Characterization and mapping of a novel mutant sms1 （senescence and male sterility 1） in rice

Plant senescence plays diverse important roles in development and environmental responses.However,the molecular basis of plant senescence is remained largely unknown.A rice spontaneous mutant with the character of early senescence and male sterility (sms) was found in the breeding line NT10-748.In order to identify the gene SMS1 and the underlying mechanism,we preliminarily analyzed physiological and biochemical phenotypes of the mutant.The mutant contained lower chlorophyll content compared with the wild type control and was severe male sterile with lower pollen viability.Genetic analysis showed that the mutant was controlled by a single recessive gene.By the map-based cloning approach,we fine-mapped SMS1 to a 67 kb region between the markers Z3-4 and Z1-1 on chromosome 8 using 1,074 F2 recessive plants derived from the cross between the mutant sms1 (japonica) × Zhenshan 97 (indica),where no known gene involved in senescence or male sterility has been identified.Therefore the SMS1 gene will be a novel gene that regulates the two developmental processes.The further cloning and functional analysis of the SMS1 gene is under way.     

Organelle trafficking,the cytoskeleton,and pollen tube growth

The pollen tube is fundamental for the reproduction of seed plants. Characteristically, it grows relatively quickly and uni‐directionally("polarized growth") to extend the male gametophyte to reach the female gametophyte. The pollen tube forms a channel through which the sperm cells move so that they can reach their targets in the ovule. To grow quickly and directionally, the pollen tube requires an intense movement of organelles and vesicles that allows the cell's contents to be distributed to sustain the growth rate. While the various organelles distribute more or less uniformly within the pollen tube, Golgi‐released secretory vesicles accumulate massively at the pollen tube apex, that is, the growing region. This intense movement of organelles and vesicles is dependent on the dynamics of the cytoskeleton,which reorganizes differentially in response to external signals and coordinates membrane trafficking with the growth rate of pollen tubes.     

MS1 is essential for male fertility by regulating the microsporocyte cell plate expansion in soybean

Male sterility is an essential trait in hybrid seed production, especially for monoclinous and autogamous food crops. Soybean male-sterile ms1 mutant has been known for more than 50 years and could be instrumental in making hybrid seeds. However, the gene responsible for the male-sterile phenotype has remained unknown. Here, we report the map-based cloning and characterization of the MS1 gene in soybean. MS1 encodes a kinesin protein and localizes to the nucleus, where it is required for the male meiotic cytokinesis after telophase Ⅱ. We further substantiated that MS1 colocalizes with microtubules and is essential for cell plate formation in soybean male gametogenesis through immunostaining. Both ms1 and CRISPR/Cas9 knockout mutants show complete male sterility but are otherwise phenotypically normal, making them perfect tools for producing hybrid seeds.The identification of MS1 has the practical potential for assembling the sterility system and speeding up hybrid soybean breeding.     

一种紫色水稻的遗传及其在光敏不育系育种中应用的研究

研究表明,本院获得的一种紫色水稻的植株色遗传受控于C、 A、Pl3个独立基因座位上显性基因的互补作用,另有一独立的显性基因对Pl基因的表达起抑制作用。由于该抑制基因在籼稻中的高频率存在,因而,紫稻与一般绿稻品种杂交F1多表现为绿株。紫稻光敏不育(株)系的不育性表达和配合力表现,均可达到与普通光敏不育系相似的水平。本文还讨论了选育籼型紫稻光敏不育系设想的可行性。 Abstract:The inheritance of a purple rice in crosses to green rices was investigated.A group of dominant and interactive genes,C,A and Pl,was found to control the expression of the trait and the other independent inhibitor I-Pl-1 to inhibit the effect of the gene Pl.Because of the wide existence of the gene I-Pl-1 in indica rice,most of F1 plants of the crosses between purple rice and green cultivars were green.The primary study indicated that,for the purple photoperiod-sensitive genic male-sterile lines,the degrees of the sterility and its stability in the sterile stage,and of the fertility in the fertile stage,and the combining ability levels of the purple rice were as high as the degrees and levels for the green rice.A tentative idea on breeding purple photoperiod-sensitive male-sterile lines of indica rice was suggested and its feasibility and advantage was discussed.     

应用荧光原位杂交技术检测人βE珠蛋白基因在转基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态.检测结果表明,外源人βE珠蛋白基因已稳定地整合于小鼠染色体上.FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合后进行染色体定位检测。 Abstract:To determine the integration site of human βE globin gene in the chromosomes of transgenic mice, transgenic mice carrying human βE globin gene were injected intraperitoneally with colchicines, then, bone marrow cells wereisolated and metaphase chromosomes were prepared, the traditional FISH method was improved to detect the integration site of humanβE globin gene in transgenic mice when combined with G-banding. Human t3E globin gene can bedetected in different position of different chromosomes in transgenic mice and FISH signals showed that two mice were heterozygous of human 13E globin gene and one was homozygous. Human t3E globin gene was integrated into thechromosomes of transgenic mice in a random pattern and the results demonstrated that FISH can be used to investigate the integration site of foreign genes in transgenic mice.     

