白5 (abscisic acid-insensitive 5,ABI5)是种子中大量表达的碱性亮氨酸拉链类型(basic leucine zipper,b ZIP)转录因子,在调节种子萌发和幼苗早期生长的脱落酸(abscisic acid,ABA)信号中起着核心作用。油菜素内酯(brassinosteroid,BR)是一种新型植物内源激素,具有调节植物生长发育和逆境胁迫响应等诸多生理功能。近期研究发现,油菜素内酯胁迫条件下,BR信号通路中BIN2 (BRASSINOSTEROID INSENSITIVE2)和BES1 (BRI1-EMS-SUPPRESSOR 1)通过抑制ABI5表达,促进拟南芥(Arabidopsis thaliana)种子萌发。为进一步探究BR胁迫下ABI5功能,本研究分析了种子萌发期ABI5表达特性,鉴定出拟南芥ABI5基因缺失突变体abi5-1并对BR胁迫下其功能进行解析。结果表明:ABI5在拟南芥干种子中大量表达并响应萌发期BR胁迫;正常条件下,abi5-1与野生型幼苗下胚轴无明显差异;BR胁迫下,abi5-1幼苗下胚轴明显长于野生型。本研究结果揭示了ABI5调控BR胁迫下拟南芥下胚轴生长,为深入了解ABI5调节植物发育的分子机制提供了依据。     

碳离子束辐照拟南芥介导外源基因转移的研究

采用700keV或4.0MeV碳离子束辐照拟南芥种子,通过对样品的显微摄影,发现随着辐照剂量的增加,碳离子束对种子表面的损伤逐渐加剧,特别是在4.0MeV碳离子束辐照下,当剂量达到1×1014ions·cm-2后,种皮局部逐渐被刻蚀殆尽,甚至造成种皮局部破裂。对拟南芥种子进行台盼蓝染色后的显微观测显示,碳离子束辐照可以导致拟南芥种皮细胞着染,在剂量较大的情况下,部分皮下细胞也可着染,表明碳离子束可作用到皮下细胞,为外源基因提供导入的通道。GUS基因导入后的组织化学检测表明:质粒pCAMBIA1301能够进入4.0MeV碳离子束辐照后的拟南芥种子,并在种子和幼芽中获得瞬间表达。     

黄梁木NcEXPA8基因提高拟南芥种子萌发速度的研究

为探究NcEXPA8基因的分子功能,该文以在黄梁木形成层区域中高表达的扩展蛋白基因NcEXPA8为研究对象,研究其在黄梁木种子萌发过程中的表达及其过表达对拟南芥种子萌发的影响。该文以黄梁木和拟南芥野生型(WT)(Col-0)种子以及转NcEXPA8基因的拟南芥T3代纯合体种子为实验材料,利用实时荧光定量RT-qPCR分析NcEXPA8基因在黄梁木种子萌发不同阶段的表达量,并分析NcEXPA8基因和拟南芥种子萌发内源相关基因在拟南芥WT和转基因不同株系萌发种子中的表达量,且对拟南芥WT种子和转基因T3代纯合体种子在不同处理和不同时间的萌发率进行比较。结果表明:NcEXPA8基因在黄梁木种子萌发不同阶段的表达量存在差异,在种壳破裂时表达量最高,随后降低。与拟南芥WT相比,过表达NcEXPA8基因不仅显著提高了种子的萌发速度,而且提高了对赤霉素的敏感性,降低了对脱落酸的敏感性,但未影响拟南芥内源相关结构基因的表达。该研究初步分析了黄梁木NcEXPA8基因在种子萌发中的功能,但其最终确定还需在黄梁木中进行验证。     

甘蓝型油菜的BR响应及BnBZL2基因的功能分析

对甘蓝型油菜(Brassica napus L.)与拟南芥(Arabidopsis thaliana L.)中保守的油菜素甾醇(Brassinosteroids,BR)信号相关基因进行对比分析,并以甘蓝型油菜品种‘沪油15’为材料,对BR信号通路相关同源基因进行了组织表达分析。结果显示,BR合成基因与信号组分在花和幼嫩种子中表达量更高;低浓度BR处理可以促进幼苗根的生长,高浓度BR处理则起抑制作用; BR合成抑制剂(Brassinozole,BRZ)处理可抑制黑暗条件下幼苗下胚轴的伸长; BR处理可以降低BR合成基因的表达水平,而BRZ处理则相反,表明甘蓝型油菜中BR信号增加能反馈抑制BR的合成。烟草(Nicotiana tabacum L.)瞬时表达实验结果发现,与拟南芥BZR1基因同源的甘蓝型油菜BnBZL2编码蛋白定位在细胞质和细胞核中,BR处理可增加BnBZL2的核定位。蛋白质免疫印迹检测结果显示,BR处理可增加去磷酸化BnBZL2的比例。本研究进一步模拟了拟南芥bzr1-1D功能获得性突变体对BnBZL2蛋白进行点突变(BnBZL2*),并构建载体转化拟南芥,黑暗条件下转基因植株幼苗对BRZ处理不敏感,提示BnBZL2*可提高转基因植株的BR信号水平。本研究结果表明甘蓝型油菜中存在与拟南芥相似且保守的BR信号通路和调控机制。     

