Expression of Engineered Nuclear Male Sterility in Brassica napus (Genetics,Morphology, Cytology,and Sensitivity to Temperature)

A dominant genetic male sterility trait obtained through transformation in rapeseed (Brassica napus) was studied in the progenies of 11 transformed plants. The gene conferring the male sterility consists of a ribonuclease gene under the control of a tapetum-specific promoter. Two ribonuclease genes, RNase T1 and barnase, were used. The chimaeric ribonuclease gene was linked to the bialophos-resistance gene, which confers resistance to the herbicide phosphinotricine (PPT). The resistance to the herbicide was used as a dominant marker for the male sterility trait. The study presented here concerns three aspects of this engineered male sterility: genetics correlated with the segregation of the T-DNA in the progenies; expression of the male sterility in relation to the morphology and cytology of the androecium; and stability of the engineered male sterility under different culture conditions. Correct segregation, 50% male-sterile, PPT-resistant plants, and 50% male-fertile, susceptible plants were observed in the progeny of seven transformants. The most prominent morphological change in the male-sterile flowers was a noticeable reduction in the length of the stamen filament. The first disturbances of microsporogenesis were observed from the free microspore stage and were followed by a simultaneous degeneration of microspore and tapetal cell content. At anthesis, the sterile anthers contained only empty exines. In some cases, reversion to fertility of male-sterile plants has been observed. Both ribonuclease genes are susceptible to instability. Instability of the RNase T1-male sterility trait increased at temperatures higher than 25[deg] C. Our results do not allow us to confirm this observation for the barnase male-sterile plants. However, the male-sterile plants of the progeny of two independent RNase T1 transformants were stably male sterile under all conditions studied.     

Intein‐mediated protein assembly in transgenic wheat: production of active barnase and acetolactate synthase from split genes

Engineering traits by the assembly of non‐functional gene products is a promising tool for modern plant biotechnology. In this article, we describe the establishment of male sterility and herbicide resistance in wheat (Triticum aestivum) by complementing inactive precursor protein fragments through a split intein system. N‐ and C‐terminal fragments of a barnase gene from Bacillus amyloliquifaciens were fused to intein sequences from the Synechocystis sp. gene DnaB and delivered into the wheat genome via biolistic particle bombardment. Both barnase fragments were expressed under the control of a tapetum‐specific promoter. High efficiency of the split barnase system was achieved by introducing GGGGS linkers between the fusion domains of the assembled protein. Depending on the vector version that was transformed, up to 51% of primary transformed plants produced sterile pollen. In the F₁ progeny, the male‐sterile phenotype segregated with both barnase gene fragments. Expression of the cytotoxic barnase in the tapetum did not apparently affect the vegetative phenotype and remained stable under increased temperatures. In addition, the reconstitution of sulphonylurea resistance was achieved by DnaE intein‐mediated assembly of a mutated acetolactate synthase (ALS) protein from rice. The impacts of the technical advances revealed in this study on the concepts for trait control, transgene containment and hybrid breeding are discussed.     

The production of male-sterile wheat plants through split barnase expression is promoted by the insertion of introns and flexible peptide linkers

The successful use of transgenic plants depends on the strong and stable expression of the heterologous genes. In this study, three introns (PSK7-i1 and PSK7-i3 from Petunia and UBQ10-i1 from Arabidopsis) were tested for their ability to enhance the tapetum-specific expression of a split barnase transgene. We also analyzed the effects of introducing multiple copies of flexible peptide linkers that bridged the fusion domains of the assembled protein. The barnase fragments were assembled into a functional cytotoxin via intein-mediated trans-splicing, thus leading to male sterility through pollen ablation. A total of 14 constructs carrying different combinations of introns and peptide linkers were transformed into wheat plants. The resulting populations (between 41 and 301 independent plants for each construct) were assayed for trait formation. Depending on which construct was used, there was an increase of up to fivefold in the proportion of plants exhibiting male sterility compared to the populations harboring unmodified constructs. Furthermore, the average barnase copy number in the plants displaying male sterility could be reduced. The metabolic profiles of male-sterile transgenic plants and non-transgenic plants were compared using gas chromatography–mass spectrometry. The profiles generated from leaf tissues displayed no differences, thus corroborating the anther specificity of barnase expression. The technical advances achieved in this study may be a valuable contribution for future improvement of transgenic crop systems.     