应用荧光原位杂交技术检测人βE珠蛋白基因在转基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态.检测结果表明,外源人βE珠蛋白基因已稳定地整合于小鼠染色体上.FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合后进行染色体定位检测。 Abstract:To determine the integration site of human βE globin gene in the chromosomes of transgenic mice, transgenic mice carrying human βE globin gene were injected intraperitoneally with colchicines, then, bone marrow cells wereisolated and metaphase chromosomes were prepared, the traditional FISH method was improved to detect the integration site of humanβE globin gene in transgenic mice when combined with G-banding. Human t3E globin gene can bedetected in different position of different chromosomes in transgenic mice and FISH signals showed that two mice were heterozygous of human 13E globin gene and one was homozygous. Human t3E globin gene was integrated into thechromosomes of transgenic mice in a random pattern and the results demonstrated that FISH can be used to investigate the integration site of foreign genes in transgenic mice.     

培育具有安全选择标记或无选择标记的转基因植物

转基因植物中选择标记的安全性已成为植物基因工程研究的热点之一。从两个方面可以解决转基因植物中的选择标记问题。一是选用安全的正向选择标记,主要是与糖代谢和激素代谢相关的基因。二是构建能去除选择标记基因的转化系统,主要有共转化系统、双T-DNA边界载体系统、位点特异性重组系统和转座子系统等。这些植物基因工程的方法将有助于培育安全的转基因植物。 Abstract:The bio-safety of selective markers in transgenic plants has been a hot spot in the field of plant genetic engineering.To solve the problem of selective markers in the transgenic plants,two means of producing transgenic plants have been developed.One is the utilization of bio-safe positive selective markers which are genes mainly related to metabolism of auxins and carbohydrates.The other is the establishment of transformation systems allowing marker genes to be eliminated from the transgenic plants,which include co-ransformation,double T-DNA border vectors,site-specific recombination and transposition.All these approaches of plant genetic engineering will benefit breeding transgenic plants with bio-safety.     

A novel hybrid seed production technology based on a unilateral cross-incompatibility gene in maize

Hybrid seed production technology(SPT) using genic recessive male sterility is of great importance in maize breeding. Here, we report a novel SPT based on a maize unilateral cross-incompatibility gene Zm Ga1F with an extremely low transgene transmission rate(TTR). Proper pollen-specific Zm Ga1F expression severely inhibits pollen tube growth leading to no fertilization.The maintainer line harbors a transgene cassette in an ipe1 male sterile background containing IPE1 to restore ipe1 male fertili...     

转基因植物中筛选标记基因的利用及消除

在基因转移过程中,人们常常使用标记基因来筛选转化细胞或组织。常用的筛选标记基因尤其是抗生素抗性基因的使用往往对环境及植物体的生长发育产生不良影响,且影响基因多重转化。为了消除这些弊端,一种全新的发展策略即获取无选择标记的转基因植物应运而生。本文主要综述转基因植物中有关筛选标记基因及其消除方法。 Abstract:Selective marker gene is usually used to select transformed cells or tissue during gene transfer.However,the use of selective marker gene,especially antibiotic-resistant gene,is harmful to environment,plant development and affects multi-transformation.A new strategy that offers a approach for the elimination of those disadvantages caused by the selectable marker gene is developed.We summarized correlative marker genes used in transgenic plants and some methods of its removal.     

Molecular strategies for gene containment in transgenic crops

The potential of genetically modified (GM) crops to transfer foreign genes through pollen to related plant species has been cited as an environmental concern. Until more is known concerning the environmental impact of novel genes on indigenous crops and weeds, practical and regulatory considerations will likely require the adoption of gene-containment approaches for future generations of GM crops. Most molecular approaches with potential for controlling gene flow among crops and weeds have thus far focused on maternal inheritance, male sterility, and seed sterility. Several other containment strategies may also prove useful in restricting gene flow, including apomixis (vegetative propagation and asexual seed formation), cleistogamy (self-fertilization without opening of the flower), genome incompatibility, chemical induction/deletion of transgenes, fruit-specific excision of transgenes, and transgenic mitigation (transgenes that compromise fitness in the hybrid). As yet, however, no strategy has proved broadly applicable to all crop species, and a combination of approaches may prove most effective for engineering the next generation of GM crops.     

