拟南芥冷诱导型启动子CBF 3的克隆及活性检测

目的：构建冷诱导型启动子CBF3基因的植物表达载体,并将其转入烟草。方法：以拟南芥基因组DNA为模板,通过特异PCR扩增,克隆冷诱导表达启动子CBF3（C-repeat binding factor）。用CBF3启动子替换pBI121载体上的35S启动子构建新的载体pBC-GUS,通过农杆菌介导的叶盘法转化烟草。结果：获得了转基因烟草,转基因烟草的GUS组织化学染色及PCR分析结果表明,在低温诱导下,CBF3启动子可增强GUS基因表达。结论：CBF3启动子可应用于植物抗冷基因工程研究。     

LE-ACS6启动子在LE-ACS6::GUS转基因拟南芥中的特异性

通过对不同发育时期LE-ACS6::GUS转基因拟南芥中GUS表达特异性的研究,证明LE-ACS6基因的启动子在拟南芥中也表现启动参与第一系统乙烯合成的关键酶基因的活性.在转基因拟南芥中,LE-ACS6启动子还表现响应外源生长素处理、伤害处理等多种刺激因子的特点.     

AtSTP3启动子控制的外源基因在转基因水稻中的特异表达研究

利用PCR技术从哥伦比亚型拟南芥基因组DNA中分离了AtSTP3绿色组织特异表达的启动子,序列分析表明,扩增片段(1774bp)与已报道序列的相应区域同源性达99.9%。将其与GUS报告基因融合在一起,构建了植物表达载体,并由农杆菌介导法导入水稻品种‘中花11’中。对转基因水稻植株中的GUS活性进行定性与定量测定结果表明,AtSTP3启动子可驱动GUS报告基因在转基因水稻植株叶片中特异性表达,而在根和种子等器官中不表达或表达活性极弱,AtSTP3启动子表现出明显的组织特异性。     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.     

拟南芥2-甲基-6-叶绿基-1,4-苯醌甲基转移酶启动子的分离及表达特性分析

维生素E是一类人体必需的脂溶性抗氧化剂, 具有重要的生理功能。2-甲基-6-叶绿基-1,4-苯醌甲基转移酶(MPBQ MT)是天然维生素E合成途径中的关键酶之一, 催化MPBQ甲基化, 生成DMPBQ。从拟南芥分离了MPBQ MT基因1018bp的启动子序列, 构建了含该启动子和GUS报告基因的植物表达载体, 通过农杆菌介导转化拟南芥, 获得了转基因植株。GUS组织化学染色结果表明, 在MPBQ MT启动子驱动下, 报告基因GUS在拟南芥的茎、叶、花萼、雄蕊、种荚均有表达, 且在茎、叶、种荚中表达量较高, 而在根、花瓣和种子中则没有观察到GUS基因的表达, 表明MPBQ MT基因可能仅在拟南芥幼嫩茎、叶、种荚等绿色组织中特异性高表达。     

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     

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.     

Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in Arabidopsis

The experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space. For this purpose, a binary vector, 962-UAS::GUS, was constructed in which the ALCR activator and β-glucuronidase (GUS) reporter gene were placed under the control of upstream activator sequence (UAS) elements and the alcA response element, respectively. Three different GAL4/VP16-UAS enhancer trap lines of Arabidopsis were transformed, resulting in transgenic plants in which GUS activity was detected only on ethanol induction and exclusively in the predicted tissues of the enhancer trap lines. As a library of different enhancer trap lines with distinct green fluorescent protein (GFP) patterns exist, transformation with a similar vector, in which GUS is replaced by another gene, would enable the control of the time and place of transgene expression. We have constructed two vectors for easy cloning of the gene of interest, one with a polylinker site and one that is compatible with the GATEWAY™ vector conversion system. The method can be extended to other species when enhancer trap lines become available.     

FLP-mediated recombination for use in hybrid plant production

We have studied the feasibility in Arabidopsis of using a site-specific recombination system FLP/FRT, from the 2 microm plasmid of yeast, for making plant hybrids. Initially, Arabidopsis plants expressing the FLP site-specific recombinase were crossed with plants transformed with a vector containing kanamycin-resistance gene (npt) flanked by FRT sites, which also served to separate the CaMV35S promoter from a promoterless gusA. Hybrid progeny were tested for excision of the npt gene and the positioning of 35S promoter proximal to gusA. GUS activity was observed in the progeny of all crosses, but not in the progeny derived from the self-pollinated homozygous parents. We then induced male sterility in Arabidopsis plants using the antisense expression of a pollen- and tapetum-specific gene, bcp1, flanked by FRT sites. Upon cross-pollination of flowers on the same male-sterile plants with pollen from FLP-containing plants, viable seeds were produced and the progeny hybrid plants developed normally. Molecular analyses revealed that the antisense expression cassette of bcp1 had been excised in these plants. These results show for the first time that a site-specific recombinase can be used to restore fertility in male-sterile plants, providing an alternative method for the production of hybrid seeds and plants.     

