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1.
I. Saalbach T. Pickardt D. R. Waddell S. Hillmer O. Schieder K. Müntz 《Euphytica》1955,85(1-3):181-192
Summary Epicotyl explants were co-cultivated with Agrobacterium tumefaciens EHA101 to transfer a chimeric 2S albumin gene construct carried in the binary Ti plasmid vectors pGSGLUC1 or pGA472 into the grain legume Vicia narbonensis. This gene encoding the sulphur-rich Brazil nut albumin was under the control of either the CaMV 35S promoter which permits gene expression in all organs, or the Vicia faba legumin B4 promoter which elicits seed-specific gene expression. After callus formation and selection for kanamycin resistance, somatic embryos were induced which, in the case of transformation with the vector pGSGLUC1, were screened for GUS activity. Embryos that produced GUS were in addition analysed for 2S albumin formation. Selected transgenic embryos were cloned by multiple shoot regeneration. Rooted and fertile plants were obtained by grafting transgenic shoots on the appropriate seedlings. R1 and R2 generations were raised and analysed for GUS as well as 2S albumin gene expression.Expression of the 35S promoter/2S albumin gene fusion took place in all organs of the transgenic plants including the cotyledons of seeds, whereas seed-specific gene expression was found in transformants with the legumin promoter/2S albumin gene fusion. The 2S albumin accumulated in the 2S protein fraction of transgenic seeds and its primary translation product was processed into the 9 and 3 kDa polypeptide chains. The foreign protein was localised in the protein bodies of the grain legume. Analysis of the R2 plants indicated Mendelian inheritance of the 2S albumin gene. In homozygous V. narbonensis plants the amounts of 2S albumin were twice that present in the corresponding heterozygous plants. Whereas only low level formation of the foreign protein was achieved if the gene was under the control of the 35S promoter, approximately 3.0% of the soluble seed protein was 2S albumin if seed-specific gene expression was directed by the legumin B4 promoter. Some of these transformants exhibited a three-fold increase in the methionine content of the salt-soluble protein fraction extracted from seeds.Abbreviations 35S
cauliflower mosaic virus 35S protein gene
- GUS
-glucuronidase
- NPTII
neomycin phosphotransferase II
- LeB4
Vicia faba legumin B4 gene
- 2S albumin
Brazil nut (Bertholletia excelsa) 2S albumin
- ER
endoplasmic reticulum
- rER
rough endoplasmic reticulum
- HPLC
high pressure liquid chromatography 相似文献
2.
Bt基因转化玉米培育抗玉米螟自交系 总被引：7，自引：0，他引：7
采用基因枪法将苏云金杆菌(简称Bt)的杀虫毒蛋白基因(CryIA)导入东北春玉米自交系铁7922的幼胚中,诱导愈伤组织.抗虫基因的表达载体是pBI121,包含Bt杀虫毒蛋白基因和新霉素磷酸转移酶(NPTⅡ)基因,筛选标记bar由CaMV 35S启动子驱动.通过对后代植株的除草剂草丁膦(PPT)筛选、分子鉴定和ELISA法检测,证明外源基因已经整合到玉米基因组中并可以稳定遗传.对转基因株系进行田间接玉米螟虫卵,调查不同生长时期的危害指数,最终筛选出一批抗玉米螟自交系. 相似文献
3.
Summary Little work has been reported on genetic transformation with maize inbred lines, especially elite inbred lines used in breeding. In this work, 7 self-pollinated inbred lines and 4 hybrid lines have been screened. The results revealed that calli derived from immature embryos from two inbred lines X333 and X301 were compact, hyperhydric and unsuitable for transformation, but the calli induced from other inbred lines and all the hybrid lines were friable and yellow and could be used for genetic transformation. The sb401 gene isolated from potato (Solanum berthaultii) encodes a protein with a high lysine content. Maize calli from 5 self-pollinated inbred lines and 4 hybrid lines were transformed using particle-bombardment with different plasmids to simultaneously introduce the sb401 lysine rich gene and the selectable gene hpt respectively. Two hundred and sixty-eight regenerated plants were obtained from these genotypes. Co-insertion was confirmed in 29 regenerated plants by PCR and Southern blot analysis. Transgene segregation of the R1 plants was observed and one marker-free transgenic maize line was recovered. Analysis of the crude protein content in mature seeds of R1 transgenic plants also showed an increase from 36.8% to 48.2%. This study thus provides a workable system for generating transgenic maize free from selectable marker genes and generates valuable resources for obtaining marker free transgenic maize with a high-lysine protein content. 相似文献
4.
