外源ble基因在杜氏盐藻中的瞬时表达

利用电击法将带有ble基因的pSP124S转入杜氏盐藻细胞内进行瞬时表达．研究了外源基因在盐藻内的存留及表达情况,确定了合适的电击转化条件,发现利用电击法可以使大量的质粒导入盐藻细胞,质粒在细胞中逐渐降解但至少96h内可以检测得到。外源启动子能够使ble基因有效转录,转录至少可以持续72h,ble基因能够在盐藻细胞中正确翻译,可以作为盐藻遗传转化研究的筛选标记。     

外源ble基因在杜氏盐藻中的瞬时表达

利用电击法将带有ble基因的pSP124S转入杜氏盐藻细胞内进行瞬时表达,研究了外源基因在盐藻内的存留及表达情况,确定了合适的电击转化条件,发现利用电击法可以使大量的质粒导入盐藻细胞,质粒在细胞中逐渐降解但至少96h内可以检测得到,外源启动子能够使ble基因有效转录,转录至少可以持续72h,ble基因能够在盐藻细胞中正确翻译,可以作为盐藻遗传转化研究的筛选标记。     

SOEing 法构建EGFP真核表达载体及其在杜氏盐藻中的表达

通过重叠区扩增基因拼接法（Gene splicing by overlap extension,SOEing）构建含有杜氏盐藻（Dunaliella salina）硝酸盐还原酶（NR）基因5′-上游序列（Pnr）and 3′-端序列（Tnr）的EGFP真核表达载体,并将其转化杜氏盐藻。利用改进的SOEing法,将杜氏盐藻NR基因Pnr与报告基因EGFP cDNA融合,并与pEGM-7zf克隆载体连接,顺序将盐藻NR基因Tnr序列与融合片段相连,构建含Pnr-EGFP-Tnr表达盒的盐藻真核表达载体p7NET。电击法转化杜氏盐藻,在盐藻转化株中观察到了EGFP的瞬时表达。此研究为转基因杜氏盐藻研究和成功建立杜氏盐藻生物反应器奠定了实验基础。     

核基质结合区提高报告基因在稳定转化的杜氏盐藻中的表达

前期的研究从杜氏盐藻（Dunaliella salina）中分离出一核基质结合区（matrix attachment region,MAR）片段——DSM1.实验证实,它在体外能与核基质结合且具有MAR的典型特征.为研究其在转基因盐藻中的作用,构建了RbcS启动子驱动、氯霉素乙酰转移酶（chloramp henicol acetyltransferase,CAT）基因为报告基因及表达盒两侧含DSM1 MAR的表达载体.电击法转化盐藻,随机挑选20株稳定转化的盐藻藻株,分析CAT酶活性.结果表明,在稳定转化的盐藻细胞中,MAR能使报告基因CAT的表达水平比对照藻株提高1-5倍,不同藻株之间个体表达的差异性也有所降低     

绿色荧光蛋白基因作为报告基因在水稻基因转化中的应用研究

本研究中 ,构建了含有编码绿色荧光蛋白的改进型基因质粒pJPM5。用基因枪法分别把pJPM5和另一带有绿色荧光蛋白基因的质粒pSBG70 0转入水稻TNG6 7愈伤组织。用South ern杂交法证实了转基因的存在 ,而且表明多数转基因植株含有 1到 8个拷贝的转基因。取 2个月的转基因植株上的叶片用于分析绿色荧光蛋白基因表达。用SLM - 80 0 0荧光分析仪定量测定绿色荧光蛋白。多数转基因植株具有很高的绿色荧光蛋白信号。虽然水稻植株有少量自发荧光 ,但是绿色荧光蛋白基因表达出的绿色荧光蛋白信号比植株的自发荧光强得多 ,其测定不会受自发荧光的太大影响。在荧光显微镜下观察到了绿色荧光蛋白基因的表达。借助观察分析绿色荧光蛋白基因的瞬时表达 ,本研究还发现基因枪法转化中 ,如果两枪的气压为90 0psi& 135 0psi,比两枪的气压都为 90 0psi或者 135 0psi更好 ,因其能使质粒进入更多的细胞。研究结果表明 ,绿色荧光蛋白基因可以作为水稻 (甚至小麦、玉米 )转基因研究中的报告基因。研究还显示 ,MAR序列能明显增强绿色荧光蛋白基因的表达能力 (这一结果在另文讨论 ) .     

