A binary vector for transferring genomic libraries to plants.

The transformation of mutant plants with a complete recombinant library derived from wild-type DNA followed by assay of transformed plants for complementation of the mutant phenotype is a promising method for the isolation of plant genes. The small genome of Arabidopsis thaliana is a good candidate for attempting this so-called shotgun transformation. We present the properties of an A. thaliana genomic library cloned in a binary vector, pC22. This vector, designed to introduce genomic libraries into plants, contains the oriV of the Ri plasmid pRiHR1 by which it replicates perfectly stably in Agrobacterium. Upon transfer of the library from E. coli to A. tumefaciens large differences in transfer efficiencies of individual recombinant clones were observed. There is a direct relation between transfer efficiency and stability of the recombinant clones both in E. coli and A. tumefaciens. The stability is independent of the insert size, but seems to be related to the nature of the insert DNA. The feasibility of shotgun transformation and problems of statistical sampling are discussed.     

Gene rescue in plants by direct gene transfer of total genomic DNA into protoplasts.

To study the possibility of gene rescue in plants by direct gene transfer we chose the Arabidopsis mutant GH50 as a source of donor DNA. GH50 is tolerant of chlorsulfuron, a herbicide of the sulfonylurea class. Tobacco protoplasts were cotransfected with genomic DNA and the plasmid pHP23 which confers kanamycin resistance. A high frequency of cointegration of the plasmid and the genomic DNA was expected, which would allow the tagging of the plant selectable trait with the plasmid DNA. After transfection by electroporation the protoplasts were cultivated on regeneration medium supplemented with either chlorsulfuron or kanamycin as a selective agent. Selection on kanamycin yielded resistant calluses at an absolute transformation frequency (ATF) of 0.8 x 10(-3). Selection on chlorsulfuron yielded resistant calluses at an ATF of 4.7 x 10(-6). When a selection on chlorsulfuron was subsequently applied to the kanamycin resistant calluses, 8% of them showed resistance to this herbicide. Southern analysis carried out on the herbicide resistant transformants detected the presence of the herbicide resistance gene of Arabidopsis into the genome of the transformed tobacco. Segregation analysis showed the presence of the resistance gene and the marker gene in the progeny of the five analysed transformants. 3 transformants showed evidence of genetic linkage between the two genes. In addition we show that using the same technique a kanamycin resistance gene from a transgenic tobacco could be transferred into sugar beet protoplasts at a frequency of 0.17% of the transformants.     

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。     

The Arabidopsis genes RPW8.1 and RPW8.2 confer induced resistance to powdery mildew diseases in tobacco

Plant disease resistance (R) gene products recognize pathogen avirulence (Avr) gene products and induce defense responses. It is not known if an R gene can function in different plant families, however. The Arabidopsis thaliana R genes RPW8.1 and RPW8.2 confer resistance to the powdery mildew pathogens Erysiphe orontii, E. cichoracearum, and Oidium lycopersici, which also infect plants from other families. We produced transgenic Nicotiana tabacum, N. benthamiana, and Lycopersicon esculentum plants containing RPW8.1 and RPW8.2. Transgenic N. tabacum plants had increased resistance to E. orontii and O. lycopersici, transgenic N. benthamiana plants had increased resistance to E. cichoracearum, but transgenic L. esculentum plants remained susceptible to these pathogens. The defense responses induced in transgenic N. tabacum and N. benthamiana were similar to those mediated by RPW8.1 and RPW8.2 in Arabidopsis. Apparently, RPW8.1 and RPW8.2 could be used to control powdery mildew diseases of plants from other families.     

单子叶植物表达载体的构建及农杆菌介导的玉米遗传转化的研究

构建了单子叶植物表达载体pCUA-tr-cat-als,其中含有豌豆过氧化氢酶基因cat和突变的乙酰乳酸合成酶基因als,分别由玉米ubi启动子和花椰菜花叶病毒35S启动子启动。以玉米昌7-2种子苗的茎尖分生组织为受体,用农杆菌介导法首次将目的基因定向转入玉米叶绿体中。以als基因作为选择标记,以氯磺隆为选择剂进行筛选获得一定数量的转基因植株。经PCR分析,可初步确定目的基因已经整合到玉米基因组中。     

