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1.
马铃薯质体表达载体构建及GFP基因在块茎中的瞬时表达 总被引：2，自引：0，他引：2
利用高等植物质体基因组在进化中高度保守的特点, 根据烟草质体基因组全序列设计合成引物, PCR扩增并克隆了马铃薯质体的trnI-trnA基因片段。将测序正确的trnI基因和trnA基因作为定点整合外源基因的同源重组片段, 构建成包含Prrn-gfp-aadA-TpsbA表达盒的马铃薯质体定点转化载体pBMLSIA-GFP, 酶切鉴定表明, 所构建载体符合预期设计。采用该载体对马铃薯块茎进行基因枪法转化, 结果表明, GFP基因可在质体特异性启动子Prrn及终止子TpsbA的调控下在马铃薯块茎中瞬时高量表达, 经基因枪轰击后马铃薯块茎在紫外投射仪下产生很强的绿色荧光, 在距离为6 cm, 压力1 100 psi轰击2枪的条件下产生的绿色荧光最强。马铃薯块茎可溶性蛋白SDS-PAGE电泳分析表明, GFP蛋白表达量约占总可溶性蛋白的15.4%~30.2%, 均达到了较高的表达水平。该载体对后期马铃薯质体转化体系的建立和其他功能基因导入马铃薯质体进行性状改良具有重要应用价值。 相似文献
2.
除草剂抗性基因bar导人烟草叶绿体 总被引：15，自引：0，他引：15
将编码Phosphinothricin acetyltrans ferase的bar基因与水稻叶绿体psbA基因的启动子和终止子构建成表达盒,连同烟草叶绿体基因组同源片段rpl2-trnH-psbA和trnK-ORF509A以及选择标记基因aadA一起构建成烟草叶绿体转化载体pTZBA。基因枪法转化烟草叶片,经壮观霉素筛选获得转化再生植株。分别以1.0 kb的叶绿体同源片段trnK-OR 相似文献
3.
烟草叶绿体转化载体的构建及转基因植株的获得 总被引：12，自引：2，他引：10
选择烟草叶绿体基因组同源片段ｒｐ１２－ｔｒｎＨ－ｐｓｈｂＡ和ｔｒｎＫ－ＯＲＦ５０９Ａ,及ａａｄＡ抗壮观霉素基因,构建烟草叶绿体转化载体ｐＴＲＺ,制备ｐＴＲＺＤＮＡ金粉子弹,通过基因枪轰击烟草幼苗叶片,经壮观霉素筛选获得了愈伤组织和转化再生植株,对烟草叶绿体转化植株进行ＰＣＲ和Ｓｏｕｔｈｅｒｎ分析,结果证明其中Ｎｏ１３,１６,２３为整入了外源ａａｄＡ的叶绿体转基植株,同时其子代呈现壮观霉素抗性ａａＤ 相似文献
4.
Summary Semilooper resistant transgenic castor plants were produced through Agrobacterium-mediated genetic transformation method. Two castor cultivars, Jyothi and VP1 were transformed using the super-binary vector
pTOK233 carrying gus A and hpt genes. Putative transformants were regenerated following selection on the hygromycin containing medium. GUS positive primary
transformants, when subjected to Southern analysis, revealed stable integration of gus A into their genomes. In the T1 generation, a monogenic segregation ratio of 3 GUS positive: 1 GUS negative plants was observed. Furthermore, transformation
experiments were carried out with the Agrobacterium pSB111 super-binary vector carrying a synthetic delta endotoxin gene cryIAb and the herbicide resistance gene bar both driven by cauliflower mosaic virus 35S promoter. Putative transformants were regenerated through selection on the phosphinothricin
containing medium and Basta tolerant transformants were subjected to molecular analysis. PCR analysis revealed the presence
of both bar and cryIAb genes in the Basta tolerant primary transformants. Southern analysis of PCR positive plants with cryIAb probe showed a 3 Kb band upon HindIII digestion and a > 6 Kb band with BamHI digestion, thus suggesting stable integration of cryIAb intact expression cassette and independent nature of the transformants. The primary transformants subjected to ELISA disclosed
varied levels of Cry protein. These transgenics expressing cryIAb – when bioassayed against freshly hatched semilooper larvae – induced substantial (> 88%) insect mortality. Southern analysis
of 2T1 plants revealed the presence of cryIAb gene, indicating stable inheritance of the transgene into the next generation. In T1, all the Southern-positive plants for cryIAb invariably exhibited tolerance to Basta, denoting co-segregation of both bar and cryIAb genes. Transgenics, expressing cryIAb exhibited ample resistance against the castor semilooper. 相似文献
5.