杜氏盐藻外源基因稳定表达系统的构建

建立了一种单细胞海水绿藻--杜氏盐藻(Dunaliella salina Teod.)的外源基因稳定表达系统.通过电激法将携带乙肝病毒表面抗原基因(HBsAg)和氯霉素乙酰转移酶基因(CAT)的质粒转入盐藻细胞内,CAT基因为筛选基因.PCR和Southern杂交结果显示,HBsAg基因已经整合到盐藻基因组中.Northern杂交结果表明,转化成功细胞内的该基因已转录成mRNA.HBsAgELISA和Western杂交检测证明,HBsAg蛋白在转化的盐藻细胞内稳定地表达.同时,PCR和Southern杂交显示,CAT基因也已整合到盐藻基因组中.且CATELISA检测证明,CAT蛋白在转化体中也已稳定地表达.进一步对转化盐藻进行无氯霉素筛选培养,60代后,HBsAg基因依然稳定地存在并表达.本实验第一次报道了外源基因在杜氏盐藻细胞内的稳定表达.     

甘油对杜氏盐藻电击转化的影响

通过在HEPES电击缓冲液中添加不同浓度的甘油,讨论了甘油对电转前细胞存活率的影响;通过在盐藻培养基中添加不同浓度的甘油,讨论了甘油对盐藻细胞生长的影响;使用含有不同浓度甘油的HEPES缓冲液介导质粒载体转入盐藻细胞,比较了甘油对于转化率的影响。实验结果表明,在电击缓冲液中添加0.5mol/L甘油能有效提高细胞存活率,促进转化细胞恢复生长,从而获得最佳转化效果。因此,0.5mol/L甘油可作为杜氏盐藻电击转化过程中一种良好的稳渗剂。     

静态荧光光谱法研究杜氏盐藻的生长规律

测量了杜氏盐藻不同生长期的静态荧光光谱,得出了杜氏盐藻生长规律曲线。通过与传统的分光光度计法和血球计数板法研究盐藻生长规律的比较发现,该方法反映的是活盐藻细胞浓度的变化情况,更能反映盐藻实际生长规律;不仅操作简便,灵敏度高,而且可以实现远距离非接触测量。     

G418和氯霉素作为转基因盐藻的抗生素筛选标记

寻找可以作为筛选标记的抗生素是进行盐藻转基因的前提,尤其是分子更小,反应更灵敏的抗生素,通过降低培养液中NaCl浓度至0.25mol/L以后,进行抗生素筛选,得到了一种可以作为筛选标记的抗生素-G418,700μg/ml G418在第5d即比较完全地杀死野生型盐藻细胞,由于未知的原因,实验中氯霉素表现出的抑制浓度是400μg/ml,作用时间是10d以上,不同于已有报道。在含1.5mol/L NaCl的盐藻培养基中,氯霉素,潮霉素,壮观霉素,G418,氨苄青霉素,卡那霉素都没有表现出杀死盐藻细胞的能力,这说明G418在低盐培养基中可以作为一种快速有效的筛选转基因盐藻的抗生素筛选标记。     

Expression of CaMV35S-GUS gene in transgenic rice plants

Summary To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter in a monocotyledonous plant, rice (Oryza sativa L.), a transgenic plant and its progeny expressing the CaMV35S-GUS gene were examined by histochemical and fluorometric assays. The histochemical study showed that -glucuronidase (GUS) activity was primarily localized at or around the vascular tissue in leaf, root and flower organs. The activity was also detected in the embryo and endosperm of dormant and germinating seeds. The fluorometric assay of various organs showed that GUS activity in transgenic rice plants was comparable to the reported GUS activity in transgenic tobacco plants expressing the CaMV35S-GUS gene. The results indicate that the level of expression of the CaMV 35S promoter in rice is similar to that in tobacco, a dicotyledonous plant, suggesting that it is useful for expression of a variety of foreign genes in rice plants.     

