Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley

In order to evaluate the long-term stability of transgene expression driven by the B(1)- and D-hordein promoters in transgenic barley ( Hordeum vulgare L., 2 n=2 x=14), we analyzed plants from 15 independent transgenic barley lines [6 for uidA and 9 for sgfp(S65T)] produced via microprojectile bombardment of immature embryos; 4 were diploid and 11 were tetraploid. The expression and inheritance of transgenes were determined by analysis of functional transgene expression, polymerase chain reaction and fluorescence in situ hybridization (FISH). Ability to express transgenes driven by either B(1)- or D-hordein promoter was inherited in T(4) and later generations: T(4) (2 lines), T(5) (8 lines), T(6) (3 lines), T(8) (1 line) and T(9) (1 line). Homozygous transgenic plants were obtained from 12 lines [5 for uidA and 7 for sgfp(S65T)]; the remaining lines are currently being analyzed. The application of the FISH technique for physical mapping of chromosomes was useful for early screening of homozygous plants by examining for presence of the transgene. For example, one line expressing uidA, and shown to have doublet fluorescence signals on a pair of homologous chromosomes was confirmed as a homozygous line by its segregation ratio; additionally this line showed stable inheritance of the transgene to T(9) progeny. The expression of transgenes in most lines (14 out of 15 lines) driven by hordein promoters was stably transmitted to T(4) or later generations, although there was a skewed segregation pattern (1:1) from the T(1) generation onward in the remaining line. In contrast, transgene silencing or transgene loss under the control of the maize ubiquitin promoter was observed in progeny of only 6 out of 15 lines.     

Association of transgene integration sites with chromosome rearrangements in hexaploid oat

Transgene loci in 16 transgenic oat (Avena sativa L.) lines produced by microprojectile bombardment were characterized using phenotypic and genotypic segregation, Southern blot analysis, and fluorescence in situ hybridization (FISH). Twenty-five transgene loci were detected; 8 lines exhibited single transgene loci and 8 lines had 2 or 3 loci. Double FISH of the transgene and oat C- and A/D-genome-specific dispersed and clustered repeats showed no preferences in the distribution of transgene loci among the highly heterochromatic C genome and the A/D genomes of hexaploid oat, nor among chromosomes within the genomes. Transgene integration sites were detected at different locations along individual chromosomes, although the majority of transformants had transgenes integrated into subtelomeric and telomeric regions. Transgene integration sites exhibited different levels of structural complexity, ranging from simple integration structures of two apparently contiguous transgene copies to tightly linked clusters of multiple copies of transgenes interspersed with oat DNA. The size of the genomic interspersions observed in these transgene clusters was estimated from FISH results on prometaphase chromosomes to be megabases long, indicating that some transgene loci were significantly larger than previously determined by Southern blot analysis. Overall, 6 of the 25 transgene loci were associated with rearranged chromosomes. These results suggest that particle bombardment-mediated transgene integration may result from and cause chromosomal breakage and rearrangements. Received: 29 July 1999 / Accepted: 9 November 1999     

Irregular patterns of transgene silencing in allohexaploid oat

An irregular pattern of transgene silencing was revealed in expression and inheritance studies conducted over multiple generations following transgene introduction by microprojectile bombardment of allohexaploid cultivated oat (Avena sativa L.). Expression of two transgenes, bar and uidA, delivered on the same plasmid was investigated in 23 transgenic oat lines. Twenty-one transgenic lines, each derived from an independently selected transformed tissue culture, showed expression of both bar and uidA while two lines expressed only bar. The relationship of the transgenic phenotypes to the presence of the transgenes in the study was determined using (1) phenotypic scoring combined with Southern blot analyses of progeny, (2) coexpression of the two transgenic phenotypes since the two transgenes always cosegregated, and (3) reactivation of a transgenic phenotype in self-pollinated progenies of transgenic plants that did not exhibit a transgenic phenotype. Transgene silencing was observed in 19 of the 23 transgenic lines and resulted in distorted segregation of transgenic phenotypes in 10 lines. Silencing and inheritance distortions were irregular and unpredictable. They were often reversible in a subsequent generation of self-pollinated progeny and abnormally segregating progenies were as likely to trace back to parents that exhibited normal segregation in a previous generation as to parents showing segregation distortions. Possible causes of the irregular patterns of transgene silencing are discussed.     

