根癌农杆菌介导法获得烟草转人骨形成蛋白—3成熟肽基因植株

利用冻融法将质粒pCAMBIA-hBMP-3m直接转入根癌农杆菌LBA4404,以烟草无菌苗叶盘为外植体,通过农杆菌介导法进行遗传转化,获得了在含25mg/L潮霉素（Hy-gromycin,Hyg）的筛选培养基上再生的抗性植株,经PCR检测呈阳性,初步鉴定并筛选整合了人骨形成蛋白－3成熟肽基因的转基因植株。     

Transgenic Tobacco Expressing Pinellia ternata Agglutinin Confers Enhanced Resistance to Aphids

Tobacco leaf discs were transformed with a plasmid, pBIPTA, containing the selectable marker neomycin phosphotransferase gene (nptII) and Pinellia ternata agglutinin gene (pta) via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic tobacco plants were regenerated. PCR and Southern blot analyses confirmed that the pta gene had integrated into the plant genome and northern blot analysis revealed transgene expression at various levels in transgenic plants. Genetic analysis confirmed Mendelian segregation of the transgene in T1 progeny. Insect bioassays showed that transgenic plants expressing PTA inhibited significantly the growth of peach potato aphid (Myzus persicae Sulzer). This is the first report that transgenic plants expressing pta confer enhanced resistance to aphids. Our study indicates that the pta gene can be used as a supplement to the snowdrop (Galanthus nivalis) lectin gene (gna) in the control of aphids, a sap-sucking insect pest causing significant yield losses of crops.     

CMO与BADH双基因表达载体构建及在烟草中的表达

本研究的目的是将甜菜碱合成关键酶CMO与BADH基因构建到同一表达载体中,利用转基因方法将该表达载体导入植物体内,完善植物体内的甜菜碱合成途径,提高植物的抗旱性和耐盐性。以pC1303质粒为基础,构建了均由35S启动子驱动的CMO基因和BADH基因的植物双基因表达载体pC35SC35SB1303。利用冻融法将其导入农杆菌LBA4404中,通过农杆菌介导法分别将CMO基因、BADH基因以及该双基因表达载体导入烟草中,PCR检测和Northern杂交分析表明,外源基因已整合到受体植物基因组中并正常表达。对转基因植株及对照植株甜菜碱含量的检测结果表明,转双基因植株的甜菜碱含量明显高于转BADH基因植株、转CMO基因植株及对照植株。     

Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene

Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes. Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome. Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene. To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase. Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase. 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA. In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T₁ progeny. Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing. This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype.     

人干扰素α-2b基因在烟草中的表达研究

应用PCR的技术从质粒pAIFN中扩增人干扰素α-2b(Human interferon α-2b,HuIFN α-2b)编码基因,将其连接到pBI121双元载体构建植物真核表达载体pBIFN;用冻融法将该载体转染根癌农杆菌LBA4404;并用叶盘浸染法转化烟草叶片,经转化的烟草叶片的组织培养,诱导愈伤获得再生植株。通过应用PCR,RT-PCR,Wes-tern blot和WISH/VSV方法检测获得的烟草再生植株,结果表明HuIFN α-2b基因已成功整合进烟草核基因组并表达出具有活性的HuIFN α-2b蛋白。本文对HuIFN α-2b基因在烟草核系统中的表达进行了研究,为进一步在烟草叶绿体系统中该基因的表达研究奠定了基础。     

人干扰素α-2b 基因在烟草中的表达研究

应用PCR的技术从质粒pAIFN中扩增人干扰素α-2b（Human interferon α-2b,HuIFN α-2b）编码基因,将其连接到pBI121双元载体构建植物真核表达载体pBIFN;用冻融法将该载体转染根癌农杆菌LBA4404;并用叶盘浸染法转化烟草叶片,经转化的烟草叶片的组织培养,诱导愈伤获得再生植株。通过应用PCR,RT-PCR,Wes-tern blot和WISH/VSV方法检测获得的烟草再生植株,结果表明HuIFN α-2b基因已成功整合进烟草核基因组并表达出具有活性的HuIFN α-2b蛋白。本文对HuIFN α-2b基因在烟草核系统中的表达进行了研究,为进一步在烟草叶绿体系统中该基因的表达研究奠定了基础。     

六棱大麦HVA1基因在烟草中遗传转化的研究

本研究依据HVA1基因序列克隆六棱大麦HVA1基因cDNA片段,构建Ubiquitin启动子驱动下的植物表达载体pCAMBIA1300-HVA1。然后通过三亲杂交法将重组质粒PCAMBIA1300-HVA1转入农杆菌LBA4404,并采用农杆菌介导法转化烟草。经PCR,PCR-Southern blotting和RT-PCR检测表明HVA1基因已整合进烟草基因组,并在转录水平上获得表达。功能验证的结果显示,转基因植株叶片的保水率提高了近1倍,暗示转基因烟草具有一定的抗旱潜力。     