Genetic engineering of reproductive sterility in forest trees

Containment of transgenes inserted into genetically engineered forest trees will probably be necessary before most commercial uses are possible. This is a consequence of (1) high rates of gene dispersal by pollen and seed, (2) proximity of engineered trees in plantations to natural or feral stands of interfertile species, and (3) potentially undesirable ecological effects if certain transgenes become widely dispersed. In addition to gene containment, engineering of complete or male sterility may stimulate faster wood production, reduce production of allergenic pollen, and facilitate hybrid breeding. We review the regulatory and ecological rationale for engineering sterility, potentially useful floral genes, strategies for creating sterility-causing transgenes, and problems peculiar to engineering sterility in forest trees. Each of the two primary options — ablating floral tissuesvia floral promoter-cytotoxin fusions, and disrupting expression of essential floral genes by various methods of gene suppression — has advantages and disadvantages. Because promoters from structural and enzymatic floral-specific genes often work well in heterologous species, ablation methods based on these genes probably will not require cloning of homologs from angiosperm trees. Methods that inhibit gene expression will require cloning of tree genes and may be more prone to epigenetic variability, but should allow assay of transgene efficacy in seedlings. Practical constraints include the requirement for vegetative propagation if complete sterility is engineered and the need for highly stable forms of sterility in long-lived trees. The latter may require suppression of more than one floral gene or employment of more than one genetic mechanism for sterility.     

禾谷类作物雄性不育及育性恢复表达载体的构建

通过ＰＣＲ反应扩增出了玉米花药特异启动子ＣＡ５５,将其分别与Ｂａｒｎａｓｅ和Ｂａｒｓｔａｒ基因融合,构建成了植物雄性不育基因ＣＡ５５ＢＮＮＯＳ和其育性恢复基因ＣＡ５５ＢＳＮＯＳ,再将它们分别插入到ｐＣＡＭＢＩＡ３３００中,获得了应用于禾谷类作物的基因工程雄性不育及育性恢复的表达载体。     

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。     

Male Sterility in Tomato

Male sterility research has been directed toward two goals: identifying genes required for the pollen development pathway and, more practically, identifying genetically stable lines that can be used in hybrid seed-breeding programs. The present resurgence of interest in male sterility remains true to these goals, but in addition seeks a molecular understanding of pollen development in order to genetically engineer controllable male sterility for hybrid seed production. In this review, we discuss the genetic and histochemical studies of tomato male sterile mutants that were conducted prior to 1970 in the context of gene expression and interaction. We also examine the use of molecular biological techniques in recent studies of male sterility and report on the current strategies being used for hybrid seed production.     

A biotechnology‐based male‐sterility system for hybrid seed production in tomato

Incorporating male sterility into hybrid seed production reduces its cost and ensures high varietal purity. Despite these advantages, male‐sterile lines have not been widely used to produce tomato (Solanum lycopersicum) hybrid seeds. We describe the development of a biotechnology‐based breeding platform that utilized genic male sterility to produce hybrid seeds. In this platform, we generated a novel male‐sterile tomato line by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9)‐mediated mutagenesis of a stamen‐specific gene SlSTR1 and devised a transgenic maintainer by transforming male‐sterile plants with a fertility‐restoration gene linked to a seedling‐colour gene. Offspring of crosses between a hemizygous maintainer and the homozygous male‐sterile plant segregated into 50% non‐transgenic male‐sterile plants and 50% male‐fertile maintainer plants, which could be easily distinguished by seedling colour. This system has great practical potential for hybrid seed breeding and production as it overcomes the problems intrinsic to other male‐sterility systems and can be easily adapted for a range of tomato cultivars and diverse vegetable crops.     

Engineered selective plant male sterility through pollen‐specific expression of the EcoRI restriction endonuclease

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.     

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.     

Enabling technologies for manipulating multiple genes on complex pathways

Many complex biochemical pathways in plants have now been manipulated genetically, usually by suppression or over-expression of single genes. Further exploitation of the potential for plant genetic manipulation, both as a research tool and as a vehicle for plant biotechnology, will require the co-ordinate manipulation of multiple genes on a pathway. This goal is currently very difficult to achieve. A number of approaches have been taken to combine or `pyramid' transgenes in one plant and have met with varying degrees of success. These approaches include sexual crossing, re-transformation, co-transformation and the use of linked transgenes. Novel, alternative `enabling' technologies are also being developed that aim to use single transgenes to manipulate the expression of multiple genes. A chimeric transgene with linked partial gene sequences placed under the control of a single promoter can be used to co-ordinately suppress numerous plant endogenous genes. Constructs modelled on viral polyproteins can be used to simultaneously introduce multiple protein-coding genes into plant cells. In the course of our work on the lignin biosynthetic pathway, we have tested both conventional and novel methods for achieving co-ordinate suppression or over-expression of up to three plant lignin genes. In this article we review the literature concerning the manipulation of multiple genes in plants. We also report on our own experiences and results using different methods to perform directed manipulation of lignin biosynthesis in tobacco.     