根癌农杆菌介导获得稗草Ecppc转基因小麦的研究 总被引：1，自引：0，他引：1
采用携带pUbi-Ecppc质粒的3个根癌农杆菌菌株（LBA4404、EHA105和C58c1），对经过预培养10~12 d的春小麦品种扬麦158、Bobwhite和扬麦12的幼胚愈伤组织进行了遗传转化。对筛选中的抗生素浓度、菌液浓度、共培养温度和时间、受体基因型、菌株-质粒组合等影响转化的重要因素进行了研究。首次将单子叶野生C4植物稗草的磷酸烯醇式丙酮酸羧化酶基因（Ecppc）导入小麦受体基因型，并得到具有潮霉素（Hyg）抗性的转化植株。从816块共培养愈伤组织中转化得到34株抗性植株，其中14株PCR检测为阳性。扬麦158的转化效率达3.03%。Southern和RT-PCR分析表明外源基因已整合到小麦基因组并得到正确的转录。生理学检测显示，转基因小麦植株的光合速率和PEPC活性都有所提高。说明Ubiqintin基因启动子控制的稗草PEPC cDNA基因在小麦中可以正确表达和起到一定的生理作用。这些工作为进一步探讨PEPC对小麦光合作用及其他生理过程的影响奠定了基础。 相似文献
5.
猪传染性胃肠炎病毒纤突糖蛋白在转基因玉米中的表达 总被引：1，自引：0，他引：1
本研究拟构建携带猪传染性胃肠炎病毒纤突糖蛋白基因(TGEV-S)的转基因玉米植株并检测其表达情况。首先利用PCR方法自携带TGEV-S基因的重组质粒中扩增2.2kb的S基因片段,然后插入植物表达载体pBAC176中,该表达载体以编码除草剂草甘膦抗性的EPSP基因作为选择标记,目的基因以双增强子的CaMV35S(E35S)及其玉米Hsp70第一内含子为驱动。以授粉后10～12d约0.5～1mm大小的玉米幼胚为受体,用基因枪进行转化,经过愈伤组织诱导、草甘膦抗性筛选和分化再生培养,先后获得74株转化再生植株。利用PCR和Southern blot检测表明,T0代转基因苗有9株检测结果为阳性。T1代转基因玉米株系经PCR检测有14个转基因株系为阳性,初步确定了目的基因在这些转基因玉米中的稳定整合。进而通过间接ELISA分析初步确定目的基因在7个T1代转基因玉米株系获得了表达。研究结果为进一步研究表达TGEV-S转基因玉米的生物学活性奠定了基础。 相似文献
6.
AtFT基因植物表达载体的构建研究 总被引：1，自引：1，他引：0
FT是从拟南芥克隆得到的诱导植物开花调控基因。利用FT基因表达特点,克隆拟南芥FT基因片段,重组构建了以CaMV35S为启动子、GUS为报告基因的植物表达载体pCAMBIA2301-FT与pCAMBIA2300-FT:GUS,并采用农杆菌介导法转化橡胶树体细胞胚,通过GUS组织化学染色观察橡胶树体细胞胚瞬时表达情况,结果验证了重组构建的2个载体的有效性,为进一步研究FT基因在橡胶树中的功能及应用奠定基础。 相似文献
7.
8.
根癌农杆菌介导获得稗草Ecppc转基因小麦的研究 总被引：14，自引：0，他引：14
采用携带pUbi-Ecppc质粒的3个根癌农杆菌菌株（LBA4404、EHA105和C58c1）,对经过预培养10~12 d的春小麦品种扬麦158、Bobwhite和扬麦12的幼胚愈伤组织进行了遗传转化。对筛选中的抗生素浓度、菌液浓度、共培养温度和时间、受体基因型、菌株-质粒组合等影响转化的重要因素进行了研究。首次将单子叶野生C4植物稗草的磷酸烯醇式丙酮酸羧化酶基因（Ecppc）导入小麦受体基因型,并得到具有潮霉素（Hyg）抗性的转化植株。从816块共培养愈伤组织中转化得到34株抗性植株,其中14株PCR检测为阳性。扬麦158的转化效率达3.03%。Southern和RT-PCR分析表明外源基因已整合到小麦基因组并得到正确的转录。生理学检测显示,转基因小麦植株的光合速率和PEPC活性都有所提高。说明Ubiqintin基因启动子控制的稗草PEPC cDNA基因在小麦中可以正确表达和起到一定的生理作用。这些工作为进一步探讨PEPC对小麦光合作用及其他生理过程的影响奠定了基础。 相似文献
9.
