Reversible male sterility in transgenic tobacco carrying a dominant-negative mutated glutamine synthetase gene under the control of microspore-specific promoter

Metabolic engineering was used to disrupt glutamine metabolism in microspores in order to block pollen development. We used a dominant-negative mutant (DNM) approach of cytosolic glutamine synthetase (GS1) gene under the microspore-specific promoter NTM19 to block glutamine synthesis in developing pollen grains. We observed partial male sterility in primary transgenic plants by using light microscopy, FDA, DAPI and in vitro pollen germination test. Microspores started to die in the early unicellular microspore stage, pollen viability in all primary transgenic lines ranged from 40-50%. All primary transgenics produced seeds like control plants, hence the inserted gene did not affect the sporophyte and was inherited through the female germline. We regenerated plants by in vitro microspore embryogenesis from 4 individual lines, pollen viability of progeny ranged from 12 to 20%, but some of them also showed 100% male sterility. After foliage spray with glutamine, 100% male-sterile plants were produced viable pollen and seed set was also observed. These results suggested that mutated GS1 activity on microspores had a significant effect on normal pollen development. Back-cross progenies (T2) of DH 100% male-sterile plants showed normal seed set like primary transgenics and control plants.     

Combination of reversible male sterility and doubled haploid production by targeted inactivation of cytoplasmic glutamine synthetase in developing anthers and pollen

Reversible male sterility and doubled haploid plant production are two valuable technologies in F₁-hybrid breeding. F₁-hybrids combine uniformity with high yield and improved agronomic traits, and provide self-acting intellectual property protection. We have developed an F₁-hybrid seed technology based on the metabolic engineering of glutamine in developing tobacco anthers and pollen. Cytosolic glutamine synthetase (GS1) was inactivated in tobacco by introducing mutated tobacco GS genes fused to the tapetum-specific TA29 and microspore-specific NTM19 promoters. Pollen in primary transformants aborted close to the first pollen mitosis, resulting in male sterility. A non-segregating population of homozygous doubled haploid male-sterile plants was generated through microspore embryogenesis. Fertility restoration was achieved by spraying plants with glutamine, or by pollination with pollen matured in vitro in glutamine-containing medium. The combination of reversible male sterility with doubled haploid production results in an innovative environmentally friendly breeding technology. Tapetum-mediated sporophytic male sterility is of use in foliage crops, whereas microspore-specific gametophytic male sterility can be applied to any field crop. Both types of sterility preclude the release of transgenic pollen into the environment.     

A Brassica napus gene family which shows sequence similarity to ascorbate oxidase is expressed in developing pollen. Molecular characterization and analysis of promoter activity in transgenic tobacco plants

The genomic clone named Bp10 contains a member of a small pollen-specific gene family of B. napus. The expression of the Bp10 gene family is maximal in early binucleate microspores and declines considerably in mature trinucleate pollen. Homologues of the Bp10 genes are expressed in the pollen of other plant species. The pollen-specific expression of the gene contained in the genomic clone was confirmed in tobacco plants transformed with a chimeric Bp10 promoter/GUS construct. A promoter fragment of 396 bp is sufficient to direct a strong and correct spatial and temporal expression in transgenic plants. The Bp10 gene family codes for proteins of 62 kDa showing approximately 30% sequence identify to cucumber and pumpkin ascorbate oxidases (AAOs). However, the AAO active centres are not conserved in the Bp10 products, suggesting an evolutionary relationship but a different enzymatic activity for these proteins. Expression of a recombinant Bp10 protein in E. coli inhibits bacterial growth on minimal medium, suggesting the production of an enzymatically active polypeptide in bacteria. No AAO activity could be correlated with the expression of the recombinant protein. Moreover, substances affecting AAO activity do not appear to influence the inhibitory activity of the protein produced in bacteria. However, as indicated by the rescue of bacterial growth in the presence of sodium bicarbonate or gaseous CO2, the Bp10 protein activity could be modulated by CO2 levels.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.     