转基因水稻中外源基因的荧光原位杂交（FISH）分析

Using fluorescence in situ hybridization (FISH) with metaphase preparations, we localized a transferred barnase-psl DNA sequence onto chromosomes in 8 rice transgenic plants. All the tested rice transgenic lines showed hybridization signals on the middle and terminal regions of chromosome arms except for those close to centromeres. In two lines, two different integration sites were identified, and the other lines showed only one integration site. With the aid of Southern analysis and expression detection, we found that the barnase tended to show a higher level expression in the lines whose integration sites near the distal regions of chromosomes, while the expression level became lower in the lines whose integration sites near the centromeres. This result suggested a possible relationship between chromosomal location of transgenes and the expression level. However it showed no obvious relationship between copy numbers and expression levels. In most cases, the results of multi-color FISH showed that barnase-ps1 always integrated at the same position on the chrmosome as the reporter genes(pHctinG).     

应用荧光原位杂交技术检测人类APPSWE基因在转基因小鼠染色体上的定位及位置效应

应用荧光原位杂交技术研究了人类淀粉样前体蛋白基因瑞典型突变（APPSWE）在转基因小鼠首建、F1及F2代小鼠染色体上的整合及定位,结果在2只首建转基因小鼠中,分别观察80个分裂相,出现杂交信号的核型分别为34及36个,检出率为42.5%和45%;1只F1及1只F2代转基因小鼠中,分别观察100个分裂相,出现杂交信号的核型分别为33及30个,检出率为33%和30%。转基因分别整合在8号、1号、17号和2号染色体上,提示转基因APPSWE已稳定整合到转基因小鼠的染色体上,并通过生殖细胞遗传给予子代,证实转基因在小鼠染色上的整合可能是随机的多点整合,同时,对不同整合位点的转基因小鼠进行了表型研究,结果发现不同整合位点对表型具明显影响。     

Integration and inheritance of transgenes in crop plants and trees

Transgene integration and inheritance have been investigated in a number of crop plants and few tree species. Transgene integration is predominantly a random process, whether mediated by Agrobacterium or particle bombardment. Depending on the genomic position of the integrated transgene and structure of the integration site as well as copy number of the transgene in the genome, its expression may be stable or variable. Therefore, integration patterns would affect the mode of transgene inheritance in plants, regardless of the method of gene transfer. So far, both Mendelian and non-Mendelian inheritance of transgenes has been reported across several generations (T1–T3) of crop plants. In few tree species (apple, poplar, plum, and American chestnut), mostly Mendelian inheritance of the transgenes has been observed in the T1 or BC1 generations. However, detailed studies in the transgenic papaya trees showed Mendelian segregation of the transgene in the T1 generation but non-Mendelian inheritance in the T2 generation. Variation in transgene inheritance was also detected in transgenic apple and plum trees. Long generation cycles in many economically important tree species preclude investigation of inheritance of transgenes in the tree progeny. Production of early flowering trees, either by genetic modification or by environmental modulation, would facilitate the study of transgene inheritance across generations of transgenic trees. In order to overcome problems of randomness of transgene integration, targeted transgene insertions by homologous or site-specific recombination or by designer recombinases or nucleases offer prospects for stable integration of transgenes in predetermined locations in the plant genome. And perhaps, that might provide a platform for stable expression and Mendelian inheritance of transgenes in plants.     