超量表达IPT和KN1基因对转基因烟草生长发育的影响

为了比较异戊烯转移酶基因IPT和玉米homeobox基因KNI超量表达对植物生长发育的影响,将35S：：IPT和35S：：KNI分别导入烟草。观察发现,过量表达IPT和KNI基因对植株再生频率、形态、生长周期和顶端优势等方面的影响均相似,但在生根和开花结籽方面,35S：：IPT转基因植物所受影响更显著。细胞分裂素含量检测表明,35S：：IPT转基因植物叶片中细胞分裂素的含量高于35S：：KNI转基因植物。     

盐生杜氏藻(Dunaliella salina)cDNA文库构建及功能基因筛选

采用Qiagen公司的植物总RNA提取技术、Clontech公司的CreatorTM技术平台以及SMARTTM技术进行cDNA文库构建.从杜氏藻中提取出了高质量的总RNA,通过PowerScript反转录酶反转录杜氏藻的总RNA,采用LD-PCR、酶处理等方法对cDNA进行等比例扩增、纯化,同时使用CHROMASPIN-400柱子将cDNA分段化,最后将长片段连入pDNR-LIB质粒,1.5 kV,25 μ F电转化大肠杆菌JM109,得到含1.5×106个克隆子的原始文库,滴度为1.5×106cfuml-1.结合酶切和PCR,对该文库的质量进行了鉴定和统计,文库的平均片段插入长度为1.5kb.采用烯醇酶和UDP葡萄糖脱氢酶的EST作为同源探针,对文库中的功能基因进行筛选,并采用放射性原位杂交法,对扩增文库进行了初筛和复筛,得到了含这两条基因全编码序列的cDNA,烯醇酶为1.8kb,UDP葡萄糖脱氢酶为1.9kb,为今后对该种进行大规模功能基因组学研究奠定基础.     

The use of the luciferase reporter system forin planta gene expression studies

The properties of the firefly luciferase (LUC) make it a very good nondestructive reporter to quantify and image transgene promoter activity in plants. The short half-life of the LUC mRNA and protein, and the very limited regeneration of the LUC protein after reacting with luciferin, enables monitoring of changes in gene activity with a high time resolution. However, the ease at which luciferase activity is measuredin planta, using a light sensitive camera system (2D-luminometer), contrasts sharply with the complications that arise from interpreting the results. A variegated pattern of luciferase activity, that is often observed inin planta measurements, might either be caused by differences in influx, availability of the substrates (luciferin, oxygen, ATP) or by local differences in reporter gene activity. Here we tested the possible contribution of differences in the availability of each substrate to the variegatedin planta luciferase activity, and we show whenin planta luciferase activity is measured under substrate equilibrium conditions and can be related to the promoter activity of the reporter gene. Furthermore, we demonstrate the effects of protein stability, apparent half-life of luciferase activity, regeneration of luciferase and pH on thein vivo andin vitro luciferase measurements. The combined results give the prerequisites for the correct utilisation of the luciferase reporter system, especially forin vivo gene expression studies in plant research.     

A METHOD FOR THE CRYOCONSERVATION OF DUNALIELLA SALINA (CHLOROPHYCEAE): EFFECT OF GLYCEROL AND COLD ADAPTATION1

The unicellular green alga Dunaliella salina Teod. was frozen according to the following procedure: 3 days cold adaptation at 4°C, addition of 3.5 M glycerol as a cryoprotectant, slow cooling to –40°C, immersion in liquid nitrogen, and rapid thawing. The survival rate was higher when cells were grown, before freezing, in the presence of 2 M NaCl instead of 1 M NaCl (78 and 48% survival, respectively). This difference is probably due to the intracellular amount of glycerol, which increases with external NaCl concentration and, therefore, may enhance cell protection. Although cells grown in 4 M NaCl accumulated a large amount of glycerol in response to osmotic stress, they did not withstand freezing. The use of cryoprotectant was absolutely necessary for the cells to recover from storage at –196°C. Glycerol was used because it is naturally produced by Dunaliella salina and therefore is not toxic. Provided it was added slowly to avoid osmotic shock, 3.5 M glycerol gave better results than 1M glycerol (48 and 18% survival, respectively). Cold adaptation in the dark increased postthaw viability. Cells grown in 1 M or 2 M NaCl had a survival rate of 48 and 78%, respectively, when cold-adapted, against 10 and 42% when not cold-adapted. This adaptation could be due to the synthesis, at low temperature, of specific proteins because two bands (28–29 kDa) appeared when electrophoretically separated proteins from cold-adapted cells and control cells were compared. Also, it could be due to the degradation of starch that occurs in the dark and leads to glycerol accumulation. Our procedure has never been used to cryopreserve microalgae and could enhance reported survival rates.