用DDRT-PCR技术对太谷核不育小麦基因差别表达的研究

采用DDRT—PCR技术对太谷核不育小麦的一对近等基因系进行了差别表达分析,以找出与太谷核不育基因Tα1表达有关的基因并研究其引起雄性不育的机制。共用30对随机引物进行差异显示,从展示的近1000条cDNA片段中找出30条特异表达的cDNA片段,其中包括可育特异和不育特异,这些片段可能与Tα1基因的表达有关。     

Mitochondrial genome organization and cytoplasmic male sterility in plants

Plant mitochondrial genomes are much larger and more complex than those of other eukaryotic organisms. They contain a very active recombination system and have a multipartite genome organization with a master circle resolving into two or more subgenomic circles by recombination through repeated sequences. Their protein coding capacity is very low and is comparable to that of animal and fungal systems. Several subunits of mitochondrial functional complexes, a complete set of tRNAs and 26S, 18S and 5S rRNAs are coded by the plant mitochondrial genome. The protein coding genes contain group II introns. The organelle genome contains stretches of DNA sequences homologous to chloroplast DNA. It also contains actively transcribed DNA sequences having open reading frames. Plasmid like DNA molecules are found in mitochondria of some plants Cytoplasmic male sterility in plants, characterized by failure to produce functional pollen grains, is a maternally inherited trait. This phenomenon has been found in many species of plants and is conveniently used for hybrid plant production. The genetic determinants for cytoplasmic male sterility reside in the mitochondrial genome. Some species of plants exhibit more than one type of cytoplasmic male sterility. Several nuclear genes are known to control expression of cytoplasmic male sterility. Different cytoplasmic male sterility types are distinguished by their specific nuclear genes(rfs) which restore pollen fertility. Cytoplasmic male sterility types are also characterized by mitochondrial DNA restriction fragment length polymorphism patterns, variations in mitochondrial RNAs, differences in protein synthetic profiles, differences in sensitivity to fungal toxins and insecticides, presence of plasmid DNAs or RNAs and also presence of certain unique sequences in the genome. Recently nuclear male sterility systems based on (i) over expression of agrobacterialrol C gene and (ii) anther specific expression of an RNase gene have been developed in tobacco andBrassica by genetic engineering methods.     

Cytoplasmic male sterility: a window to the world of plant mitochondrial-nuclear interactions

Mitochondrial function depends on the coordinate action of nuclear and mitochondrial genomes. The genetic dissection of these interactions presents special challenges in obligate aerobes, because the viability of these organisms depends on mitochondrial respiration. The plant trait cytoplasmic male sterility (CMS) is determined by the mitochondrial genome and is associated with a pollen sterility phenotype that can be suppressed or counteracted by nuclear genes known as restorer-of-fertility genes. Here, I review the nature and the origin of the genes that determine CMS, together with recent investigations that have exploited CMS to provide new insights into plant mitochondrial-nuclear communication. These studies have implicated mitochondrial signaling pathways, including those involved in regulating cell death and nuclear gene expression, in the elaboration of CMS. The molecular cloning of nuclear genes that restore fertility (i.e. restorer-of-fertility genes) has identified genes encoding pentatricopeptide-repeat proteins as key regulators of plant mitochondrial gene expression.     

TA29-barnase基因转化菜心

利用根癌农杆菌导入法, 以菜心带柄子叶为外植体, 对TA29-barnase基因转化菜心进行研究。获得转化植株,进行PCR、Southern blotting杂交和半定量RT-PCR检测, 表明目的基因已经整合到转化植株中, 并且目的基因在转基因植株花蕾中得到表达, 但是表达水平在不同转基因植株间存在差别; 转基因植株开花后, 均表现雄性不育, 不能产生花粉或产生没有活力的少量花粉, 自交不能结实; 用未转化植株正常花粉对雄性不育植株进行授粉, 能够正常结实; 保持系(未转化植株)与不育株杂交后代中雄性不育株与可育株的比例为1:1, 在杂交后代植株子叶期, 喷洒10 mg/L的PPT可以完全杀死可育株; 利用其他菜心品种为父本与不育株进行杂交, 获得的F1植株在生长势和产量方面表现优势, 表明开展菜心优势育种具有一定的潜力。     

Misregulation of spermatogenesis genes in Drosophila hybrids is lineage‐specific and driven by the combined effects of sterility and fast male regulatory divergence