抗除草剂转基因大豆后代遗传分析

分析了来源于农杆菌介导的4个独立的大豆转化系的后代遗传特性。分别采用种子切片GUS染色方法和除草剂涂抹以及喷洒方法检测gus报告基因和抗除草剂bar基因在后代的表达。其中3个转化系T1代gus基因和bar基因能够以孟德尔方式3：1连锁遗传,说明这2个基因整合在大豆基因组的同一位点。这3个转化系在T2代获得了纯合的转化系,并能够稳定遗传至T5代。有一个转化系在T1代GUS和抗除草剂检测都为阴性,但通过Southern杂交证明转基因存在于后代基因组,显示发生了转基因沉默。为了证明转基因沉默是转录水平还是转录后水平,T1代植物叶片接种大豆花叶病毒（SMV）并不能抑制转基因沉默,说明该转化系基因沉默可能不是发生在转录后水平。     

转不可翻译PVY^N CP基因烟草的抗病性分析

我们曾报道表达不可翻译PVY^N CP基因的转基因烟草抗病性是由RNA介导的,其抗病性类似于转录后的基因沉默(PTGS)。本研究以这类不同抗性的T0代转基因烟草植株为材料,对自交后的T1代转基因植株的遗传和抗病性进行了分析,并选取部分T1代抗病株系自交留种。对T2代RNA介导抗病性转基因植株进行了分子分析和一系列抗病性研究。结果表明,含1—2个转基因拷贝的T0代感病植株,在T1代中的Km抗性分离符合单位点插入的3：1的遗传规律;含3个或3个以上转基因拷贝的T0代中抗或高抗植株,在T1代中的Km抗性分离符合多位点插入的15：1或63：1的遗传规律。大多数T1、T2代转基因植株的抗病性与转基因拷贝数成正相关,转基因在T1、T2代植株中能够转录表达,且转基因植株之间转基因mRNA在细胞质中的积累水平与转基因植株的抗病性成负相关。转基因植株的抗病性能够在T1、T2代中遗传,且T2代转基因植株的抗病性具有以下特征：1)既抗病毒粒体又抗病毒RNA的侵染,且这种抗病性不受接种物剂量的影响;2)抗病谱较窄,只对PVY的某些株系具有高度抗病性;3)与传毒方式无关,既抗摩擦接种又抗带毒蚜虫接种;4)与植株的发育阶段没有关系。     

High-resolution structural analysis of biolistic transgene integration into the genome of wheat

Transformation of plant genomes by biolistic methods has become routine over the past decade. However, relatively little is known about how transgenes are physically integrated into the host genome. Using a high-resolution physical mapping technique, fluorescence in situ hybridization on extended DNA fibers (fiber-FISH), 13 independent transgenic wheat lines were analyzed to determine the structural arrangement of stably inherited transgenes in host-plant chromosomes. Twelve transgenic lines were transformed with a single plasmid and one line was co-transformed with two separate plasmids, which co-segregated genetically. Three basic integration patterns were observed from the fiber-FISH experiments: Type I, large tandemly repeated integration; Type II, large tandem integrations interspersed with unknown DNA; and Type III, small insertions, possibly interspersed with unknown DNA. Metaphase FISH showed that the integration of transgenes was in both hetero- and euchromatic, as well as proximal, interstitial and distal, regions of the chromosomes. In the transgenic plants, the type of promotor used, rather than the chromosomal site of transgene integration, was most critical for transgene expression. The integration of the transgenes was not associated with detectable chromosomal rearrangements. Received: 25 August 2000 / Accepted: 31 October 2000     

The distribution of transgene insertion sites in barley determined by physical and genetic mapping

The exact site of transgene insertion into a plant host genome is one feature of the genetic transformation process that cannot, at present, be controlled and is often poorly understood. The site of transgene insertion may have implications for transgene stability and for potential unintended effects of the transgene on plant metabolism. To increase our understanding of transgene insertion sites in barley, a detailed analysis of transgene integration in independently derived transgenic barley lines was carried out. Fluorescence in situ hybridization (FISH) was used to physically map 23 transgene integration sites from 19 independent barley lines. Genetic mapping further confirmed the location of the transgenes in 11 of these lines. Transgene integration sites were present only on five of the seven barley chromosomes. The pattern of transgene integration appeared to be nonrandom and there was evidence of clustering of independent transgene insertion events within the barley genome. In addition, barley genomic regions flanking the transgene insertion site were isolated for seven independent lines. The data from the transgene flanking regions indicated that transgene insertions were preferentially located in gene-rich areas of the genome. These results are discussed in relation to the structure of the barley genome.     