An investigation of the role of the anther tapetum during microspore development using genetic cell ablation

The effects on anther development of a fusion of the Arabidopsis anther-specific apg gene promoter to a ribonuclease (barnase) in transgenic tobacco plants were examined. Contrary to expectations, viable pollen grains were produced by these plants despite the demonstration that ribonuclease expression in the microspores and tapetum caused targeted cell ablation. Transformed plants were reduced in male fertility due to ablation of a proportion of pollen dependent on apg-barnase locus number. Plants were otherwise phenotypically normal and fully female fertile, confirming the anther-specific nature of the apg promoter. In microspores inheriting an apg-barnase locus following meiosis, loss of cell viability, as judged by fluorescein diacetate staining, occurred during mid to late microspore development. Microspores not inheriting a transgene went on to mature into viable pollen grains. Premature degeneration of the tapetum was also observed as a result of apg-barnase expression, but this did not appear to disrupt the subsequent microspore and pollen developmental programmes. This was substantiated by observations of microspore development in plants in which the tapetum was rescued from ablation by crossing in a second transgene encoding a tapetum-specific inhibitor of the ribonuclease. It was determined that tapetum cell disruption occurs at the early to mid uninucleate microspore stage in apg-barnase transformants. The data presented show that after this point in microspore development the tapetum is no longer essential for the production of viable pollen in tobacco.     

Rice salT promoter is activated in Papaver somniferum and Nicotiana tabacum transgenic cells in the absence of exogenous ABA

With the aim of modifying secondary metabolism in Opium poppy (Papaver somniferum) and tobacco (Nicotiana tabacum) cells, gene transfer was performed using the sam1 gene from Arabidopsis thaliana under the control of the salT promoter. This promoter is induced by ABA in rice and in tobacco and we have shown that it is also induced in poppy cells (gus gene). Putatively transformed poppy and tobacco cell lines with the sam1 gene were obtained. In the absence of exogenous inducer we noticed the expression of the transgene resulting in a significant increase of SAM-S activity in all tested transformants of poppy and in half the transgenic tobacco cell lines tested. Addition of ABA to the culture medium failed to enhance the expression of the transgene in both species and resulted in a decrease of the sam1 gene expression in some cell lines. Since the salT promoter is induced by exogenous ABA in both species (gus reporter gene), we suggest a partial sam1 transgene inactivation in certain cell lines. These results show that the efficiency of a regulatory sequence may be different when fused with a reporter gene (gus) compared to fusion with a gene belonging to the housekeeping family (sam1).     

Directed microspore-specific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes

A major challenge for future genetically modified (GM) crops is to prevent undesired gene flow of transgenes to plant material intended for another use. Recombinase-mediated auto excision of transgenes directed by a tightly controlled microspore-specific promoter allows efficient removal of either the selectable marker gene or of all introduced transgenes during microsporogenesis. This way, transgene removal becomes an integral part of the biology of pollen maturation, not requiring any external stimulus such as chemical induction by spraying. We here show the feasibility of engineering transgenic plants to produce pollen devoid of any transgene. Highly efficient excision of transgenes from tobacco pollen was achieved with a potential failure rate of at most two out of 16 800 seeds (0.024%). No evidence for either premature activation or absence of activation of the recombinase system was observed under stress conditions in the laboratory. This approach can prevent adventitious presence of transgenes in non-GM crops or related wild species by gene flow. Such biological containment may help the deployment and management of coexistence practices to support consumer choice and will promote clean molecular farming for the production of high-value compounds in plants.     

TM2, a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants

Nuclear matrix attachment regions (MARs) are thought to influence the expression of the flanking genes. TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro. In this study, we investigated the effect of TM2 on transgene expression under the control of three different promoters in stably transformed rice calli and plants. The presence of TM2 flanking the transgene increased the expression of constructs based on the constitutive CaMV 35S and maize ubiquitin gene promoters in both resistant calli and transformed plants. The GUS expression directed by the photosynthetic-tissue-specific PNZIP promoter was also increased in photosynthetic tissues of transformants. However, TM2 did not change the gene expression pattern controlled by the PNZIP promoter. The effect of TM2 in transgenic plants was stronger than that in transgenic calli based on all three promoters. Our results indicate that TM2, as a novel strong MAR, can be used to increase the transgene expression levels in the whole plant or in particular tissues of monocotyledons.     