Hybrid male sterility is a common outcome of crosses between different species. Gene expression studies have found that a number of spermatogenesis genes are differentially expressed in sterile hybrid males, compared with parental species. Late‐stage sperm development genes are particularly likely to be misexpressed, with fewer early‐stage genes affected. Thus, a link has been posited between misexpression and sterility. A more recent alternative explanation for hybrid gene misexpression has been that it is independent of sterility and driven by divergent evolution of male‐specific regulatory elements between species (faster male hypothesis). The faster male hypothesis predicts that misregulation of spermatogenesis genes should be independent of sterility and approximately the same in both hybrids, whereas sterility should only affect gene expression in sterile hybrids. To test the faster male hypothesis vs. the effect of sterility on gene misexpression, we analyse spermatogenesis gene expression in different species pairs of the Drosophila phylogeny, where hybrid male sterility occurs in only one direction of the interspecies cross (i.e. unidirectional sterility). We find significant differences among genes in misexpression with effects that are lineage‐specific and caused by sterility or fast male regulatory divergence.     

Molecular Strategies for Addressing Gene Flow Problems and Their Potential Applications in Abiotic Stress Tolerant Transgenic Plants

Transgenic plant technology provides a powerful tool to improve abiotic stress tolerance of crop plants. However, introgression of stress tolerance genes into weedy relatives may increase the potential for persistence and invasiveness, resulting in undesirable ecological consequences. A variety of gene confinement strategies have been developed to reduce unwanted transgene movement. In this review, we discuss some of these strategies, such as male and female sterility, GeneSafe?, parthenocarpy, chloroplast transformation and gene deletor technologies. In the case of the gene deletor technology, all transgenes from pollen, seeds, fruits or other organs may be eliminated once the transgene functions are no longer needed at the stage when the presence of the transgene becomes a cause for ecological or public concern. The gene deletor and other technologies can be useful to reduce unintended dispersal of stress tolerance genes and thus may facilitate commercialization of transgenic crops with enhanced tolerance to abiotic stresses.     

The effect of genotype on pollen transmission of mitochondria in rapeseed (Brassica napus)

Summary Crossing experiments were conducted to determine whether parental genotype affected the rate of transmission of paternal mitochondria to progeny in rapeseed (Brassica napus). Progeny were screened either by RFLP analysis of mitochondrial (mt) DNA or by means of a mt marker that causes male sterility. To date we have transferred paternal mitochondria to progeny in only cross, i.e. a specific female line crossed to a specific male line. The male line carries the polima cytoplasm, the mitochondria of which confer a characteristic malesterile flower morphology when in a napus nuclear background. This line is male fertile due to a restorer gene carried on an extra chromosome from a closely related species, Brassica juncea. The female line has a Brassica campestris cytoplasm with a chloroplast mutation conferring resistance to triazine herbicides. Progeny with mixtures of parental mtDNA display a range of plant phenotype from complete male fertility through varying proportions of male-sterile sectors to complete male sterility. The male sterility or fertility of flowers on a sector of a plant reflects the mt population of that sector, and such sectors will give rise to stably fertile or sterile progeny. These experiments suggest that maternal inheritance of mitochondria in higher plants is due to genes active in both the pollen parent and the egg parent.     

Development of a novel recessive genetic male sterility system for hybrid seed production in maize and other cross‐pollinating crops

We have developed a novel hybridization platform that utilizes nuclear male sterility to produce hybrids in maize and other cross‐pollinating crops. A key component of this platform is a process termed Seed Production Technology (SPT). This process incorporates a transgenic SPT maintainer line capable of propagating nontransgenic nuclear male‐sterile lines for use as female parents in hybrid production. The maize SPT maintainer line is a homozygous recessive male sterile transformed with a SPT construct containing (i) a complementary wild‐type male fertility gene to restore fertility, (ii) an α‐amylase gene to disrupt pollination and (iii) a seed colour marker gene. The sporophytic wild‐type allele complements the recessive mutation, enabling the development of pollen grains, all of which carry the recessive allele but with only half carrying the SPT transgenes. Pollen grains with the SPT transgenes exhibit starch depletion resulting from expression of α‐amylase and are unable to germinate. Pollen grains that do not carry the SPT transgenes are nontransgenic and are able to fertilize homozygous mutant plants, resulting in nontransgenic male‐sterile progeny for use as female parents. Because transgenic SPT maintainer seeds express a red fluorescent protein, they can be detected and efficiently separated from seeds that do not contain the SPT transgenes by mechanical colour sorting. The SPT process has the potential to replace current approaches to pollen control in commercial maize hybrid seed production. It also has important applications for other cross‐pollinating crops where it can unlock the potential for greater hybrid productivity through expanding the parental germplasm pool.