转基因在玉米中的遗传分离与整合特性的研究

用ＰＣＲ和ＤＮＡ分子杂交方法研究了转基因Ｂｔ在３种不同方法获得的８个转化体后代中的遗传分离、整合性质及其稳定性,结果表明：（１）转基因在大多数转化体后中呈简单的孟德尔遗传;在Ｒ１至Ｒ２代,部分家系中转化体比例偏低,有的发生转基因丢失,但到Ｒ３代以后均趋于正常的孟德尔遗传方式,在群体中固定下来;（２）转基因在不同转化体中的整合类型有一定差异,但整合的位点和拷贝数都较少,且多呈串联或紧密连锁的整合;（     

Development of enhancer trap lines for functional analysis of the rice genome

Enhancer trapping has provided a powerful strategy for identifying novel genes and regulatory elements. In this study, we adopted an enhancer trap system, consisting of the GAL4/VP16-UAS elements with GUS as the reporter, to generate a trapping population of rice. Currently, 31 443 independent transformants were obtained from two cultivars using Agrobacterium-mediated T-DNA insertion. PCR tests and DNA blot hybridization showed that about 94% of the transformants contained T-DNA insertions. The transformants carried, on average, two copies of the T-DNA, and 42% of the transformants had single-copy insertions. Histochemical assays of approximately 1000 T0 plants revealed various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues. The expression pattern of the reporter gene in T1 families corresponded well with the T0 plants and segregated in a 3 : 1 Mendelian ratio in majority of the T1 families tested. The frequency of reporter gene expression in the enhancer trap lines was much higher than that in gene trap lines reported previously. Analysis of flanking sequences of T-DNA insertion sites from about 200 transformants showed that almost all the sequences had homology with the sequences in the rice genome databases. Morphologically conspicuous mutations were observed in about 7.5% of the 2679 T1 families that were field-tested, and segregation in more than one-third of the families fit the 3 : 1 ratio. It was concluded that GAL4/VP16-UAS elements provided a useful system for enhancer trap in rice.     

两例水稻极端异常分离现象的遗传分析

遗传异常分离既是自然界非常普遍的现象, 也是生物进化的动力之一。产生异常分离的原因可能与配子体或孢子体的选择有关。利用6个以类病变(lmi)和矮杆突变体(d6)为亲本的杂交组合(F2或F3), 对该类病变和矮杆基因的遗传规律及异常分离现象作初步的分析。结果显示, lmi×02428和d6×93-11的F2群体以及F3株系中存在极端异常分离的现象; LMI基因附近的分子标记ST8-1和D6基因附近的ST7-1、ST7-2、RM5490的带型分离同样也极显著偏离期望比; 偏分离因子与类病斑LMI和矮杆基因D6紧密连锁, 分别位于第8染色体分子标记ST8和ST8-2之间以及第7染色体分子标记ST7-1和ST7-3之间。异常分离现象还与杂交的组合有直接的关系。     

A Novel Two T-DNA Binary Vector Allows Efficient Generation of Marker-free Transgenic Plants in Three Elite Cultivars of Rice (Oryza sativa L.)