拟南芥高迁移率族蛋白B族基因表达模式分析

为了解高迁移率族蛋白B族(HMGB)基因在拟南芥中的表达模式及作用方式,该研究克隆了拟南芥中5个编码HMGB的基因:AtHMGB1、AtHMGB2、AtHMGB3、AtHMGB4、AtHMGB5,并运用荧光实时定量PCR方法检测野生型拟南芥中以上5种基因在不同器官中的表达及在外源植物激素(ABA、2,4-D)处理前后的表达差异,选取AtHMGB2、AtHMGB4和AtHMGB5分别转化拟南芥并筛选出超表达株系,随即检测ABA诱导下超表达AtHMGB的转基因拟南芥的表型。研究证实:在野生型拟南芥中AtHMGB2在拟南芥各个器官中的表达量远高于其它家族成员,AtHMGB4和AtHMGB5在花、果荚和根中的表达略高于茎和叶;在ABA处理前后AtHMGB家族成员的表达水平有显著差异,其中AtHMGB2的表达被ABA显著负调控;ABA诱导下超表达AtHMGB2的转基因拟南芥与野生型相比出现萌发及生长迟缓现象,但超表达AtHMGB4与AtHMGB5的转基因拟南芥在ABA诱导下的种子萌发和幼苗生长与野生型相比差异不大。研究发现,AtHMGB家族成员在转录水平上响应ABA的方式各有不同,对理解AtHMGB家族成员的生物学功能提供了新的基础。     

质粒DNA物理形态和其它因素对获得可育转基因小麦的影响

本工作分析了不同形态质粒DNA和未成熟胚高渗透压处理对基因枪小麦转化体系的适用性.高渗处理对瞬间表达和转基因小麦的再生均有明显的促进作用.轰击之前对质粒DNA进行变性处理导致瞬间表达反应大幅度下降,但稳定转化频率(指从100个轰击未成熟胚得到的再生可育转基因植株数)与双链DNA相差不大.使用单链质粒DNA、线性双链质粒DNA和环状双链质粒DNA均可以得到转基因小麦植株.迄今已得到26个不同的转基因冬小麦株系和4个不同的转基因春小麦株系.这些转基因小麦大多数已产生种子,几个春小麦株系已获第二代种子.     

质粒DNA物理形态和其它因素对获得可育转基因小麦的影响

The applicability of hyperosmotic treatment and different configurations of plasmid DNA for stable transformation of wheat mediated by particle bombardment was investigated. Hyperosmotic treatment increased the frequency of transient expression and had also a positive effect on stable transformation. Denaturation of plasmid DNA prior to bombardment led to dramatic reduction of transient expression. However, there were no marked differences between single-stranded and double-stranded DNA in stable transformation. Single-stranded plasmid DNA, double-stranded plasmid DNA in linear state and double-stranded plasmid DNA in circular state could all be used to produce transgenic wheat plants. A total of 26 independent transgenic plants of winter wheat genotype Florida and 4 independent transgenic plants of spring wheat genotype Veery were obtained. Most transgenic plants have set seeds. T2 seeds of some spring wheat transgenic plants have also been harvested.     

离子注入法将外源DNA直接导入小麦的研究

通过离子束介导法将外源GUS基因直接导入小麦成熟种子.组织化学染色结果表明GUS基因的瞬时表达率可以达到70%以上.当代(Ru)PCR分析结果表明,阳性植株的频率与离子注入剂量有关,适宜的注入剂量为7×106离子/cm2.PCR-Southern和Southern B1ot分析结果表明外源基因已整合到小麦基因组中,说明离子束介导外源DNA直接导入小麦是可行的.另外还探讨了用离子束介导创造小麦远缘分子杂种的可能性.     

不同离子剂量介导外源薄荷全DNA转化对拟南芥菜生长发育的影响

在离子束介导外源全DNA转化中,外源DNA包含有供体的所有遗传信息,对受体的影响是多方面的,因此转化效果无法用统一的转化率来衡量,这也增加了转化过程中最佳转化条件的选择难度。本论文的主要工作是根据不同离子剂量介导外源全DNA转化的拟南芥菜在遗传和生理上的变化选择合适的转化剂量。剂量实验结果表明:拟南芥菜种子的剂量-存活率曲线为"马鞍型",在"鞍桥"上选择0.5×1017ions/cm2、1.5×1017ions/cm2和2.5×1017ions/cm2三个有代表性的剂量介导薄荷全DNA转化拟南芥菜,三个转化当代群体的出芽、成苗长势以及表型变异情况有明显的不同,综合分析表明:1.5×1017ions/cm2为离子束介导外源全DNA转化拟南芥菜的合适剂量。     

Gene transfer method for transient gene expression, stable transformation, and cotransformation of suspension cell cultures.

