用基因枪法将GUS基因导入烟草脱外壁花粉及其瞬间表达

以β－葡糖苷酸酶（β－ｇｌｕｃｕｒｏｎｉｄａｓｅ,ＧＵＳ）基因作为报告基因,用基因枪法将其导入烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）脱外壁花粉和作为对照系统的完整花粉,探讨了启动子及不同轰击条件对外源基因瞬间表达的影响,比较了二者的转化频率。结果表明：玉米花粉特异启动子能启动ＧＵＳ基因在脱外壁花粉和完整花粉中表达,而ＣａＭＶ３５Ｓ启动子则不能。靶距离越近,着弹率越高,ＧＵＳ基因瞬间表达频     

芸苔属脱外壁花粉的分离与人工萌发

芸苔属青菜（Ｂｒａｓｓｉｃａ ｃｈｉｎｅｎｓｉｓ）与紫菜苔（Ｂ． ｃａｍ ｐｅｓｔｒｉｓｖａｒ． ｐｕｒｐｕｒｅａ）的花粉经低温水合、热激、渗激三步程序,分离出大量具萌发能力的脱外壁花粉,脱外壁率可高达６０％ 以上。在含有碳源与氮源及Ｒｏｂｅｒｔｓ培养基盐成分的碱性ＰＥＧ 培养基中,首次使芸苔属脱外壁花粉萌发,萌发率可达３３％ ～４１％ 。在扫描电镜下观察了花粉脱外壁与萌发的过程。讨论了不同植物花粉脱外壁的方法与花粉壁生物学特点的对应关系,以及外壁对花粉萌发的可能作用     

植物遗传工程

921489单子叶植物LHCp启动子在转基因水稻中的表达〔英〕/Tada,Y.…2 EMBOJ一1991,10(7)一1803~1808〔译自DBA,1991,10(18),91-10498〕 通过电激将与编码水稻(Or夕za夕at苏公a)捕光叶绿素a/b光系统一n(LHCPll)结合蛋白启动子相连的户葡糖昔酸酶(GUS)基因(位于质粒PLHC4.4中)导入水稻。转化原生质体和愈伤培养物中由LHCP启动子控制的GUS基因表达远低于由花椰菜花叶病毒(CaMV)355启动子控制的,而在绿色器官中,LHCP启动子启动的LHCP-GUS活性比CaMV35S启动子启动尸的高10倍。在叶、茎和花组织中检测到LHCP一GUS基因的表…     

拟南芥atslA基因启动子的克隆和功能分析

通过PCR扩增,从拟南芥中克隆出atslA基因启动子(包括叶绿体转运肽),将此启动子与GUS基因相连构建植物瞬时表达载体,用基因枪法将之导入烟草进行瞬时表达。GUS基因检测分析表明,atslA基因启动子能特异的启动GUS基因在烟草叶片中高效表达。     

拟南芥ats1A基因启动子的克隆和功能分析

通过PCR扩增,从拟南芥中克隆出ats1A基因启动子(包括叶绿体转运肽),将此启动子与GUS基因相连构建植物瞬时表达载体,用基因枪法将之导入烟草进行瞬时表达。GUS基因检测分析表明,ats1A基因启动子能特异的启动GUS基因在烟草叶片中高效表达。     

番茄Lehsp23.8启动子的热诱导表达调控序列鉴定

番茄线粒体小分子热激蛋白(Lehsp23.8)启动子是典型的热诱导启动子。为了研究热激条件下该启动子的调控序列,本研究将不同长度的Lehsp23.8启动子序列与gus基因融合,构建5′缺失植物表达载体。然后用农杆菌介导法转化烟草,PCR及Southern blotting结果表明融合基因已经整合到烟草基因组中。GUS组织化学染色结果表明:不同长度Lehsp23.8启动子转基因植株热激处理后,在幼苗根、茎、叶以及花和果实中均表现出GUS活性,只是染色强弱有差异。叶片中GUS荧光活性测定结果表明:在热激处理条件下,565bp的Lehsp23.8启动子介导的GUS表达最强;而255bp的Lehsp23.8启动子介导的GUS表达最弱。说明Lehsp23.8启动子中255bp的序列即能满足该启动子的热激表达,-565bp～-255bp之间存在明显的增强子元件,而-871bp～-565bp之间的片段具有一定的抑制作用。     

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.     