玉米Ubi－1启动子在可育转基因玉米植株中的表达活性

本工作将玉米泛素基因－1启动子（Ubi-1)与大肠杆菌β－葡萄糖苷酸酶基因（gus,uidA)的编码区融合,通过基因枪粒子轰击方法转化来自水成熟胚盾片组织的I－型愈伤组织,经PPT选择获得可育的玉米转基因植株,并采用组织化学方法分析了Ubi-1启动子驱动的gus基因在不同组织,细胞中的表达活性,发现gus基因在除花药壁以外的其它所试组织中均可以有效表达。Ubi：GUS在花粉,卵细胞中T1代转基因植株未成熟胚中的表达显示该启动子在植株发育的早期阶段即具有活性。对T0代转基因植株的花粉进行GUS组织化学染色,gus基因呈1：1分离,显示外源基因在转基因植株中以孟德尔方式遗传。同时发现,使用玉米本身的启动子Ubi-1可以降低外源基因在转基因玉米中的拷贝数,进而避免基因沉默现象的发生。目前已得到第二代转基因种子。     

Antisense-mediated suppression of transgene expression targeted specifically to pollen

A potential problem in the field release of transgenic plants is the spread of foreign gene products via pollen. Therefore, the use of the tomato pollen-specific lat52 gene promoter was investigated as a means of targeting antisense RNA to pollen without affecting transgene expression elsewhere in the plant. A transgenic tobacco line T115, which showed GUS expression in pollen, leaves and roots were retransformed with a construct containing the pollen-specific lat52 promoter driving the GUS encoding uid A gene in antisense orientation. From 24 independent transformants obtained, 19 showed a significant reduction in pollen GUS activity. Of these lines, four showed a reproducible antisense effect in pollen in the next generation, while it was shown in one line that GUS activity in leaves and roots was also unaffected. To ascertain the effectiveness of the antisense strategy to downregulate very high levels of pollen expression, a lat52-gus antisense construct was introduced into tobacco lines containing lat52-gus, which had pollen GUS activity of up to 250 times greater than in line T115. Results showed that 30 out of 34 independent lines exhibited a significant antisense effect in pollen, confirming the effectiveness of pollen-targeted antisense strategy to reduce undesirable expression in pollen independent of expression level in pollen.     

Male Sterile Tobacco Plants Obtained by Genetical-Engineering

Chimaeric TA29-barnase gene was obtained by fusing tobacco anther-specific promoter TA29, barnase gene and NOS terminator and was inserted into a plant expression yector containing bromoxynil-resistant bxn gene and then introduced into tobacco plants. Some transgenic tobacco plants were resistant to 1.0 × 10-3 mol/L bromoxynil. At about 26℃, all of the 9 transgenic plants were male fertile; However, at 20℃, 7 out of 11 transgenic plants were male sterile.     

Activities of CaMV 35S and nos promoters in pollen: implications for field release of transgenic plants

The expression of foreign genes in pollen may pose potentialproblems in the field release of transgenic plants, since pollenrepresents a route whereby foreign genes and their productsmay escape into the wider environment. The possible risks posedby cross-hybridization with wild relatives have been extensivelyexplored, but problems that may arise due to the expressionof foreign gene products in pollen have not been so widely studied.The activities of the CaMV 35S and nos promoters in pollen inpopulations of stably transformed plants and in transient expressionanalysis are described. These promoters are commonly used inall areas of plant molecular biology research and their expressionpatterns will be of interest to those involved in field releasestudies. The results show that both promoters had no detectablepollen activity in Arabidopsis, but both showed activity intobacco pollen. The CaMV 35S-gus gene fusion showed heritableexpression levels in tobacco pollen of up to a maximum of 64.6pmol 4-MU min^–1 mg ^–1 total protein. nos promoteractivity in transgenic tobacco pollen was highly variable, withGUS activities ranging from undetectable levels up to 2561 pmol4-MU min^–1 mg^–1 total protein within the transgenicpopulation. Histochemical staining of anther sections from 10–12mm buds revealed that the CaMV 35S promoter had some activityin the vascular bundle, stomium and tapetum, while GUS expressionfrom the nos promoter in sporophytic tissues was confined entirelyto the stomium. Key words: CaMV 35S promoter, nos promoter, pollen, transgenic plant release     