美洲商陆抗真菌蛋白转化烟草的研究和抗病性检测

本研究为美洲商陆抗病毒蛋白(PaAFP)基因首次对植物遗传转化的研究,转入烟草中研究此蛋白对烟草立枯病的抗性。从美洲商陆叶片中获得美洲商陆抗真菌蛋白前体蛋白基因cDNA序列,构建植物表达载体pCAMBIA1300-PaAFP,通过三亲杂交法将其导入根癌农杆菌LBA4404受体菌,转染烟草获得了大量再生转基因植株。PCR、Southern杂交、RT-PCR以及Tris-Tricine-SDS-PAGE检测结果表明目的基因已经整合到烟草基因组中,并且已经得到转译。转基因植株苗期抗立枯病试验表明,转基因烟草植株对立枯丝核菌表现出了抗性。     

转人α-乳清白蛋白基因烟草中半胱氨酸含量的提高

将人α-乳清白蛋白基因构建到植物表达载体上,通过农杆菌介导采用叶盘法将人α-乳清白蛋白基因导入到烟草中,经过PCR和Southern blot分析表明:人α-乳清白蛋白基凶已经整合到烟草基因组中;经过RT-PCR葙GUS组织化学染色证明:人α-乳清白蛋白基因得到了表达.测定了9株转基因烟草叶片半胱氨酸含量,大部分植株有着明显的提高,最高幅度达到了318.02%,半胱氨酸含量平均提高了166.40%.     

Obtaining of transgenic French bean plants (Phaseolus vulgaris L.) resistant to the herbicide pursuit by Agrobacterium-mediated transformation

The transgenic plants of French bean (Phaseolus vulgaris) resistant herbicide Pursuit and kanamycin have been obtained. The genetic transformation was carried out with Agrobacterium tumefaciens strain LBA4404 containing binary vector carrying mutant ahas/als and selective nptII genes. Integration of the transgenes into plant genome was confirmed by polymerase chain reaction.     

Activity of T-DNA borders in plant cell transformation by mini-T plasmids.

By using a binary vector system, we examined the requirements for border sequences in T-DNA transformation of plant genomes. Mini-T plasmids consisting of small replicons with different extents of pTiT37 T-DNA were tested for plant tumor-inducing ability in Agrobacterium tumefaciens strain LBA4404 containing helper plasmid pAL4404 (which encodes virulence genes needed for T-DNA transfer). Assays of these bacteria on carrot disks, Kalanchoë leaves, and SR1 Nicotiana tabacum plantlets showed that mini-T plasmid containing full length T-DNA including left and right borders was highly virulent, as were mini-T plasmids containing all onc (oncogenicity) genes and only the right border. In contrast, mini-T plasmids containing all onc genes and only the left border induced tumors only rarely, and a mini-T plasmid containing all onc genes but no T-DNA borders was completely avirulent. Southern hybridization analyses of tumor DNA showed that T-DNA border sequences delimited the extent of the two-border mini-T plasmid transferred and integrated into the plant genome. When only one T-DNA border was present, it formed one end of the transferred DNA, and the other end mapped in the vector sequences. The implications of these results for the mechanism of T-DNA transfer and integration are discussed.     

The natural transformation of the soil bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly depends on homologous sequences in the recipient cells

The nptII(+) gene present in the genome of transgenic potato plants transforms naturally competent cells of the soil bacteria Pseudomonas stutzeri and Acinetobacter BD413 (both harboring a plasmid with an nptII gene containing a small deletion) with the same high efficiency as nptII(+) genes on plasmid DNA (3x10(-5)-1x10(-4) transformants per nptII(+)) despite the presence of a more than 10(6)-fold excess of plant DNA. However, in the absence of homologous sequences in the recipient cells the transformation by nptII(+) dropped by at least about 10(8)-fold in P. stutzeri and 10(9)-fold in Acinetobacter resulting in the latter strain in < or =1x10(-13) transformants per nptII(+). This indicated a very low probability of non-homologous DNA fragments to be integrated by illegitimate recombination events during transformation.     

Phenotypes of Tobacco Plants Expressing Genes for the Synthesis of Growth Regulators

The expression of genes for synthesis of auxin (iaaM and iaaH) and cytokinins (ipt) was studied in tobacco plants transformed by two Agrobacterium tumefaciens strains C 58 and LBA 4404. The strain LBA 4404 carried binary vector plasmid pCB 1334 (ipt gene) and plasmid pCB 1349 (iaaM, iaaH and ila genes). Both plasmids carried reportered gene for npt II. Obtained plants expressed incorporated genes. New proteins with molecular masses of about 74, 40, 26, 25, 21 and 17 kDa for wild plasmid pTi C58; 60, 36, 31.5, 27, 26 and 17 kDa for binary vector plasmid pCB 1334 and 74, 49, 36, 31.5, 26 and 25 kDa for binary vector plasmid pCB 1349 were found in the patterns of soluble proteins. Significant changes in the content of chlorophylls, especially chlorophyll a, were detected in the plants carrying ipt gene and in plants transformed by the wild strain C58 of A. tumefaciens. Tobacco plants expressing ipt gene and genes from T-DNA of pTi C58 plasmid were dwarf, and in comparison to the controls, they had thicker stems, and the surface of the leaf blades was reduced to 20 - 50 %. Adventitious roots, growing from the stem, were typical for transformants overproducing auxins. Regenerants and transformants expressing genes from T-DNA of plasmid pTi C58 differed in the shape of the flowers and their fertility. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants

To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV 16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV 16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.     