A new resistance (R) gene to powdery mildew has been identified and characterized in a population derived from the wild potato species, Solanum neorossii under natural infection in the greenhouse. The segregation of resistance has revealed that this R gene is controlled by a single monogenic and dominant gene designated Rpm-nrs1. Analysis of the DNA sequence on an internal transcribed spacer (ITS) region of the pathogen genome suggests that the pathogen
causing the powdery mildew disease is either Golovinomyces orontii or G. cichoracearum. The resistance locus was localized to the short arm of chromosome 6 where several disease R genes already identified in potato and tomato are known to reside. The resistance locus cosegregated in 96 progeny with three
AFLP markers and one PCR marker. The sequences of the two cosegregating AFLP markers are highly homologous to Mi-1 conferring resistance to nematode, potato aphid and whitefly and Rpi-blb2 conferring resistance to late blight. The results in this study will facilitate the cloning of this gene conferring resistance
to powdery mildew. 相似文献
6.
We recently mapped the Pp523 locus that includes a single, dominant gene conferring resistance to downy mildew expressed in adult plants to a 75.1 cm
long linkage group on a genetic linkage map of Brassica oleracea L. More recently, we identified a new AFLP marker 2.8 cm downstream from the resistance gene. The five DNA markers within
an 8.5 cm region encompassing the Pp523 gene were cloned and sequenced. Three of these markers were transformed into SCARs (sequence characterised amplified regions),
however, two among them were monomorphic and were analysed as CAPS (cleaved amplified polymorphic sequence) markers among
the mapping population. Searched against genomic databases, the five B. oleracea DNA-marker sequences matched Arabidopsis thaliana L. gene sequences that delimit a conserved syntenic region in the top arm end of chromosome 1 of this last species. Considering
the close genetic relatedness between both species, the information on this specific genomic region in A. thaliana is particularly useful for the construction of a fine-scale map of the corresponding genomic region in B. oleracea. The identified SCAR and CAPS markers can be used for marker assisted selection (MAS) in breeding programs aimed at the introgression
of the Pp523 resistance locus, allowing the reliable indirect identification of plants harbouring the resistance gene with a margin of
error of approximately six in ten-thousand selected plants. 相似文献
7.
小麦编码高分子量谷蛋白亚基基因的转化 总被引：17，自引：0，他引：17
以小麦的幼穗和幼胚作为转化受体,首次用抗除草剂草甘麟的EPSPs基因作为选择标记,通过基因枪法共转化,将小麦编码高分子量谷蛋白亚基的基因1Dx5和1Dy10转移到普通小麦京花1号中,获得33株再生植株,经PCR初步检测有12株同时扩增到了3个处在不同质粒上的外源基因EPSPs, 1Dx5和1Dy10的目标片段。将部分PCR检测为阳性的转化体 相似文献
8.
探究适用于水稻的真空渗透转化装置及方法,旨在解决目前水稻转基因效率低、转化体变异多、周期长等问题。以抽穗期的‘云资粳41号’幼穗作为受体,应用自制的水稻真空渗透装置对其短暂抽真空渗透浸染导入外源基因。本试验共计转化水稻30株,收获19860粒T0代种子。研究发现不同浓度除草剂对T0代种子的萌发影响不显著,但对T1代材料的生长发育却具有明显抑制作用,因此选择苗期喷施法进行抗性转化子筛选,最佳筛选浓度为20 mg/L。经除草剂抗性筛选,获得抗性苗135株。经PCR分子检测,最终获得阳性苗114株,表明外源DNA已成功导入到受体水稻基因组中。本研究首次应用真空渗透法快速实现了水稻外源基因的转化,为水稻及其他作物的基因转化提供了一种快速、有效的新装置和新途径。 相似文献
9.
10.