抗逆调节转录因子CBF1基因提高多年生黑麦草的抗旱能力 总被引：16，自引：0，他引：16
通过逆境诱导型启动子rd29B为驱动,分别构建出含有抗逆调节转录因子CBF1基因的表达载体pBAC122,pBAC127,其中pBAC127以CaMV35S启动子驱动的bar基因作为选择标记。用高压氦气基因枪PDS1000/He分别将表达载体导入多年生黑麦草(Lolium perenne)品种Topgun的幼胚、成熟胚和愈伤组织。经除草剂Bialaphos抗性筛选和植株再生,获得了36棵转基因植株。经PCR,Dot-blotting的分子检测,CBF1基因已整合到多年生黑麦草部分转基因株系的基因组中。用5种不同浓度的除草剂涂抹黑麦草叶片,非转基因植株表现为不抗,而转基因植株最高可以抗到135~200 mg/L。叶片脯氨酸含量测定表明,经干旱处理或使用15%PEG处理,转基因植株叶片脯氨酸含量比未处理时显著提高,部分转基因植株提高幅度明显高于非转基因植株。经过25 d人工温室干旱处理,有3棵植株显示出存活迹象,复水后,有1棵植株(C122-7)恢复正常生长。从而表明,利用逆境诱导型启动子(rd29B)来调控外源CBF1基因的表达,能显著改良黑麦草的抗旱能力。 相似文献
10.
The European corn borer (ECB), Ostrinia nubilalis (Hübner), is a major pest of maize (Zea mays L.) in Central Europe. In order to compare transgenic Bt maize hybrids with their non‐transgenic counterparts and commercial hybrids, field trials and a laboratory bioassay were conducted. The field experiments were performed at four locations with natural and manual infestation of ECB larvae in 1998 and 1999. Transgenic Bt hybrids showed significantly lower means than their corresponding non‐transgenic counterparts and commercial hybrids for all resistance traits (damage rating of stalks, number of larvae per plant, and percentage of damaged plants or ears under infestation). Bt hybrids containing the CryIA(b) gene under the control of green tissue and pollen‐specific promoters (event 176) showed a significantly higher percentage of damaged ears than Bt hybrids carrying the CryIA(b) gene under the control of a constitutive promoter (Mon810). Bt and non‐Bt hybrids showed no significant differences for all agronomic traits, except for plant height under insecticide protection and grain yield reduction under infestation, whereas Bt hybrids had significantly lower means than their non‐transgenic counterparts and other commercial hybrids. All resistance traits were significantly correlated with grain yield reduction. The laboratory bioassay confirmed the level of antibiosis of Bt hybrids against neonate ECB larvae. Bt hybrids showed the highest level of ECB resistance and therefore are an attractive method of preventing ECB damage within an integrated pest‐management system. 相似文献
11.
Anneli Ritala Reino Aikasalo Kristian Aspegren Marjatta Salmenkallio-Marttila Satu Akerman Leena Mannonen Ulrika Kurtén Riitta Puupponen-Pimiä Teemu H. Teeri Veli Kauppinen 《Euphytica》1955,85(1-3):81-88
Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for -glucuronidase (GUS).Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T0). This chimeric plant passed the transferred nptII gene to its T1 progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T0 spikes and 15 T1 offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T2 and T3 plants were produced by isolating embryos from green grains of transgenic T1 and T2 plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T2 offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed.Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin
R
or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile. 相似文献
12.
Spatio-temporal expression of an insecticidal gene (Cry1Ac) in pre existing transgenic lines of transgenic cotton was studied. Seasonal decline in expression of Cry1Ac differed significantly among different cotton lines tested in the field conditions. The leaves of the Bt cotton plants were found to have the highest levels of toxin expression followed by squares, bolls, anthers and petals. Expression
of the gene decreased consistently with the age of plants. Toxin expression in fruiting parts was not enough to confer full
resistance against bollworms. The reduction in efficacy of transgenic cotton plants late in the season was attributed to reduction
in promoter activity. For this purpose, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (rbcS) promoter was isolated from Gossypium arboreum that was further cloned upstream of an insecticidal gene (Cry1Ac) in expression vector pCAMBIA 1301. A local cotton cultivar NIAB-846 was transformed with Cry1Ac driven by rbcS promoter. The same cotton cultivar was also transformed with Cry1Ac gene driven by 35SCaMV promoter to compare the expression pattern of insecticidal gene under two different promoters. The
results showed that rbcS is an efficient promoter to drive the expression of Cry1Ac gene consistent throughout the life of cotton plant as compared to 35S promoter. The use of tissue specific promoter is also
useful for addressing the biosafety issues as the promoter activity is limited to green parts of plants, hence no gene expression
in roots, cotton seed and other cotton products and by products. 相似文献
13.