A pilot binary vector was constructed to assess the potential of the 2 T-DNA system for generating selectable marker-free progeny plants in three elite rice cultivars (ZhongZuo321, Ariete and Khao Dawk Mali 105) known to exhibit contrasting amenabilities to transformation. The first T-DNA of the vector, delimited by Agrobacterium tumefaciens borders, contains the hygromycin phosphotransferase (hpt) selectable gene and the green fluorescent protein (gfp) reporter gene while the second T-DNA, delimited by Agrobacterium rhizogenes borders, bears the phosphinothricin acetyl transferase (bar) gene, featuring the gene of interest. 82-90% of the hygromycin-resistant primary transformants exhibited tolerance to ammonium glufosinate mediated by the bar gene suggesting very high co-transformation frequency in the three cultivars. All of the regenerated plants were analyzed by Southern blot which confirmed co-integration of the T-DNAs at frequencies consistent with those of co-expression and allowed determination of copy number for each gene as well as detection of two different vector backbone fragments extending between the two T-DNAs. Hygromycin susceptible, ammonium glufosinate tolerant phenotypes represented 14.4, 17.4 and 14.3% of the plants in T1 progenies of ZZ321, Ariete and KDML105 primary transformants, respectively. We developed a statistical model for deducing from the observed copy number of each T-DNA in T0 plants and phenotypic segregations in T1 progenies the most likely constitution and linkage of the T-DNA integration locus. Statistical analysis identified in 40 out of 42 lines a most likely linkage configuration theoretically allowing genetic separation of the two T-DNA types and out segregation of the T-DNA bearing the bar gene. Overall, though improvements of the technology would be beneficial, the 2 T-DNA system appeared to be a useful approach to generate selectable marker-free rice plants with a consistent frequency among cultivars.     

转新型双抗虫基因棉花的遗传分析

首次用含合成的BtCrylAc活性杀虫蛋白嵌合基因及慈菇蛋白酶抑制剂B(API-B)基因表达框的双抗虫基因植物表达载体,通过土壤根癌杆菌介导转化棉花品种冀合321,获得一批抗虫的转化再生棉花植株。利用叶片涂抹卡那霉素、叶片离体养虫和PCR扩增等检测方法对6个不同转双抗虫基因株系的抗虫性进行遗传分析。结果显示,转化株系自交的T1抗虫性状遗传较为复杂;农杆菌介导获得的转基因抗虫棉在早期世代不易选到纯合系,但是随着对抗虫性状进行单向选择,到T4和T5就能获得抗虫纯合系。利用抗虫性稳定的转化后代材料和转化受体进行田间杂交,发现F2抗虫性分离完全符合一对或两对显性基因的分离规律,并证明了DR248和DR193两个材料为外源基因双拷贝插入。转化株系的Southern杂交也证明了上述结果。     

无选择标记和载体骨干序列的Xa21转基因水稻的获得

利用双右边界T-DNA载体通过根癌农杆菌介导法将水稻白叶枯病广谱抗性基因Xa21导入杂交稻重要恢复系C418中。T0代共获得27个独立转基因株系,通过田间抗性鉴定与PCR分析,有17个株系的Xa21基因分子鉴定为阳性,且对白叶枯病原菌P6生理小种具有抗性。通过对17个株系的后代植株进行田间抗性鉴定,分子标记辅助选择及Southern杂交分析,结果显示4个株系的T1代植株中能分离出无潮霉素标记基因的Xa21转基因植株。无选择标记Xa21转基因株系的获得率为15%。PCR检测还表明,这些无选择标记的Xa21转基因植株不带有载体骨架序列。通过对转基因后代进一步的抗性鉴定与PCR辅助选择,获得了无选择标记和载体骨架序列的转基因Xa21纯合的抗白叶枯病水稻。     

虾夷扇贝遗传连锁图谱的初步构建

用AFLP标记首次构建了虾夷扇贝遗传连锁图谱。用56对引物组合对父母本和52个F^₁代个体进行遗传连锁分析, 共得到1 855个标记, 其中多态位点为598(32.2%)个, 而354个符合孟德尔1: 1分离比。用这些标记和23个偏分离标记(0.01相似文献   

Evidence for the location of foreign genes in three different chromosomes in a plant obtained from direct DNA transfer

Tobacco mesophyll protoplasts were treated with plasmids, pCT2 (17.1 kbp) or pCT2T3 (18.3 kbp), which contained a chimeric aminoglycoside phosphotransferase II (APH(3′)II) gene and an intact nopaline synthase gene. Expression of two marker enzymes, APH(3′)II and nopaline synthase, were analyzed in transformed plants. Four out of 16 transformants obtained by pCT2T3 possessed both enzymes. Upon self-pollination, the progeny of one of transformants (T2) segregated to 153∶4 in terms of resistant and susceptible character to kanamycin, suggesting insertion of foreign genes into three independent chromosomes. The kanamycin resistant character in the rest of transformants showed 3∶1 segregation. DNA blot analysis of the T2 transformant and progenies indicated the presence of two marker genes.     