Alterations of Root and Fiber in Transgenic Cotton Plants with Chimeric Ph/P-ipt Gene Expression

The seed-specific phaseolin promoter (Ph/P) was fused to an ipt gene, then was cloned to a plant expression vector containing a gus gene driven by a 35S promoter. Cotton (Gossypium hirsutum L.) plants were transformed through pollen tube pathway methods. After seed germination, histochemical staining of the roots demonstrated that 32 GUS positive plants were obtained and three of which contained the chimeric Ph/P-ipt transgene as confirmed by PCR analysis. An immunosorbent assay showed that two of the three transgenic cotton lines contained higher levels of zeatin equivalents in seeds than the control. Seedling development of these two transgenic lines differed from the control in a reduction of the shoot growth, showing a stunted phenotype as expected, but a surprisingly developed root system with a 3-4 fold fast-growing lateral roots. In addition, fibers (seed-hairs) of the two transgenic cotton lines were considerably shorter than those of the control. These results indicate that genetic engineering may be used to manipulate the development of cotton plants, particularly cotton fibers.     

种子特异表达ipt转基因棉花根和纤维的改变

将种子特异表达的菜豆蛋白启动子（Ｐｈ／Ｐ）与ｉｐｔ基因融合,构建了植物表达载体。该载体含有由３５Ｓ启动子驱动的ｇｕｓ报告基因。应用该载体通过花粉管通道法转化棉花（Ｇｏｓｓｙｐｉｕｍ ｈｉｒｓｕｔｕｍ Ｌ．）,种子萌发后剪取幼根进行ＧＵＳ染色,获得ＧＵＳ阳性植株２３棵。ＰＣＲ检测证明有３棵ＧＵＳ阳性植株中含有Ｐｈ／Ｐ－ｉｐｔ基因,并进一步用地高辛标记的ＤＮＡ探针作杂交验证了上述结果。分析表明２棵转基     

A pFBP6::Barnase Construct Resulted in Stigma and Style Ablation and Floral Abscission in Transgenic Tobacco

The potential for transgene dispersal through pollen, fruit, and seed is an important argument against the release of genetically modified plants. One approach toward addressing the concerns of gene flow from transgenic crops into closely related wild species involves in the use of tissue-specific promoters to engineer male and/or female sterility. In this study, we investigated the potential of Barnase ectopic expression for engineering floral sterility. A 2.6?kb promoter region of floral binding protein 6 (FBP6) from Petunia hybrida was isolated and fused to a reporter gene encoding ??-glucuronidase (GUS). The construct was introduced into tobacco plants where GUS staining was detected ubiquitously throughout the various tissues. The expression pattern of FBP6 resembled AG promoters, i.e., weak promoter activity was found in vegetative tissues, and strong activity was found in the various floral organs including the carpels and stigma. Meanwhile,The pFBP6::Barnase construct was then cotransformed into tobacco along with the Barstar gene, encoding an enzymatic inhibitor of Barnase, which was expressed at low but ubiquitous levels. Although cotransformed tobacco plants showed near normal vegetative growth, 74% of transgenic plants exhibited stigma and style ablation, and 98% of flower buds abscised before opening. Further analyses confirmed that stigma and style ablation prevented fertilization of the flower, and abscission of the bud followed rapidly. Thus, this approach has advantages for those ornamental/landscaping species where the pollen and fruit represent pollutants of the urban environment (e.g., platanus and poplar).