参与水稻光合作用的PsbR3基因启动子的功能分析

本实验旨在研究水稻光合作用蛋白中各基因的表达模式. 采用RT-PCR和定量real-time PCR数据分析水稻不同组织的mRNA表达水平.结果显示,PsaK和PsbR3基因仅在茎、叶等绿色组织表达,而胚、胚乳部分均不表达.通过其启动子克隆、植物表达载体构建,以及农杆菌介导转化后,GUS组织染化分析和GUS荧光定量分析表明,两启动子均为组织特异性优势表达,PsbR3启动报告酶GUS在叶片中的表达活性为Actin启动子的3.29倍,而PsaK启动报告酶GUS在叶片中的表达活性低于Actin启动子的.这些初步结果提示,PsbR3启动子决定水稻绿色组织茎叶的优势表达,PsbR3基因可能参与水稻光合作用.     

环形电极介导的小麦基因转化

用环形电极电激法有效地将外源DNA导入完整的小麦幼胚组织中。电激的物理参数采用770V／cm场强、800μF电容、100μg／mL质粒DNA(含有bar和GUS双标记基因),幼胚被电激3次。经PCR和Southern杂交分析表明,外源基因已稳定整合到小麦基因组中,转化频率为7．5％,高于相同处理条件下基因枪法的转化频率(4．2％)。     

甘蓝型油菜BcNA1基因启动子在转基因烟草中对GUS基因表达的调控

通过PCR扩增,从甘蓝型油菜（Brassica napus)品种H165中克隆了种子贮藏蛋白基因BcNAl的启动子,将此启动子与GUS基因相连构建了植物表达载体,利用农杆菌介导法将其导入烟草,对转基因烟划GUS基因检测分析表明,BcNAl基因启动子能特异地启动GUS基因在种子中的表达;而且GUS酶的活性随着转基因烟草种子的发育而变化。同时还间接证明BcNA1基因的启动子在转基因烟草中的遗传传递方式符合盂德尔遗传定律。     

Exine-Detached Pollen of Nicotiana tabacum as an Electroporation Target for Gene Transfer

In developing alternative systems for plant transformation the authors investigated the use of male gametophyte as the foreign gene receptor. However, delivery of foreign DNA into pollen is difficult because of the existence of a thick exine, therefore a new experimental system was developed using exine-detached pollen (EDP) of Nicotiana tabacum as an electroporation target which was also compared with germinating pollen (GP) and pollen grains (P). A transient GUS expression assay was conducted to analyze the effects of different electroporation conditions and promoter activity. The pollen-specific promoter Zml3 from Zea mays mediated high level of GUS gene expression but CaMV 35S only had very low activity in both EDP and GP. The optimal field strength for gene transfer was obtained at 750 V/cm for EDP and 1250 V/cm for GP when the time constant of pulse was 13 ms. The GUS activity in EDP had a 5-fold increase as compared with GP and P respectively. The level of GUS gene expression was slightly increased when adding 10 % PEG into the electroporation buffer. This result indicates that pollen deprived of exine responds much better to foreign gene transfer than the previously used intact pollen grains and may be a better vector to introduce, via pollen tube, genes into the egg cell and offsprings.     