Allelic interaction of F1 pollen sterility loci and abnormal chromosome behaviour caused pollen sterility in intersubspecific autotetraploid rice hybrids

The intersubspecific hybrids of autotetraploid rice has many features that increase rice yield, but lower seed set is a major hindrance in its utilization. Pollen sterility is one of the most important factors which cause intersubspecific hybrid sterility. The hybrids with greater variation in seed set were used to study how the F(1) pollen sterile loci (S-a, S-b, and S-c) interact with each other and how abnormal chromosome behaviour and allelic interaction of F(1) sterility loci affect pollen fertility and seed set of intersubspecific autotetraploid rice hybrids. The results showed that interaction between pollen sterility loci have significant effects on the pollen fertility of autotetraploid hybrids, and pollen fertility further decreased with an increase in the allelic interaction of F(1) pollen sterility loci. Abnormal ultra-structure and microtubule distribution patterns during pollen mother cell (PMC) meiosis were found in the hybrids with low pollen fertility in interphase and leptotene, suggesting that the effect-time of pollen sterility loci interaction was very early. There were highly significant differences in the number of quadrivalents and bivalents, and in chromosome configuration among all the hybrids, and quadrivalents decreased with an increase in the seed set of autotetraploid hybrids. Many different kinds of chromosomal abnormalities, such as chromosome straggling, chromosome lagging, asynchrony of chromosome disjunction, and tri-fission were found during the various developmental stages of PMC meiosis. All these abnormalities were significantly higher in sterile hybrids than in fertile hybrids, suggesting that pollen sterility gene interactions tend to increase the chromosomal abnormalities which cause the partial abortion of male gametes and leads to the decline in the seed set of the autotetraploid rice hybrids.     

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.     

Expression of <Emphasis Type="Italic">Aprotinin</Emphasis> in Anther Causes Male Sterility in Tobacco var <Emphasis Type="Italic">Petit havana</Emphasis>

Expression of many proteinases has been documented during anther development. Although their roles are not completely understood, their inhibition could possibly result in impairment of anther development leading to male sterility. We proposed that such an impairment of anther development can be engineered in plants resulting in male sterile plants that can be used for hybrid seed production. Here, we report that anther-specific expression of Aprotinin gene (serine proteinase inhibitor) in tobacco has resulted in male sterility. Southern analysis and zymogram analysis confirmed the integration and expression of Aprotinin gene in the anthers of the transgenic plants. Transverse sections of anthers of transgenic male sterile plants showed damaged tapetum. The pollen germination in the transgenic plants ranged between 2% and 65% that confirmed the impairment in pollen production leading to male sterility and low seed yield. Thus, inhibition of serine proteinases that are expressed during anther development has resulted in impaired pollen production and male sterility, though the exact role of these proteinases in anther development still has to be elucidated.     

Comparing relative rates of pollen and seed gene flow in the island model using nuclear and organelle measures of population structure

We describe a method for comparing nuclear and organelle population differentiation (F(ST)) in seed plants to test the hypothesis that pollen and seed gene flow rates are equal. Wright's infinite island model is used, with arbitrary levels of self-fertilization and biparental organelle inheritance. The comparison can also be applied to gene flow in animals. Since effective population sizes are smaller for organelle genomes than for nuclear genomes and organelles are often uniparentally inherited, organelle F(ST) is expected to be higher at equilibrium than nuclear F(ST) even if pollen and seed gene flow rates are equal. To reject the null hypothesis of equal seed and pollen gene flow rates, nuclear and organelle F(ST)'s must differ significantly from their expected values under this hypothesis. Finite island model simulations indicate that infinite island model expectations are not greatly biased by finite numbers of populations (>/=100 subpopulations). The power to distinguish dissimilar rates of pollen and seed gene flow depends on confidence intervals for fixation index estimates, which shrink as more subpopulations and loci are sampled. Using data from the tropical tree Corythophora alta, we rejected the null hypothesis that seed and pollen gene flow rates are equal but cannot reject the alternative hypothesis that pollen gene flow is 200 times greater than seed gene flow.     

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.     

Pollen and seed flow under different predominant winds in wind-pollinated and wind-dispersed species <Emphasis Type="Italic">Engelhardia roxburghiana</Emphasis>

In most plants, the contributions of pollen and seed flow to their genetic structures are generally difficult to disentangle. For typical wind-pollinated and wind-dispersed species Engelhardia roxburghiana in a 20-ha natural forest plot in lower subtropic China, because the prevailing wind directions change during its pollen release and seed dispersal seasons, we could compare its genetic structures in different directions, which could result primarily from pollen or seed flow. Furthermore, because the plot has undergone from an open to a closed canopy stage historically, we also examined forest canopy effects on gene flow in different generations and different directions. Using 522 E. roxburghiana individuals mapped in the plot, our results revealed that greater pollen flow led to biased gene flow in the pollen dispersal-predominant direction (pollen direction), while greater seed flow generated less spatial genetic structure in the seed dispersal-predominant direction (seed direction). The results predicted from generalized additive models indicated that canopy closure enhanced resistance to gene flow from the old generation to the new generation. Analyses by landscape genetic models for the new generation revealed that gene flow associated with pollen direction was more strongly affected by canopy than with seed direction. Our study is new by proposing an alternative way to separate effects of the pollen and seed flow on spatial variation patterns in E. roxburghiana. To our knowledge, our study is also the first attempt to use landscape genetic models to represent canopy effects for different dispersal vectors in spatial scales only up to a few hundred meters.     

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.