André M. Almeida Enrique Villalobos Susana S. Araújo Barbara Leyman Patrick Van Dijck Luís Alfaro-Cardoso Pedro S. Fevereiro José M. Torné Dulce M. Santos 《Euphytica》2005,146(1-2):165-176
Summary Trehalose (a non-reducing disaccharide) plays an important role in abiotic stress protection. It has been shown that using
trehalose synthesis genes of bacterial origin, drought and salt tolerance could be achieved in several plants. A cassette
harboring the AtTPS1 gene under the control of the CaMV35S promoter and the Bialaphos resistance gene was inserted in the binary plasmid vector
pGreen0229 and used for Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum). T0 plants obtained were analyzed by PCR for the presence of AtTPS1 gene. Thirty lines were positive and seeds were germinated on media with 6 mg/l PPT to obtain T1 plants that were grown in
the greenhouse to obtain T2 seeds that were germinated on selective media. Lines which seeds showed a 100 % survival rate
were considered homozygous transgenic T1 lines. Three lines were selected and gene expression confirmed by northern and western
blots. Transgenic seeds were germinated on media with different concentrations of mannitol (0, 0.25, 0.5 and 0.75 M) and sodium
chloride (0, 0.07, 0.14, 0.2, 0.27 and 0.34 M) to score their tolerance to osmotic stress. Assays were conducted to test the
tolerance of transgenic plants to drought (measurement of water percentage as a consequence of water withdrawal), desiccation
(measurement of water loss as a consequence leaf detaching) and temperature stresses (germination at 15 ∘C and 35∘C). Transgenic tobacco plant lines registered higher germination rates under osmotic and temperature stress situations than
did wild-type plants. Responses to drought and desiccation stresses were similar for all plant lines. It can hence be suggested
that the heterologous expression of TPS1 gene from Arabidopsis can be used successfully to increase abiotic stress tolerance in model plants and probably in other crops. 相似文献
14.
抗双链RNA依赖性蛋白激酶PKR基因表达载体构建及在玉米中的遗传转化 总被引：1，自引：0，他引：1
玉米病毒性病害和杂草严重影响其产量和品质。以pCAMBIA5300为基础载体,应用In-Fusion克隆技术构建了双价植物表达载体pCAMBIA5300-Ubi-PKR-CaMV35S-EPSPS,其中含有抗双链RNA依赖性蛋白激酶PKR基因和磷酸烯醇式丙酮酸莽草酸-3-磷酸合酶EPSPS基因,分别由玉米泛素Ubi启动子和花椰菜花叶病毒35 S启动子启动。以玉米种子黄化苗的茎尖分生组织为受体,用农杆菌介导法进行遗传转化,将抗双链RNA依赖性蛋白激酶基因PKR和抗除草剂草甘膦基因EPSPS导入玉米自交系掖478中,获得转基因植株及其子代。 相似文献
15.
Summary In some cereal species that are still recalcitrant to stable transformation and regeneration, transient expression in isolated protoplasts is a useful tool for the study of gene expression and regulation. We have successfully applied these techniques to barley protoplasts derived from developing endosperm, aleurone, leaves and roots in order to characterize functionally cis-acting motives in two gene promoters, corresponding to trypsin inhibitor BTI-CMe and to sucrose synthase Ss1. Gene specificity is maintained in transient expression assays with protoplasts isolated from these different barley tissues and the pattern of expression parallels the mRNA levels observed for the corresponding genes in the same tissues.Abbreviations CaMV35S
35S-cauliflower mosaic virus promoter
- GUS
-glucuronidase
- MU
4-methyl umbelliferone
- NOS
nopaline synthase gene
- PEG
polyethyleneglycol 相似文献
16.
Summary This article reports the culture and plant regeneration of Tripsacum dactyloides. Mature embryos of Tripsacum dactyloides dactyloides were used to obtain embryogenic callus cultures. Currently, 180 normal plants have been regenerated from these cultures. Callus was initiated on MS medium supplemented with dicamba (10 mol or 20 mol) and sucrose (3% or 6%), and plants were regenerated on hormone free MS medium containing 2% sucrose. No significant differences were found in callus initiation frequency or in embryogenic response of cultures on the four combinations of sucrose and dicamba tested. The embryogenic cultures have been maintained for 9 months (12 subcultures) and have retained regeneration capacity. Plants regenerated from tissue culture of maize-by-Tripsacum hybrids could be useful in maize improvement. 相似文献
17.