35S驱动Pib基因启动区和编码区的转基因初步分析

将包含Pib基因启动区及下游完整编码区的9.9 kb DNA片段克隆到双元载体pPZP2Ha3(+)中, 构建了35S驱动的正义表达载体pNAR701(20.3 kb); 同时将Pib基因编码区6 986~9 392 bp之间的DNA片段, 克隆到双元载体pPZP2Ha3(-)中, 构建了35S驱动的反义表达载体pNAR703(12.8 kb); 用农杆菌介导法转入中感稻瘟病水稻品种R109中。PCR、Southern blot鉴定以及转基因T0代种子的潮霉素抗性鉴定证明, 目的基因已经整合到R109基因组中, 并能在后代稳定遗传。Northern blot分析表明含有启动区及下游完整编码的Pib基因片段在35S驱动下能够在转基因后代中表达。对T1代苗期转基因植株和分蘖期离体叶片进行抗稻瘟病初步分析, 结果显示pNAR701转基因植株对稻瘟病生理小种ZD1和ZG1的抗性较对照增强, 而转反义片段的pNAR703转基因植株对稻瘟病的抗性较对照减弱。     

Cell Specific,Cross-Species Expression of Myrosinases in Brassica Napus,Arabidopsis Thaliana and Nicotiana Tabacum

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.     

Regulatory sequences of Arabidopsis drive reporter gene expression in nematode feeding structures.

In the quest for plant regulatory sequences capable of driving nematode-triggered effector gene expression in feeding structures, we show that promoter tagging is a valuable tool. A large collection of transgenic Arabidopsis plants was generated. They were transformed with a beta-glucuronidase gene functioning as a promoter tag. Three T-DNA constructs, pGV1047, p delta gusBin19, and pMOG553, were used. Early responses to nematode invasion were of primary interest. Six lines exhibiting beta-glucuronidase activity in syncytia induced by the beet cyst nematode were studied. Reporter gene activation was also identified in galls induced by root knot and ectoparasitic nematodes. Time-course studies revealed that all six tags were differentially activated during the development of the feeding structure. T-DNA-flanking regions responsible for the observed responses after nematode infection were isolated and characterized for promoter activity.     

A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency

We represent here the GST-MAT vector system. The R recombinase gene of the site-specific recombination system R/RS from Zygosaccharomyces rouxii was fused to the chemical inducible promoter of the glutathione-S-transferase (GST-II-27) gene from Zea mays. Upon excision, the isopentenyltransferase (ipt) gene that is used as a selectable marker gene is removed. When the cauliflower mosaic virus 35S promoter (CaMV 35S) was used to express R recombinase, 67% of the marker-free transgenic plants had more than three transgene copies. Because the CaMV 35S promoter transiently and efficiently excised the ipt gene before callus and adventitious bud formation, the frequency of emergence of the ipt-shooty explants with a single T-DNA copy might be reduced. In this study we show that the GST-MAT vector efficiently produced transgenic ipt-shooty explants from 37 (88%) out of 42 differentiated adventitious buds and marker-free transgenic plants containing the GUS gene from five (14%) out of 37 ipt-shooty lines. Furthermore, the GST-MAT vector also induced two marker-free transgenic plants without the production of ipt-shooty intermediates. Southern blot analysis showed that six (86%) out of seven marker-free transgenic plants had a single GUS gene. This result suggests that the GST-MAT vector is useful to generate high frequency, marker-free transgenic plants containing a single transgene.     

Alterations of Root and Fiber in Transgenic Cotton Plants with Chimeric Ph/P-ipt Gene Expression

The seed-specific phaseolin promoter (Ph/P) was fused to an ipt gene, then was cloned to a plant expression vector containing a gus gene driven by a 35S promoter. Cotton (Gossypium hirsutum L.) plants were transformed through pollen tube pathway methods. After seed germination, histochemical staining of the roots demonstrated that 32 GUS positive plants were obtained and three of which contained the chimeric Ph/P-ipt transgene as confirmed by PCR analysis. An immunosorbent assay showed that two of the three transgenic cotton lines contained higher levels of zeatin equivalents in seeds than the control. Seedling development of these two transgenic lines differed from the control in a reduction of the shoot growth, showing a stunted phenotype as expected, but a surprisingly developed root system with a 3-4 fold fast-growing lateral roots. In addition, fibers (seed-hairs) of the two transgenic cotton lines were considerably shorter than those of the control. These results indicate that genetic engineering may be used to manipulate the development of cotton plants, particularly cotton fibers.     

种子特异表达ipt转基因棉花根和纤维的改变

将种子特异表达的菜豆蛋白启动子（Ｐｈ／Ｐ）与ｉｐｔ基因融合,构建了植物表达载体。该载体含有由３５Ｓ启动子驱动的ｇｕｓ报告基因。应用该载体通过花粉管通道法转化棉花（Ｇｏｓｓｙｐｉｕｍ ｈｉｒｓｕｔｕｍ Ｌ．）,种子萌发后剪取幼根进行ＧＵＳ染色,获得ＧＵＳ阳性植株２３棵。ＰＣＲ检测证明有３棵ＧＵＳ阳性植株中含有Ｐｈ／Ｐ－ｉｐｔ基因,并进一步用地高辛标记的ＤＮＡ探针作杂交验证了上述结果。分析表明２棵转基