Three recombinant inbred line populations from the crosses RL6071/Thatcher, RL6071/RL6058 (Thatcher Lr34), and Thatcher/RL6058, were used to study the genetics of stem rust resistance in Thatcher and TcLr34. Segregation of stem
rust response in each population was used to determine the number of genes conferring resistance, as well as the effect of
the leaf rust resistance gene Lr34 on stem rust resistance. The relationship between resistance in seedling and adult plants was also examined, and an attempt
was made to identify microsatellite markers linked to genes that were effective in adult plants. In field plot tests at least
three additive resistance genes segregated in the RL6071/RL6058 population, whereas two resistance genes segregated in the
RL6071/Thatcher population. The presence of the gene Lr34 permitted the expression of additional stem rust resistance in Thatcher-derived lines both at the seedling and adult plant
stages. Seedling resistance to races TPMK and RKQQ was significantly associated with resistance in adult plants, whereas seedling
resistance to races QCCD and QCCB may have made a minor contribution. The seedling resistance genes Sr16 and Sr12 may have contributed to resistance in adult plants. A molecular marker linked to resistance in adult plants was identified
on chromosome 2BL. 相似文献
11.
Development and Validation of SCAR Markers Co-Segregating with an Agropyron Elongatum Derived Leaf Rust Resistance Gene Lr24 in Wheat 总被引：1，自引：0，他引：1
Summary An Agropyron elongatum-derived leaf rust resistance gene Lr24 located on chromosome 3DL of wheat was tagged with six random amplified polymorphic DNA (RAPD) markers which co-segregated with the gene. The markers were identified in homozygous resistant F2 plants taken from a population segregating for leaf rust resistance generated from a cross between two near-isogenic lines (NILs) differing only for Lr24. Phenotyping was done by inoculating the plants with pathotype 77-5 of Puccinia triticina. To enable gene-specific selection, three RAPD markers (S1302609, S1326615 and OPAB-1388) were successfully converted to polymorphic sequence characterized amplified region (SCAR) markers, amplifying only the critical DNA fragments co-segregating with Lr24. The SCAR markers were validated for specificity to the gene Lr24 in wheat NILs possessing Lr24 in 10 additional genetic backgrounds including the Thatcher NIL, but not to 43 Thatcher NILs possessing designated leaf rust resistance genes other than Lr24. This indicated the potential usefulness of these SCAR markers in marker assisted selection (MAS) and for pyramiding leaf rust resistance genes in wheat. 相似文献
12.
Anne-marie Wolters Evert Jacobsen Mary O'Connell Guusie Bonnema K. Sree Ramulu Hans de Jong Herman Schoenmakers Jelle Wijbrandi Maarten Koornneef 《Euphytica》1994,79(3):265-277
Protoplast fusion can be used to produce somatic hybrids of species that cannot be obtained by sexual hybridization. The possibility to introgress genes from Solanum species into the cultivated tomato species Lycopersicon esculentum, and to obtain novel cytoplasm-nucleus combinations (cybrids) was considered as an important strategy to extend the genetic variation available for tomato breeding. Somatic hybrids between L. esculentum and other Lycopersicon species, as well as between L. esculentum and Solanum or Nicotiana species, have been produced. Specific mutants, genotypes with antibiotic resistances, and metabolic inhibition by iodoacetate or iodoacetamide and irradiation were used for the selection of hybrids. In addition, the improvement of protoplast culture techniques and the use of the favourable tissue culture traits derived from species such as L. peruvianum, which have been introduced into tomato by classical breeding, allowed the efficient recovery of somatic hybrids. However, the occurrence of somatic incongruity in fusion combinations of L. esculentum and Solanum and even more in L. esculentum and Nicotiana, did not allow the production of true cybrids and/or fertile hybrids, indicating the importance of both cytoplasm-nucleus and nucleus-nucleus interactions in somatic incongruity. Another problem with fusions between distantly related species is the strongly reduced fertility of the hybrids and the very limited homoeologous recombination between chromosomes of the parental species. Partial genome transfer from donor to recipient through microprotoplast (+) protoplast fusion, and the production of monosomic or disomic chromosome addition lines, light overcome some of these problems. In symmetric somatic hybrids between L. esculentum and S. tuberosum the occurrence of limited somatic and meiotic recombination was demonstrated. Fertile progeny plants could be obtained, though at a low frequency, when embryo rescue was performed on a large scale after backcrossing hexaploid somatic tomato (+) potato hybrids with a tetraploid potato genotype. The potential value of genomic in situ hybridization (GISH) and RFLPs for the analysis of the genome/chromosome composition of the hybrids has been demonstrated for intergeneric somatic hybrids between Lycopersicon and Solanum.Abbreviations cpDNA
chloroplast DNA
- mtDNA
mitochondrial DNA 相似文献
13.