High level of GUS gene expression driven by pollen-specific promoters in electroporated lily pollen protoplasts

Gene constructs that contained the -glucuronidase (GUS) gene under the control of a pollen-specific Zm13 promoter from maize and a LAT52 promoter from tomato were introduced by electroporation into pollen protoplasts isolated from bicellular pollen grains of Lilium longiflorum. After 20 h in culture, the pollen protoplasts exhibited the apparent expression of GUS in a fluorometric assay. The GUS activity induced under the control of the Zm13 promoter was over 10 000 times higher than activity in the control (with no DNA or without electroporation). By contrast, the GUS gene was nearly silent in the lily microspore protoplasts and generative cell protoplasts. The GUS activity driven by the Zm13 and LAT52 promoters was also detected by a cytochemical assay. The frequency of blue-staining pollen protoplasts was about 70% in the case of the Zm13 promoter. The efficiency of gene transfer by electroporation was much higher than by particle bombardment. This protoplast-specific electroporation system is suitable for rapid and reliable examination of pollen-specific promoters, being as good as the particle bombardment system.     

β-Glucuronidase gene and green fluorescent protein gene expression in de-exined pollen of Nicotiana tabacum by microprojectile bombardment

 Gene constructs containing the β-glucuronidase (GUS) gene or green fluorescent protein (GFP) gene under the control of pollen-specific promoter Zm13-260 from maize were introduced by particle bombardment into de-exined pollen of Nicotiana tabacum. The de-exined pollen exhibited transient expression of the GUS or GFP gene as indicated by histochemical and fluorescent assay, respectively. The frequency of de-exined pollen transformation with the GUS or GFP gene was approximately 6 and 3 times higher, respectively, than that of pollen with intact walls, indicating that pollen deprived of the exine barrier responded better to foreign gene transfer than did the original. Cytological observation of GUS-expressing pollen grains showed that introduced gold particles were visible in the cytoplasm and vegetative nucleus as well as in the generative nucleus. GFP-expressing pollen tubes were observed in the style even after pollination. Received: 28 October 1997 / Revision accepted: 13 April 1998     

Expression of a foreign gene in electroporated pollen grains of tobacco

Summary The incorporation of genetically engineered DNA into pollen and subsequent fertilization of eggs by the transformed pollen would be a convenient method for producing genetically engineered seed. This method of pollen transformation would circumvent the need for other types of gene transfer methods such as the use of Agrobacterium tumefaciens, which has a limited host range and thus a limited capability for genetically engineering plants. It would also avoid the problems associated with the regeneration of some plants from tissue, cell, or protoplast culture after receiving foreign DNA. To this end, the genetically engineered plasmid DNA vector pBI221 containing the gene encoding -glucuronidase (GUS) was introduced by electroporation into germinating pollen grains of tobacco (Nicotiana gossei L.). Transient expression of the GUS gene was demonstrated by the presence of GUS activity in fluorometric assays of pollen extracts 24 h after the introduction of pBI221 via electroporation. Intact pBI221 was detected by Southern blotting procedures as a distinct DNA band in pollen extracts 1 h after electroporation. In addition, pBI221 was detected as a diffuse band of higher molecular weight DNA 24 h after electroporation, suggesting that some of the pBI221 was incorporated into the genome of the pollen.     

Reporter gene introduction and transient expression in protoplasts of Porphyra yezoensis

The transient expression of foreign genes in the protoplasts of Porphyrayezoensis was examined using three recombinant vectors, pYez-Rub-GUS, pYez-Rub-GFP and pYez-Rub-LUC, which were constructed with the promoter sequence of the ribulose-bisphosphate-carboxylase / oxygenase (Rubisco) gene as a promoter and the bacterial β-glucuronidase (GUS), mutant of green fluorescent protein (S65T-GFP) and firefly luciferase (LUC) genes, respectively, as reporter genes. When the pYez-Rub-GUS was introduced into protoplasts by electroporation, cells stained dark blue by indigotin were observed after the histochemical GUS assay. GUS activity was also detected by quantitative enzyme assays with a chemiluminescent substrate. When the pYez-Rub-GFP was electroporated into protoplasts, the expression of GFP could be detected in vivo observations with fluorescence microscopy. However, the rates of gene expression cells to the total number of cells were different between the GUS and GFP genes. LUC activity was also detected by assay with a chemiluminescent substrate after the introduction of pYez-Rub-LUC into protoplasts, although the activity levels were considerably lower. Relatively high expression rates of introduced GUS genes were observed 3 to 5 days after electroporation. These results show that the promoter sequence of the chloroplast Rubisco gene functions as a promoter of foreign gene expression and that transient expression occurred in protoplasts of P. yezoensis after the introduction of foreign genes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of a Novel Pollen-Specific Promoter from Wheat (Triticum Aestivum L.)