灰绿藜液泡膜焦磷酸酶基因（CgVP1）过表达提高拟南芥的耐盐性。

克隆获得包含完整开放阅读框（0RF）的灰绿藜液泡膜焦磷酸酶基因（CgVP1）cDNA序列,构建成基因表达载体pCAMBIA1301．1-CgVPl后,利用根癌农杆菌介导的花序浸染法转化拟南芥,再以抗性筛选方法获得了T3代纯合的转基因植株,经检测外源目的基因已经整合到拟南芥基因组中并能正常表达。分析结果表明,在拟南芥中过表达劬VP1基因后提高了植株抗盐胁迫的能力。     

百合查尔酮合成酶基因克隆及其转化烟草的花色表达分析

以‘西伯利亚’百合为试材,利用PCR技术克隆了查尔酮合成酶基因(CHS),构建了CHS基因的正义和反义植物表达载体,采用农杆菌介导法转化烟草叶盘,获得了转正义CHS基因的本明烟草18株,转反义CHS基因的普通烟草21株,总转化率为26.0%。高效液相色谱法(HPLC)检测结果显示,正义CHS转基因的本明烟草类黄酮含量升高14.0%～59.7%,反义CHS转基因的普通烟草类黄酮含量降低44.5%～76.4%。花色观察结果显示,正义转基因烟草的花瓣颜色未见变化,反义转基因烟草部分植株的花瓣颜色变浅。研究表明,CHS基因遗传转化是进行花色调控的有效手段之一。     

Pttkn1基因异位表达对烟草叶片形态的影响

以红花烟草(Nicotiana tabacum L.)叶片为材料,利用农杆菌介导的叶盘转化法将Pttkn1基因整合到烟草基因组中,获得了转基因植株,研究了该基因对植物形态发生和维管形成的影响,并对其进行了RT-PCR分析。结果表明,转基因烟草植株的叶片形态和叶脉发生了多种变化,包括叶片对称性丧失、出现裂片、皱缩、异位茎、杯状叶片、瘤状结构、叶脉脉序紊乱等。RT-PCR分析表明,该基因仅强烈表达于转基因烟草植株中,而在野生型和组织培养材料中均未检测到该基因的存在。表明Pttkn1基因已经成功转入烟草中,而且在叶片形态发生和维管形成过程中发挥一定的作用。     

Identification of new small non-coding RNAs from tobacco and Arabidopsis

Small non-coding RNAs (ncRNAs) have typically been searched in fully sequenced genomes using one of two approaches-experimental or computational. We developed a mixed method, using both types of information, which has the advantage of applying bio-computing methods to actually expressed sequences. Our method allowed the identification of new small ncRNAs in Arabidopsis thaliana and in the unfinished genome of Nicotiana tabacum. We constructed a N. tabacum cDNA library from small RNAs ranging from 20 to 30 nucleotides (nt). The sequences from 73 unique clones were compared to the A. thaliana genome and to all plant sequences using a pattern-matching approach (program Patbank). Thus, we selected 15 clones from the library corresponding mostly to A. thaliana or N. tabacum non-coding sequences. By Northern blot analyses, we confirmed the presence of most RNA candidates in Arabidopsis and in Nicotiana sylvestris with a size range of 21-100 nt. To gain more insight into the possible genesis of 21-24 nt sequences, stable folding of sRNAs with their flanking regions were predicted with the software MIRFOLD dedicated to the folding of microRNAs (miRNA). Stable hairpins structures were observed for some putative miRNAs.     

Plant transformation by coinoculation with a disarmed Agrobacterium tumefaciens strain and an Escherichia coli strain carrying mobilizable transgenes

Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens. We constructed E. coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an E. coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A. tumefaciens strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E. coli to the disarmed A. tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct A. tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants.     