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
14.
Morad Jafari Peyman Norouzi Mohammad Ali Malboobi Behzad Ghareyazie Mostafa Valizadeh Seyed Aboulghasem Mohammadi Mozhgan Mousavi 《Euphytica》2009,165(2):333-344
Two diploid sugar beet genotypes of agronomical importance were transformed using Agrobactrium tumefaciens harboring pBI35Scry containing a synthetic cry1Ab gene. Leaf blade with attached shoot bases, a highly regenerative tissue, were used as explant substratum for transformation.
PCR screening with cry1Ab-specific primers showed the presence of transgene in more than 50% of the regenerated kanamycin-resistant plants after treatment
with the antibiotic. A transformation rate of 8.8–12.2% (depending on genotype) was achieved as revealed by genomic DNA dot
blotting. The intact integration of transgene cassette into the genome was furthermore confirmed by Southern blot analysis.
The expression of the cry1Ab gene encoding a truncated endotoxin (67 kDa) at about 0.1% of total soluble protein was achieved in the leaves of transgenic
plants as shown by Western blot analysis. Bioassays under in vitro conditions with Spodoptera littoralis, one of the most important pests in sugar beet fields, demonstrated enhanced resistance against this pest. The inheritance
of the inserted transgene was confirmed in F1 plants obtained through crossing of T0 plants with a cytoplasmic male sterile line. Transgenic plants are currently grown in a greenhouse and will be subjected
to further bioassay analyses against other lepidopteran pests of sugar beet. 相似文献
15.
Transformation of a large number of potato varieties: genotype-dependent variation in efficiency and somaclonal variability 总被引：11，自引：0，他引：11
Paul Heeres Marja Schippers-Rozenboom Evert Jacobsen Richard G.F. Visser 《Euphytica》2002,124(1):13-22
Transformation of potato is a genotype dependent process as was shown by experiments conducted with 16 varieties. Not all
genotypes could be transformed with a single procedure hence two different procedures were attempted for all 16 varieties
in a pilot experiment. Large differences in regeneration capacity of putative transformants were observed with the two protocols.
Regeneration capacity and transformation efficiency were not correlated. All varieties were transformed with the same construct,
composed of a kanamycin resistance gene and an antisense gene coding for granule-bound starch synthase. This led to different
percentages of plants with the desired maximum effect (i.e. amylose-free starch) ranging from less than 1 percent to 23.3
percent. It was shown that variety-dependent phenotypic variation occurred ranging from 1 to 21%. Field experiments, conducted
over a number of years, using plants with different degrees of antisense effect (from no detectable effect to maximum effect)
showed that most transformants would have a decreased yield and starch content as determined by specific gravity measurements.
However, these negative effects can be overcome by selecting the proper transgenic plants. Molecular characterisation of transformants
using PCR, showed that 90% of the analysed transgenic plants, belonging to all effect classes, contained vector DNA sequences
since they contained either the NPTIII gene or the trfA gene. The other 10% of the transgenic plants had no insertion of vector
DNA.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
16.
David Gidoni Moshe Bar Baruch Leshem Nehama Gilboa Anahit Mett Julia Feiler 《Euphytica》2001,121(2):145-156
FLP site-specific recombination has been shown in transgenic plants to excise DNA sequences between target FRT sites, and thereby activate transgenes in plants. In previous reports, crossing of tobacco plants expressing FLP recombinase
from a CaMV-35s promoter with plants containing the target FRT sites, hybrid plants with deletion sectors were generated, which were infrequently transmitted to progeny. In this report
we evaluate the occurrence of recombination in F1 hybrid seed derived from crosses of different FLP and FRT-reporter target lines and the germinal transmission of recombined loci from these hybrids to F2 progeny. Twenty hybrids were generated from crosses of independent five FLP-active lines and four FRT-reporter target lines. In one hybrid, FLP deletions occurred at an early stage, prior to seed maturation, and the deletions
from this hybrid were more efficiently transmitted to F2 progeny. The demonstration of FLP-mediated recombination activity and germinal inheritance of the recombined FRT loci are supported by both molecular and enzymatic evidence.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
17.