18.
DREB1B基因在转基因小麦后代的稳定表达 总被引：2，自引：0，他引：2
本研究对基因枪导入了pBAC128F/R质粒(含有玉米Adh1内含子1的CaMV35S启动子驱动的bar基因作为选择标记,以来自拟南芥菜逆境诱导表达类型--亲水蛋白rd29b基因的启动子驱动脱水应答转录因子DREBIB基因的逆境诱导表达类型质粒)的T4代转基因小麦进行了稳定表达研究.PCR、PCR-Southem和Southern杂交分析表明,外源转录因子DREBlB基因已稳定整合到转基因株系的基因组中.半定量RT-PCR结果表明,部分转基因株系的DREB1B基因的相对表达量有所增强.叶片除草剂抗性检测结果显示有8个转基因株系可抗到150 mg/L.叶片脯氨酸含量测定结果表明,有4个株系(编号为1,18,30和76)的脯氨酸含量提高幅度较大,同一株系,干旱与未干旱处理相比,脯氨酸含量提高了3～5倍.干旱处理后的转基因植株与非转基因植株相比,脯氨酸含量高2～3倍.在干旱条件下,T4代田间小区产量统计数据分析结果表明,有4个转基因株系(编号为30,51,70和76)的产量与非转基因植株相比有显著增加.研究表明,利用逆境诱导型rd29b基因的启动子来增强外源DREB1B基因的表达,能显著改良小麦的抗旱性. 相似文献
19.
Coat protein-mediated protection against plum pox virus in herbaceous model plants and transformation of apricot and plum 总被引：6，自引：0，他引：6
Artur da Câmara Machado Hermann Katinger Margit Laimer da Câmara Machado 《Euphytica》1994,77(1-2):129-134
Summary The expression of the viral coat protein gene in transgenic plants has been shown to induce tolerance against virus infection
(Beachy et al., 1990). Transgenic plants ofNicotiana clevelandii andNicotiana benthamiana- herbaceous host plants for PPV - transformed withAgrobacterium strain LBA 4404 containing the plasmid pBinPPVm, regenerated on selection media containing kanamycin were tested for the
expression of the PPV coat protein gene by ELISA and immuno western blot. After rooting and acclimatisation plants were tested
for the protection against PPV Following the inoculation plants were investigated for symptom development and virus accumulation.
Different lines were identified, according to the different reaction to the mechanical inoculation, ranging from a complete
absence to a strong reduction of symptoms. There have not been many reports on transformation of trees in general, and in
fruit trees particularly. It is obvious that the major obstacle is the regeneration of transformed plantlets. Attempts to
improve crop plants by genetic engineering techniques will always depend very strongly on the availability of reliable protocols
for transformation, selection and regeneration (Laimer et al., 1989, 1990). Different systems involving juvenile and adult
plant material have been developed allowing the transfer of foreign genes into apricot and plum cultivars. We report the transformation
and regeneration ofPrunus armeniaca andPrunus domestica plants withAgrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker geneβ-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV), the causal agent of Sharka disease.
The marker geneGUS was used for the optical evaluation of the efficiency of different transformation systems involving cotyledons of immature
embryos as well as somatic embryos and leaf discs. The coat protein gene of PPV was used to introduce the coat protein mediated
resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. 相似文献
20.
Pollination with gamma-irradiated pollen and development of fruits,seeds and parthenogenetic plants in apple 总被引：3，自引：0，他引：3
Summary Four apple (Malus X domestica) genotypes, Erovan, Golden Delicious, R1-49 and X6677, were pollinated with a marked pollen irradiated by -rays at doses ranging from 125 to 1000 Gy. Pollination with such irradiated pollen affected fruit set, seed number and seed contents, and induced the formation of parthenocarpic fruits and the development of parthenogenetic embryos. The immature embryos extracted from seeds. 2 and 3 months after pollination, were cultured in vitro and germinated after 2 months of cold treatment (3°C). Haploid plants were obtained in all 4 genotypes, after pollination with pollen irradiated at doses from 200 to 500 Gy. The optimum conditions for induction of apple haploids, by irradiated pollen approach, have been established. 相似文献