Genetic transformation of Nicotiana africana Merxm. with plasmids containing lox recombination

An efficient genetic transformation method for african tobacco Nicotiana africana Merxm. has been established. African tobacco is a valuable source for cytoplasmic male sterility (CMS) and nuclear encoded resistance to potato virus Y (PVY). N. africana transgenic plants have been obtained using both Agrobacterium-mediated and direct transformation of leaf explants with gold particle bombardment using particle inflow gun. Plasmid vectors containing phosphinothricin resistance gene (bar gene) coding region without promoter and independent 35S promoter between lox sites (lox-bar-35S-lox) and nptII gene were used. Transgenic plants were selected according to growth capacity on the selective medium containing 50 mg/l kanamycin. PCR analyses of kanamycin-resistant plants confirmed the presence of nptII and bar genes in their genome. Agrobacterium-mediated transformation of root explants has proved to be the most efficient transformation method for N. africana.     

Agrobacterium tumefaciens–mediatated transformation of shoot-buds of chicory

Vegetative propagation of chicory via axillary shoot proliferation is one of the best ways to obtain an offspring with complete genetic stability. These shoot buds were used in transformation experiments using Agrobacterium tumefaciens strains containing binary plasmids carrying the neomycin phosphotransferase gene (nptII) and the β-glucuronidase gene (uidA). Selection was carried out on basal medium containing 100 mg l⁻¹ kanamycin. Transformed plantlets were recovered at a frequency of about 10% within four weeks after co-cultivation. The presence of the uidA gene was demonstrated by transient gene expression experiments using the histochemical GUS staining procedure. Evidence for stable transformation was shown by subculturing leaf discs on kanamycin selection medium, and Southern blot analysis confirmed the integration of the nptII and the uidA genes in the plant genome. Analysis of the progenies showed that kanamycin resistance was inherited as a single dominant trait. This method for obtaining transgenic chicory plants represents an alternative to leaf disc transformation. This revised version was published online in June 2006 with corrections to the Cover Date.     

大豆fad基因反义表达载体构建及转化烟草研究

使用PCR方法从大豆基因组DNA中扩增出大豆油酸脱饱和酶基因fad2-1,连接到pMD18-T载体中,转化大肠杆菌JM109菌株.测序后,用DNAstar软件进行同源性比对.然后将正确的序列反向克隆到表达载体pBt,并转化农杆菌菌株LBA4404,经双酶切鉴定和PCR扩增检测,获得具有该基因反向序列的农杆菌工程菌,转化...     

根癌农杆菌介导的乙肝表面抗原基因对烟草的转化

构建了植物表达载体pBRSAg,该载体具有完整的植物表达元件,CaMV35S启动子、农杆菌T-DNA左右边界、植物报告基因gus和植物选择标记基因hpt,适用于农杆菌的转化;通过冻融法将重组质粒pBRSAg转入根癌农杆菌LBA4404中,利用农杆菌介导法转化烟草叶盘,经筛选培养获得烟草植株。抗性植株经GUS染色和PCR检测为阳性,初步表明乙肝表面抗原基因在烟草中得到表达。     

Transgenic tomato (Lycopersicon esculentum L.) transformed with a binary vector in Agrobacterium rhizogenes: Non-chimeric origin of callus clone and low copy numbers of integrated vector T-DNA

Summary We transformed tomato (Lycopersicon esculentum L.) by using Agrobacterium rhizogenes containing two independent plasmids: the wild-type Ri-plasmid, and the vector plasmid, pARC8. The T-DNA of the vector plasmid contained a marker gene (Nos/Kan) encoding neomycin phosphotransferase which conferred resistance to kanamycin in transformed plant cells. Transgenic plants (R ₀) with normal phenotype were regenerated from transformed organogenic calli by the punctured cotyledon transformation method. Southern blot analysis of the DNA from these transgenic plants showed that one or two copies of the vector plasmid T-DNA, but none of the Ri-plamid T-DNA, were integrated into the plant genome. Different transgenic plants derived from the same callus clone showed an identical DNA banding pattern, indicating the non-chimeric origin of these plants. We also transformed tomato by using A. tumefaciens strain LBA4404 containing a disarmed Ti-plasmid (pAL4404), and a vector plasmid (pARC8). Transgenic plants derived via A. tumefaciens transformation, like those via A. rhizogenes, contained one to two copies of the integrated vector T-DNA. The kanamycin resistance trait in the progeny (R ₁) of most transgenic plants segregated at a ratio of 3:1, suggesting that the vector T-DNAs were integrated at a single site on a tomato chromosome. In some cases, the expression of the marker gene (Nos/Kan) seemed to be suppressed or lost in the progeny.