The metabolic profiles of transgenic cucumber lines vary with different chromosomal locations of the transgene

The metabolic profiles of five transgenic cucumber lines were compared taking into consideration their transgene integration sites. The plants analyzed were homozygous and contained transgenes integrated in a single locus on chromosomes I, II, III or IV. The transgenes were preferentially located in the euchromatic regions. Each of these locations possessed a specific metabolic profile. The number of altered compounds in the transgenic lines varied between 9 and 23 of the 47 metabolites identified. These alterations seem to be specific for each independent transgene integration. However, some changes are common: a decrease in the levels of phenylalanine, aspartate, ethanolamine and pipecolate, and an increase in the level of benzoic acid. The observed effects of transgene introduction are discussed in this paper.     

Field performance of transgenic citrus trees: Assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics

ABSTRACT: BACKGROUND: The future of genetic transformation as a tool for the improvement of fruit trees depends on the development of proper systems for the assessment of unintended effects in field-grown GM lines. In this study, we used eight transgenic lines of two different citrus types (sweet orange and citrange) transformed with the marker genes beta-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) as model systems to study for the first time in citrus the long-term stability of transgene expression and whether transgene-derived pleiotropic effects occur with regard to the morphology, development and fruit quality of orchard-grown GM citrus trees. RESULTS: The stability of the integration and expression of the transgenes was confirmed in 7-year-old, orchard-grown transgenic lines by Southern blot analysis and enzymatic assays (GUS and ELISA NPTII), respectively. Little seasonal variation was detected in the expression levels between plants of the same transgenic line in different organs and over the 3 years of analysis, confirming the absence of rearrangements and/or silencing of the transgenes after transferring the plants to field conditions. Comparisons between the GM citrus lines with their non-GM counterparts across the study years showed that the expression of these transgenes did not cause alterations of the main phenotypic and agronomic plant and fruit characteristics. However, when comparisons were performed between diploid and tetraploid transgenic citrange trees and/or between juvenile and mature transgenic sweet orange trees, significant and consistent differences were detected, indicating that factors other than their transgenic nature induced a much higher phenotypic variability. CONCLUSIONS: Our results indicate that transgene expression in GM citrus remains stable during long-term agricultural cultivation, without causing unexpected effects on crop characteristics. This study also shows that the transgenic citrus trees expressing the selectable marker genes that are most commonly used in citrus transformation were substantially equivalent to the non-transformed controls with regard to their overall agronomic performance, as based on the use of robust and powerful assessment techniques. Therefore, future studies of the possible pleiotropic effects induced by the integration and expression of transgenes in field-grown GM citrus may focus on the newly inserted trait(s) of biotechnological interest.     

Expression and Inheritance of Nine Transgenes in Rice

A total of 66 transgenic rice cell lines were produced by simultaneously transforming rice callus with nine different plasmids/genes. PCR analysis indicated that the co-transformation frequency of each gene was about 70%. All the cell lines carried at least three genes and 11 cell lines carried all nine genes. Thirty-two fertile transgenic plants (R₀) were generated from the transgenic cell lines and seeds of 32 transgenic R₁ lines and 5 R₂ lines were harvested and analyzed for gene inheritance and protein expression. Progeny segregation analysis indicated that the multiple transgenes were integrated into the same locus of the rice genome, resulting in a 3:1 segregation ratio of the transgenes. Expression analysis of all nine transgenes revealed that the transgenes were expressed in all generations (R₀, R₁, and R₂) and about half of the transgenes from each line were expressed. The expression of one transgene appears to have no effect on the expression of another transgene. Among the 66 cell lines, six lines (9.1%) expressed seven or eight transgenes out of the nine transformed genes. All together, our results showed that multiple genes could be delivered into rice cells simultaneously and cell lines expressing multiple genes could be generated. The results and procedures reported here should be useful in designing multi-plasmid transformation experiments such as those required for plant metabolic engineering.     