Exceptional Segregation of a Selectable Marker (Kan(r)) in Arabidopsis Identifies Genes Important for Gametophytic Growth and Development

Genes transformed into plants are usually inherited in a regular Mendelian manner. There are, however, transformants in which the selectable trait fails to segregate as expected. Genetic analysis of the kanamycin-resistance (Kan(R)) trait in >900 independent transformants of Arabidopsis revealed that 9% produced progeny families with an enormous deficiency of Kan(R) individuals. Self-pollination of individual Kan(R) plants from these families revealed lines that continued to segregate for a deficiency of Kan(R) seedlings. In subsequent generations, the segregation ratio in these families stabilized at ~1 Kan(R): 3 Kan(S). Molecular analyses showed that the deficiency of Kan(R) individuals reflected the complete absence of the introduced DNA. Reciprocal backcrosses to untransformed plants showed unequal transmission of the Kan(R) trait through the gametes in these exceptional lines. In five cases, this was primarily a failure of transmission through the microgametophyte (pollen) and in the other two cases, primarily a failure of transmission through the megagametophyte (embryo sac or egg). The number of seeds per silique was reduced by 50% in the latter two lines. We conclude that our exceptional transformants contain T-DNA insertions that delete or disrupt genes essential for gametophytic growth and development.     

Procedures allowing the transformation of a range of European elite wheat (Triticum aestivum L.) varieties via particle bombardment.

Ten current European wheat varieties were transformed at efficiencies ranging from 1-17% (mean 4% across varieties) following modifications in particle bombardment and tissue culture procedures. All plants surviving phosphinothricin selection were screened for uidA and bar gene activity, and for the presence of marker gene sequences by PCR analysis. A minimum of 35% plant 'escape' frequency was achieved with selection on 4 mg l(-1) gluphosinate ammonium after shoot initiation. Mean co-transformation frequency with various genes-of-interest was 66%. The estimated number of insertions of the uidA gene in 25 lines were; 1-2 in 32%, 3-5 in 52%, and 6-8 in 16% of lines. In T(1) progenies, marker genes segregated in a Mendelian fashion in 50% of 39 lines analysed, as determined by transgene activity assays. Based on PCR analysis, it appeared that in some lines the occurrence of distorted segregation was due to poor transmission of the transgenes.     

水稻Dwarf1移码突变的新突变体鉴定

从一批水稻品种"中花11"组织培养苗里分离到一个矮化突变株"C6PS",它的T2代群体株高呈现3:1分离。利用该群体矮化单株与"珍汕97"、"牡丹江8"构建2个F2群体F2(CZ)、F2(CM),两个群体中高株与矮株均呈现3:1分离,证明该性状变异为单基因控制。"C6PS"表现型与已经报道的Dwarf1隐性突变体"d1"相似,以D1附近标记RM430检测F2(CZ)群体基因型,结果显示群体表型与RM430基因型呈极显著相关(P=0.0001),将该基因初步定位于Dwarf1附近。对"C6PS"及"中花11"进行D1序列分析显示,突变株中D1基因在其第九个外显子与第九个内含子的剪接位点上发生6个碱基的缺失,根据缺失两侧序列设计C6PS-D1L/R标记,在T2代群体该标记与表型呈现共分离,表明"C6PS"是一种新的Dwarf1突变体。cDNA测序显示突变体d1基因转录产物发生26个碱基的缺失,导致移码产生终止突变,从而无法翻译出有功能的Gα蛋白,因此,它是一个Gα功能缺失突变体。叶倾斜度检测显示"C6PS"对油菜素内酯响应比野生型"中花11"弱。     