The genetics of resistance to Ascochyta blight (Ascochyta fabae f. sp. fabae) was studied in two populations of faba bean (Vicia faba). Plants of a resistant population, ILB 752, and a susceptible one, NEB 463, were screened for their reaction to the pathogen
and the results were quantified on a scale of 0–5. Crosses were made between plants both within and between accessions and
the F1 and F2 generations assessed in a field trial 21 and 45 days after inoculation. Disease scores were greater at 45 days than at 21
days and they were not significantly affected by the presence of susceptible spreader rows in part of the trial. ILB 752 carried
a major dominant gene conferring resistance while NEB 463 carried the recessive allele for susceptibility. Furthermore, a
minority of plants of NEB 463 appeared to carry at least one pair of complementary recessive genes, also conferring resistance.
Most of the plants of ILB 752 were homozygous for the dominant resistance gene and a few were heterozygous. Reciprocal crosses
behaved identically, indicating the absence of maternal effects in the expression of Ascochyta blight resistance in faba beans.
The results show that it is important to confirm the level of heterozygosity for the resistance genes in this partially outbreeding
species before crossing is commenced. The major dominant gene for resistance, identified in ILB 752, has clear potential for
use in breeding for Ascochyta blight resistance. The minor genes identified in NEB 463 also show the potential for accumulating
resistance through mass selection.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
18.
Stability of cassava plants at the DNA level after retrieval from 10 years of in vitro storage 总被引：2，自引：0，他引：2
Summary Cassava (Manihot esculenta Crantz) germplasm collections are conventionally maintained by continuous vegetative propagation in the field. Tissue culture techniques provide a more convenient way to conserve germplasm. The cassava in vitro gene bank held in trust at CIAT comprises nearly 6000 accessions. A study was carried out to determine whether any DNA rearrangements resulting from in vitro storage under slow growth could be detected by molecular analysis in retrieved plants. RFLPs with homologous probes, RAPDs with twenty primers and DNA fingerprinting with M13 probe were tested to detect variation at DNA level in cassava plants after ten-years in vitro storage. The molecular marker data obtained in this study supports the stability of the cassava germplasm under the in vitro storage conditions described in this work. 相似文献
19.
在研究和确定Cry1Ab和Cry1C蛋白对甜菜夜蛾和斜纹夜蛾协同杀虫作用的基础上,利用同源重组技术进行Cry1Ab和Cry1C之间的结构域交换获得高效融合蛋白,利用农杆菌介导的子叶节法将融合抗虫基因表达载体导入大豆中,从而获得抗虫转基因大豆新材料。采用PCR及蛋白试纸条检测法对获得的大豆再生植株进行鉴定,测得结果初世代阳性植株为121株。对部分后代转基因植株的遗传分析表明外源基因能够稳定遗传。室内接种斜纹夜蛾鉴定表明,含有重组基因的转基因大豆对斜纹夜蛾有较强的抗性。因此,利用同源重组技术进行Cry1Aa和Cry1C之间的结构域交换,是获得高抗食叶性害虫大豆的有效途径。 相似文献
20.
Summary
Lycopersicon pimpenellifolium L3707, resistant to the late blight oomycete Phytophthora infestans was crossed with the susceptible Lycopersicon pimpenellifolium 14377 or the susceptible Lycopersicon esculentum ZH. Progeny F1 and F2 generations were scored at the 5-leaf stage for resistance against 175 field and recombinant isolates of the pathogen. F1 plants exhibited various levels of moderate resistance and F2 plants segregated 3:6:7 resistant/moderately resistant/susceptible. The data support the hypothesis that race-non-specific resistance in L3707 is controlled by two independent genes: a partially-dominant gene and a dominant epistatic gene. 相似文献