PSG076 is a pollen-specific gene isolated from wheat. The 1.4-kb promoter upstream of the ATG start codon was isolated by inverse-PCR (IPCR). To determine its activity, the PSG076 promoter was fused with the ??-glucuronidase (GUS) reporter gene and introduced into tobacco. Histochemical analysis in transgenic tobacco showed that GUS activity was detected in late bicellular pollen grains and increased rapidly in mature pollen. GUS activity was also detected in pollen tubes of transgenic tobacco. No GUS activity was found in other floral and vegetable tissues. These results indicate that the PSG076 promoter directs pollen-specific activity at late stages of pollen development and pollen tube growth. Deletion analysis showed that a 0.4?kb fragment of the promoter was enough to confer pollen-specific expression.     

Transformation system of rice suspension-cultured microcolonies by Electroporation

For establishing a transformation system of rice (Oryza sativa), after three days of culture embryogenic suspension-cultured cell clusters were enzymatically macerated for 2 hours in electroporation buffer containing 2% cellulase and filtered through 550, 400, 250 and 100 μm stainless mesh. Filtered embryogenic microcolonies of 100–250 μm with pBI121 were electroporated at 400 V/cm for 1.2 ms. Four weeks after the electroporation, stable transformed calli were obtained at a frequency of 72% on the selection medium containing 100 mg/L kanamycin. GUS gene in the genomic DNA among 20 out of 22 putative transformed calli lines were detected by PCR analysis. The expression of GUS gene into the kanamycin-resistance calli was confirmed by spectrophotometric assay and histochemical assay of GUS activity. In a histochemical study of the transgenic rice regenerants, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in leaf vein and root apex.     

Factors influencing transient gene expression in electroporated tall fescue protoplasts

For the rapid establishment of optimal conditions for a genetic transformation system for tall fescue, several factors influencing transient gene expression were studied in protoplasts, after the reporter β-glucuronidase gene was introduced by electroporation. In a time-course study of transient gene expression, GUS activity peaked at 24 h after electroporation. Among the different field strength conditions tested, maximum GUS activity was observed at 750 V/cm. Increases in the amount of plasmid DNA to 80 μg/ml led to increased GUS activity. GUS activities increased in linear fashion with increasing protoplast densities up to 2 × 10⁶/ml. Age of suspension cells from which protoplasts were derived influenced transient expression with maximum GUS activity obtained in 3- and 5-day-old suspensions. These results show that monocot and dicot protoplasts respond similarly in electroporation.     

The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes

The Arabidopsis thaliana MALE STERILITY 2 ( MS2 ) gene product is involved in male gametogenesis. The first abnormalities in pollen development of ms2 mutants are seen at the stage in microsporogenesis when microspores are released from tetrads. Expression of the MS2 gene is observed in tapetum of wild-type flowers at, and shortly after, the release of microspores from tetrads. The MS2 promoter controls GUS expression at a comparable stage in the tapetum of transgenic tobacco containing an MS2 promoter–GUS fusion. The occasional pollen grains produced by mutant ms2 plants have very thin pollen walls. They are also sensitive to acetolysis treatment, which is a test for the presence of an exine layer. The MS2 gene product shows sequence similarity to a jojoba protein that converts wax fatty acids to fatty alcohols. A possible function of the MS2 protein as a fatty acyl reductase in the formation of pollen wall substances is discussed.