Genetic and molecular analysis of Arabidopsis thaliana (ecotype ‘Estland’) transformed with Agrobacterium

Summary Agrobacterium-mediated transformation of Arabidopsis, ecotype ‘Estland’, was established from root explants using kanamycin selection. Continuous light during callus and shoot induction phases was promotive for shoot regeneration, as compared to light/dark cycles. Use of optimized conditions for transformation led to the formation of kanamycin-resistant calluses (up to 77%) and transformed plantlets at a frequency of up to 45%. Southern analysis showed the presence of 1.2. or more T-DNA inserts in 33%, 50%, and 17% of the primary transformants, respectively. Mendelian, as well as non-Mendelian, inheritance patterns were obtained upon screening the progeny (T1) of various transformants for the expression of gus and nptII genes; the analysis of some of these transformants at the molecular level also corroborated the Mendelian inheritance pattern. Moreover, genotypes of the T1 progeny could be predicted on the basis of T2 progeny analysis.     

Tobacco PR-2d promoter is induced in transgenic cucumber in response to biotic and abiotic stimuli

The PR-2d promoter/uidA (GUS) gene construct was introduced into the cucumber (Cucumis sativus L.) genome and several transgenic lines were produced. Activation of the PR-2d promoter was investigated in these plants in response to inoculation with fungal pathogens and after salicylic acid (SA) or cold treatments. Treatment with exogenous SA increased GUS activity 2 to 11 fold over that of the control. Endogenous SA and its conjugate salicylic acid glucoside (SAG) rose in parallel after inoculation with the fungal pathogen Pseudoperonospora cubensis, with SAG becoming the predominant form. The free SA levels increased 15 fold above the basal level at 5 dpi and preceded the induction of the PR-2d promoter by five days, which occurred at 10 dpi with a 12 fold increase over the control. Inoculation with another fungal pathogen, Erysiphe polyphage, increased GUS activity 4 to 44 fold over that of the control. During normal development of flowers in the cucumber, the PR-2d/uidA gene expressed in the floral organs was similar to that of the primary host. In addition, we present the first evidence that the PR-2d promoter was induced (624 fold) under cold stress. We demonstrate that in the heterologous state the gene construct was expressed according to the signalling pattern of the native species and was stably transmitted to progeny over four generations.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium

Immature embryo-derived calli of spring wheat (Triticum aestivum L.) cv Veery5 were transformed using Agrobacterium tumefaciens strain LBA4404 carrying either binary vector pHK22 or superbinary vector pHK21, the latter carrying an extra set of vir genes--vir B, -C and -G. In both cases, transient beta-glucuronidase ( GUS) expression ranging from 35-63% was observed 3 days after co-cultivation, but 587 calli infected with pHK22/LBA4404 failed to produce a single stably transformed plant, whereas 658 calli infected with pHK21/LBA4404 gave rise to 17 transformants carrying both the GUS and bar genes. Regeneration media supplemented with 0.1 M spermidine improved the recovery of transformants from pHK21/LBA4404-infected calli from 7% to 24.2%, resulting in an increase in the overall transformation frequency from 1.2% to 3.9%. The results suggest that two important factors that could lead to an improvement in transformation frequencies of cereals like wheat are (1) the use of superbinary vectors and (2) modification of the polyamine ratio in the regeneration medium. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Of the transformed plants, 35% showed single-copy insertion of the transgene as shown by both Southern analysis and the segregation ratios.     