A new method was developed to study transient gene expression, stable transformation, and cotransformation in suspension cells, such as mouse myeloma and erythroleukemia cells. This method involves attachment of cells to a concanavalin A-coated tissue culture dish, treatment of cells with DEAE-dextran to adsorb plasmid DNA to the attached cells, and finally treatment with a 40% solution of polyethylene glycol to facilitate the uptake of DNA by the cells. Plasmids pSV2cat and pSV2neo were used as markers to optimize the conditions for transient gene expression and stable transformation, respectively, of mouse myeloma and erythroleukemia cells. This method was successfully used to obtain cotransformants of mouse myeloma cells.     

离子束介导大豆DNA导入小麦后其蛋白质和酯酶同工酶分析

对由离子束介导大豆DNA导入豫麦52获得的第四代两个转化株系9004和9002中的可育株籽粒和矮秆不育株杂交籽粒以及受体豫麦52籽粒,进行酯酶和可溶性蛋白的聚丙烯酰胺凝胶电泳分析。结果表明,可育株籽粒可溶性蛋白电泳在谱带上与受体存在差异,矮秆不育株材料的酯酶和可溶性蛋白电泳图谱谱带差异更明显。由此推测,外源大豆DNA导入受体后某些片段有可能插入受体基因组,从而导致基因突变或受体基因表达发生改变。     

Microprojectile-mediated transient and integrative transformation of rice embryogenic suspension cells: effects of osmotic cell conditioning and of the physical configuration of plasmid DNA

Microprojectile-mediated transient and integrative transformation frequencies in rice (Oryza sativa cv. Taipei 309) embryogenic suspension cells were studied as a function of various parameters. Mannitol at concentrations of 0.5 and 0.6 m was best for osmotic preconditioning of the cells for transient, but not for integrative transformation, for which sucrose yielded the best and most reliable results. Denaturation of the transforming plasmid DNA prior to bombardment improved transient and integrative transformation frequencies two to three fold. Delivery of double-stranded plasmids in linear form had no effect on transient transformation when compared to supercoiled plasmid DNA, but led to an overall two fold increase in integrative transformation frequency. This shows that optimized protocols for generating transgenic plants should not be based exclusively on transient gene expression assays. Received: 29 September 1997 / Revision received: 27 February 1998 / Accepted: 2 April 1998     

Transformation of peanut with a soybean vspB promoter‐uidA chimeric gene. I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants

Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut ( Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi‐quantitation of transient expression. One hundred and sixty hygromycin‐resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R₁ seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.     

Transient expression of the GUS gene in a unicellular marine green alga,<Emphasis Type="Italic">Chlorella</Emphasis> sp.<Emphasis Type="Italic">MACC/C95</Emphasis>, via electroporation

A transient expression system for a unicellular marine green alga,Chlorella sp.MACC/C95, was developed using a reporter GUS gene coded for by plasmid pBI121. The results demonstrated a high transformation efficiency could be achieved by using electroporation to deliver DNA into intact cells and the CaMV35S promoter to drive the foreign gene expression inChlorella sp.MACC/C95. The use of a carrier DNA coupled with osmosis treatment improved the transformation efficiency, while linearization of the plasmid had minor effects. Investigation of the effects of DNA concentration and growth phases ofChlorella sp.MACC/C95 on transformation efficiency indicated that the highest level of transient expression was observed when 6 μg mL⁻¹ of plasmid DNA and cells 2–6 days old were used.     

Particle-inflow-gun-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.): optimizing biological and physical parameters

The present study was conducted to optimize various biological and physical parameters for developing an efficient and reproducible gene transfer method for genetic transformation of buffel grass. Transformation was carried out using a helium-driven particle inflow gun (PIG). Embryogenic calli produced from mature seeds of buffel grass cv. CC-119 were separately bombarded with four plasmids, containing Actin (pAct1DX), Ubiquitin (pAHC-25; pAHC-27) and CaMV-35S (pCaMVGUS) promoters, coated on tungsten and gold particles. The efficiency of transformation was monitored through transient GUS expression. Different parameters, viz., the type of promoter, type and size of microcarrier, helium gas pressure, distance and time of bombardment, were standardized for delivering DNA into embryogenic calli. Bombardment with plasmid DNA carrying the actin promoter coated on 1.6 micro gold particles, at a helium pressure of 4 bars, a distance of 10 cm for 10 micro sec and 28 mm Hg vacuum in the chamber, produced the best result in transient GUS expression. The Actin promoter was found to be more efficient in driving expression of the GUS gene in buffel grass, followed by Ubiquitin and CaMV-35S promoters. Lower helium pressure was found to be sub-optimal, while higher pressure produced a smaller number of blue spots, probably due to excessive damage to the cells. Maximum of 385 blue spots was observed with gold particles of 1.6 micro size, whereas only 213 blue spots were recorded for tungsten particles of 1.0 micro size. The optimized parameters can be employed for genetic transformation of buffel grass with genes of agronomic importance.