转录因子DREB2A植物表达载体构建及烟草转基因研究

构建转录因子DREB2A基因表达载体,通过叶盘法进行烟草的转基因表达研究。经过抗生素卡那霉素抗性筛选-PCR鉴定-Southern杂交检测,表明目的基因已经整合到烟草基因组DNA中。转基因植株出现生长迟滞现象。     

T-DNA-mediated transfer of Agrobacterium tumefaciens chromosomal DNA into plants

Besides the well-documented integration of DNA flanked by the transfer DNA borders, occasional insertion of fragments from the tumor-inducing plasmid into plant genomes has also been reported during Agrobacterium tumefaciens-mediated transformation. We demonstrate that large (up to approximately 18 kb) gene-bearing fragments of Agrobacterium chromosomal DNA (AchrDNA) can be integrated into Arabidopsis thaliana genomic DNA during transformation. One in every 250 transgenic plants may carry AchrDNA fragments. This has implications for horizontal gene transfer and indicates a need for greater scrutiny of transgenic plants for undesired bacterial DNA.     

过量表达星星草PtSOS_1提高拟南芥的耐盐性

将星星草中分离的质膜型Na+/H+逆向转运蛋白基因PtSOS1(GenBank登录号EF440291)构建到pGWB2植物表达载体上,转化拟南芥,获得抗卡那霉素的抗性植株.PCR和Northern检测表明,PtSOS1已整合到拟南芥基因组中并过量表达.耐盐性实验表明,PtSOS1过量表达提高了拟南芥植株的耐盐性.盐分测定表明,盐胁迫下PtSOS1转基因植株中Na+积累低于野生型的,K+含量则高于野生型的,转基因植株中K+/Na+比值高于野生型.     

The CIC library: a large insert YAC library for genome mapping in Arabidopsis thaliana

A new Arabidopsis thaliana (ecotype Columbia) genomic library has been constructed in Yeast Artificial Chromosomes: the CIC library (for CEPH, INRA and CNRS). Optimization of plant culture conditions and protoplast preparation allowed the recovery of large amounts of viable protoplasts. Mechanical shearing of DNA was minimized by isolation of DNA from protoplasts embedded in agarose. Cloning of large inserts was favored by including two successive size fractionation steps (after partial Eco RI digestion and after ligation with the vector arms), which selected DNA fragments larger than 350 kb. The library consists of 1152 clones with an average insert size of 420 kb. Clones carrying chloroplast DNA and various nuclear repeated sequences have been identified. Twenty-one per cent of the clones are found to contain chloroplast DNA. Therefore, the library represents around four nuclear genome equivalents. The clones containing 5S rDNA genes, 18S-25S rDNA sequences and the 180 bp paracentromeric repeated element account for 3.6%, 8.9% and 5.8%, respectively. Only one clone was found to carry the 160 bp paracentromeric repeated element. Given the smaller size of clones carrying Arabidopsis repeated DNA, the average size of remaining clones is around 480 kb. The library was screened by PCR amplification using pairs of primers corresponding to sequences dispersed in the genome. Seventy out of 76 pairs of primers identified from one to seven YAC clones. Thus at least 92% of the genome is represented in the CIC library. The survey of the library for clones containing unlinked DNA sequences indicates that the proportion of chimeric clones is lower than 10%.     

aroA-In融合基因载体的构建、表达及对烟草的转化

PCR扩增突变的5’-烯醇丙酮酸莽草酸-3-磷酸合成酶(5’-enolpyruvylshikimate-3-phosphate synthetase,EPSPS)cDNA全长序列,插入pLitmus28得到pLEPSPS,进而通过反向PCR在EPSPS235／236aa之间将其打断为无功能的片段。选用人工构建的具有顺式和反式剪接功能的mini型蛋白内含子Ssp DnaB和Rma DnaB,插入被打断的aroA(抗除草剂基因),构建了质粒pLEBC、pLEBT、pLERC和pLERT。将4种重组质粒中的aroA-In(蛋白内含子Intein插入aroA)融合基因插入pET-32得到表达载体pETLEBC、pETLEBT、pETLERC和pETLERT,lPTG诱导后,SDS-PAGE分析显示其能在DE。中有效表达并发生相应的蛋白剪接。将aroA-cis Ssp DnaB和aroA-cis Rma DnaB融合基因分别插入pLYM中进一步构建成植物表达载体,农杆菌叶盘法转化烟草。基因组PCR分析表明融合基因整合入植物核基因组;RT-PCR分析显示其可在高等植物细胞中成功表达。结果说明蛋白内含子基因可以转化高等真核细胞,蛋白剪接技术可应用于高等植物细胞,从而为防止植物转基因扩散提供了一条新的途径。