Development of stem borer resistant transgenic parental lines involved in the production of hybrid rice

Stem borer resistant transgenic parental lines, involved in hybrid rice, were produced by Agrobacterium-mediated gene transfer method. Two pSB111 super-binary vectors containing modified cry1Ab/cry1Ac genes driven by maize ubiquitin promoter, and herbicide resistance gene bar driven by cauliflower mosaic virus 35S promoter were, used in this study. Embryogenic calli after co-cultivation with Agrobacterium were selected on the medium containing phosphinothricin. Southern blot analyses of primary transformants revealed the stable integration of bar, cry1Ab and cry1Ac coding sequences into the genomes of three parental lines with a predominant single copy integration and without any rearrangement of T-DNA. T1 progeny plants disclosed a monogenic pattern (3:1) of transgene segregation as confirmed by molecular analyses. Furthermore, the co-segregation of bar and cry genes in T1 progenies suggested that the transgenes are integrated at a single site in the rice genome. In different primary transformants with alien inbuilt resistance, the levels of cry proteins varied between 0.03 and 0.13% of total soluble proteins. These transgenic lines expressing insecticidal proteins afforded substantial resistance against stem borers. This is the first report of its kind dealing with the introduction of Bacillus thuringiensis (Bt) cry genes into the elite parental lines involved in the development of hybrid rice.     

T-DNA locus structure in a large population of soybean plants transformed using the Agrobacterium-mediated cotyledonary-node method

Designing transformation experiments for either functional genomics or crop improvement requires knowledge of the transgene locus structure, number, transmission and expression resulting from a specific transformation method. We recently reported an improvement to the soybean [Glycine max (L.) Merrill] cotyledonary-node transformation method that resulted in the efficient production of transgenic plants. To characterize the transgene loci resulting from this method, we analysed 270 independent T0 plants and 95 randomly selected T1 progenies for T-DNA locus complexity using Southern analysis. The lines were transformed with Agrobacterium tumefaciens strains LBA4404 or EHA105 carrying the binary plasmids pGPTV, pTOK233, pCAMBIA1303 or pCAMBIA1309, and regenerated in medium supplemented with or without silver nitrate (AgNO3). Analysis in the T0 generation showed that the number of hpt-hybridizing fragments per plant ranged from 1-15, with 31.5% of the lines having a single hpt-hybridizing fragment. Each primary soybean transformant had, on average, 2.0 unlinked transgene loci and that half of the segregating loci in the T1 progenies were single, simple T-DNA insertions. Of the loci containing multiple T-DNA fragments, a low frequency had tandem and inverted repeat T-DNA structures. Integration of binary plasmid backbone sequences occurred in 37% of primary transformants. A. tumefaciens strain, binary plasmid and thiol treatment had no significant effect on transgene locus structure, numbers or expression. Interestingly, exposure of soybean explants to AgNO3 throughout shoot induction and elongation increased T-DNA locus complexity in the primary transformants and decreased silencing of gusA expression in the T1 generation.     

Agrobacterium-Mediated Gene Transfer Results Mainly in Transgenic Plants Transmitting T-DNA as a Single Mendelian Factor

Forty-four independent transformed tobacco plants were obtained from a cocultivation experiment with Agrobacterium tumefaciens strains carrying modified Ti-plasmids. The transformed plants were either self-fertilized or crossed with nontransformed plants or with other transformed plants. The segregation of a phenotypic marker (kanamycin resistance) in the progenies of these plants was determined. In 40 cases out of 44, the segregation of the kanamycin resistance marker is consistent with Mendelian genetics. Among these 40 clones, 35 contain a single kanamycin resistance locus. The five others segregate two independent resistance loci. In two of the single insert clones, the segregation ratio after selfing indicates that the T-DNA insertion may have caused a recessive lethal mutation.     