Stability and inheritance of endosperm-specific expression of two transgenes in progeny from crossing independently transformed barley plants

To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T₈ plant, having uidA (or gus) driven by the barley endosperm-specific B₁-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T₄ plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F₁ progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F₂ seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F₂ plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F₃ and F₄ generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.     

Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment

The relationship between transgene copy number, rearrangement levels, inheritance patterns, expression levels, transgene stability and plant fertility was analysed in a random population of 95 independently transformed rice plant lines. This analysis has been conducted for both the selectable marker gene ( aphIV) and the unselected reporter gene ( gusA), in the presence or absence of flanking Matrix Attachment Regions (MARs) in order to develop a better understanding of transgene behaviour in a population of transgenic rice plants created by particle bombardment. In the first generation (T(0)), all the independently transformed plant lines contained and expressed the aphIV gene conferring resistance to hygromycin, but only 87% of the lines were co-transformed with the unselected gusA marker gene. Both transgenes seemed to be expressed independently. Most lines exhibited complex transgene rearrangements as well as an intact transgene expression unit for both aphIV and gusA transgenes. Transgene copy number was proportional to the quantity of DNA used during bombardment. In T(0) plants, high gusA copy number significantly decreased GUS expression levels but there was no correlation between expression level and transgene copy number across the entire population of lines. Four main factors impaired transgene expression in primary transgenic plants (T(0)) and their progeny (T(1)): (1) absence of transgene expression in T(0) plants (41% of lines), (2) sterility of T(0) plants (28% of lines), (3) non-transmission of intact transgenes to some or all progenies (at least 14% of lines), and (4) silencing of transgene expression in progeny plants (10% of lines). Transgene stability was significantly related to differences in transgene structure and expression levels. The presence of Rb7 MARs flanking the gusA expression unit had no effect on plant fertility or non-transmission of transgenes, but provided copy number-dependent expression of the transgene and improved expression levels and stability over two generations. Overall, only 7% of the plant lines without MARs and 17% of the lines with MARs initially generated, exhibited stable transgene expression over two generations.     

Allelic composition and genetic background effects on transgene expression and inheritance in white clover

Allelic composition and genetic background effects on GUS expression and inheritance using a chimeric (cauliflower mosaic virus 35Sp:uidA) transgene were investigated in white clover as a prelude to transgenic cultivar development. Stable expression and Mendelian inheritance of the uidA transgene was observed over two generations when the uidA transgene was maintained in a heterozygous state. Transgenic backcross progeny (BC1) were intercrossed to produce segregating F2 populations. GUS-positive F2 plants were test-crossed with a non-transgenic control plant to determine whether individuals were heterozygous or homozygous for the transgene. Both expected and distorted segregation ratios were observed. Distortion of the segregation ratio was not caused by transgene inactivation or rearrangement, but was influenced by genetic background. BC1, BC2 and F2 populations were found to have similar levels of uidA gene expression. Quantification of GUS expression from progeny of high and low GUS expressing plants indicate that it is possible to alter transgene expression through selection. No difference was found between the level of expression for F2 plants homozygous or heterozygous for the transgene. These results indicate that F2 plants, homozygous for a transgene, might be used to develop a transgenic cultivar. However, progeny testing to determine the influence of genetic background is a prerequisite to such a development.     

Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants

We developed a site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). We produced site-specific transgenic tobacco plants from four target lines and examined expression of the transgene in T1 site-specific transgenic tobacco plants, which were obtained by backcrossing. We found that site-specific transgenic plants from the same target lines showed approximately the same level of expression of the transgene. Moreover, we demonstrated that site-specific transgenic plants showed much less variability of transgene expression than random-integration transgenic plants. Interestingly, transgenes in the same direction at the same target locus showed the same level of activity, but transgenes in different directions showed different levels of activity. The expression levels of transgene did not correlate with those of the target gene. Our results showed that the SDI system could benefit the precise comparisons between different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.