Analysis of transgene(s) (psy+crtI) inheritance and its stability over generations in the genetic background of indica rice cultivar Swarna

Rice the major staple food crop which feeds more than half of the world’s population but, lacks pathway to synthesize and accumulate provitamin A in endosperm therefore rice eaters particularly children, and pregnant women suffer due to vitamin A deficiency. The pathway for provitamin A synthesis in rice endosperm has been engineered and transgenic rice lines have yellow endosperm, called ‘Golden Rice’. The present study aimed at studying the inheritance of transgene(s) in six transgenic events of ‘Golden Rice’ and transfer of provitamin A trait from transgenic lines to a widely grown mega rice variety Swarna. The events E1, R1 and W1 showed normal Mendelian inheritance in F₂, BC₁F₁ and BC₁F₂ generations. The event W1 was studied in BC₁F₃ as well and showed normal Mendelian inheritance of 3:1. The inheritance pattern in L1 event in BC₁F₁ and BC₁F₂ showed normal Mendelian inheritance following expected ratio 1:1 and 3:1 respectively. The two events G1 and T1 showed distorted segregation in BC₁F₂ and BC₂F₂ respectively in Swarna genetic background. In G1 event, transgene inheritance showed segregation distortion in BC₁F₂ in favour of transgene negative plants. In T1 event, inheritance followed expected Mendelian segregation in BC₁F₁, BC₂F₁ and BC₂F₂, generations. However, when tested against co-dominant inheritance 1:2:1 pattern in BC₂F₂, segregation distortion was observed with less than the expected transgene homozygotes. While against 3:1 ratio, it showed the expected segregation pattern in BC₂F₂ generation. Segregation distortion probably due to differential transmission of transgene positive/negative gametes through either/both parents which needs further study.     

Expression of a Novel Antiporter Gene from Brassica napus Resulted in Enhanced Salt Tolerance in Transgenic Tobacco Plants

Tobacco leaf discs were transformed with a plasmid pBIBnNHX1, containing the selectable marker neomycin phosphotransferase gene (nptII) and Na⁺/H⁺ vacuolar antiporter gene from Brassica napus (BnNHX1), via Agrobacterium tumefaciens-mediated transformation. Thirty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the BnNHX1 gene had integrated into plant genome and Northern blot analysis revealed the transgene expression at various levels in transgenic plants. Transgenic plants expressing BnNHX1 had enhanced salt tolerance and could grow and produce seeds normally in the presence of 200 mM NaCl. Analysis for the T₁ progenies derived from seven independent transgenic primary transformants expressing BnNHX1 showed that the transgenes in most tested independent T₁ lines were inherited at Mendelian 3:1 segregation ratios. Transgenic T₁ progenies could express _BnNHX1 and had salt tolerance at levels comparable to their T₀ parental lines. This study implicates that the BnNHX1 gene represents a promising candidate in the development of crops for enhanced salt tolerance by genetic engineering.     

Loss of decreased-rubisco phenotype between generations of wheat transformed with antisense and sense rbcS

The elite UK winter wheat cv. Riband was transformed with constructs containing rbcS in sense and antisense orientations driven by the maize ubiquitin promoter with a transformation efficiency of 1.2%. Of 77 primary transformants 31% of the sense-rbcS transformed lines and 78% of the antisense-rbcS transformed lines had decreased rubisco content compared to wild-type and marker-only controls, with decreases of up to 60%. However, in the T1 progeny which inherited the transgene, only 5% showed significantly decreased rubisco content and these effects were on the margins of significance. Five potential T2 homozygous lines from T1 parents which had transgene segregation consistent with a single locus were identified. There was no significant decrease in rubisco content relative to wild-type in any of these lines (LSD of 8% for P= 0.05). Expression of antisense rbcS transgenes in two of these T2 lines was low but was increased following exposure of the plants to 37°C for 48 h. However this did not induce a significant decrease in rubisco protein content relative to controls. Southern analysis of two antisense lines showed that they had low copy number and 1–2 insertion events. In one of the two lines there was increased methylation of the ubiquitin intron in T2 samples compared to the TO primary transformant. Further work is required to establish whether methylation occurred in all the lines which lost the phenotype, and therefore the likelihood of this being the cause. The disappearance of the decreased rubisco-content phenotype between generations may therefore be attributable to (1) greater activity of the ubiquitin promoter due to greater stress in the T0 generation plants and/or (2) increased methylation of the transgene promoter region between generations.