高羊茅和其他羊茅植株再生与遗传转化研究进展

高羊茅、紫羊茅和草地羊茅均为很重要的多年生冷季型牧草与草坪草,生物技术在其品种改良中具有很大的应用潜力。30年来,三种羊茅的组织培养、胚性培养物的长期保存以及遗传转化等研究取得了较大进展,已建立起多种植株再生体系和遗传转化技术,但作为单子叶植物,这些草种的组织培养和转基因遗传改良也还存在一些问题。本文就以上几方面的内容进行了综述。     

高羊茅转基因研究进展

高羊茅为很重要的多年生冷季型草坪草,生物技术在其品种改良中具有很大的应用潜力。本文对高羊茅植株再生体系的建立及遗传转化方面的研究进展进行了综述。同时,对高羊茅转基因研究中存在的问题和前景作了讨论。     

草坪草-高羊茅遗传育种研究进展

随着我国草坪业的快速发展 ,草坪草遗传育种工作越来越受到人们的关注。简要介绍了高羊茅的育种历史和现状 ,并对高羊茅植株再生体系的建立及遗传转化方面的研究进展进行了综述。此外 ,还提出了我国高羊茅育种的几点建议。     

农杆菌介导的高羊茅高效遗传转化和转基因植株再生

用带有质粒pDBA121(含hpt基因和bar基因)的农杆菌EHA 105转化高羊茅(Festucaarundinacea Schreb.)胚性悬浮细胞,建立了可重复的、高效的农杆菌介导的高羊茅遗传转化系统.商业用的除草剂Basta直接用于转化细胞的筛选.基因型、受体材料的类型、培养基成分和筛选剂影响农杆菌介导的转化频率.悬浮细胞的农杆菌转化效率为每克悬浮细胞再生2.85～10.9株转基因植株,大大高于基因枪法的高羊茅转化效率(2～5株).经PCR分析和Southern杂交检测表明,bar基因已整合进入高羊茅基因组,转基因植株Basta喷洒试验表明bar基因已成功地实现高水平的表达.此转化系统的建立为高效地将外源有用基因导入高羊茅并高效稳定地表达奠定了基础.     

高羊茅FaCONSTANS基因的表达及功能分析

植物由营养生长向生殖生长转变过程中光周期调控起着重要的作用。CONSTANS (CO) 是光周期途径中的特有基因,为探讨高羊茅FaCONSTANS (FaCO) 基因响应日照长短从而启动植物开花的机理,利用实时荧光定量qRT-PCR技术分析在长日照、短日照、持续光照、持续黑暗条件下FaCO基因的表达水平。构建过表达载体p1300-FaCO,利用农杆菌介导法遗传转化拟南芥,构建沉默载体p1300-FaCO-RNAi遗传转化高羊茅。结果表明,FaCO基因的表达受光周期调控,与生物钟控制的昼夜节律相关。在长日照条件下FaCO基因促进拟南芥开花,且恢复拟南芥突变体开花表型。RNAi沉默FaCO基因的高羊茅转基因植株晚花或者一直处于营养生长阶段。本研究初步探究高羊茅FaCO基因对开花过程的调控,这将有助于更进一步了解该基因的生物学功能。     

高羊茅对高温的生理生态响应

分析了昼/夜38℃/30℃高温下2种高羊茅草坪草(凌志和交战Ⅱ)与狗牙根叶片的相对含水量、叶绿素含量、细胞膜脂过氧化、抗氧化系统以及光合作用等生理生态指标的变化.结果表明:随高温时间的延长,2种高羊茅的相对含水量、叶绿素含量、净光合速率和最大光能转化效率均呈降低趋势,而且凌志高羊茅的平均降幅小于交战Ⅱ高羊茅.经过9d的高温处理,凌志高羊茅叶片超氧化物歧化酶、过氧化物酶和过氧化氢酶的平均活性比交战Ⅱ高羊茅分别高19.7%、17.9%和17.7%,而凌志和交战Ⅱ高羊茅的净光合速率分别下降了60.7%和81.9%.与交战Ⅱ高羊茅相比,凌志高羊茅具有更高的光化学转换功能,有利于减轻高温对光合器官的损伤,使其保持较高的净光合速率.在整个高温处理过程中,狗牙根的各项生理生态指标随时间变化的差异不显著.狗牙根、凌志高羊茅和交战Ⅱ高羊茅对高温胁迫的适应能力由强到弱.     

羊茅属(禾本科)新类群

描述了山东产羊茅属的2个新类群,即毛鞘羊茅和崂山小颖羊茅。毛鞘羊茅与羊茅相近,表现在叶片内卷成线状,圆锥花序紧缩成穗状,外稃背部粗糙,区别在于具有向上膜质的叶耳,叶鞘密被倒向的微毛,小穗长7-10mm,第1外稃长4-5mm,顶端具0.5-1mm长的芒或无,内稃脊光滑;崂山小颖羊茅与原变种的区别在于第1颖卵状披针形,长1.5-2mm,具2-3脉。     

高羊茅组织培养再生体系及GUS基因瞬间表达研究

以成熟种子为外值体,对高羊茅纰织培养和植株再生体系进行了优化,分析了不同浓度2．4-D、6-BA和激动素对高羊茅愈伤组织诱导和愈伤组织分化成苗的影响．结果表明：9．0mg／L 2．4-L)对愈伤组织的诱导效果最佳．0．2mg／L激动素是愈伤组织分化成苗的最适浓度．二者的诱导率和分化率分别达到68．08%和45．83％。在愈伤组织继代培养基中附加1．0mg／L 2．4-D、0．5mg／L 6-BA和1．25mg／L CuSO4;有利于胚性愈伤组织的形成,可以明显促进愈伤组织分化。同时．采用基因枪法将GUS基因导入高羊茅愈伤组织中,通过组织化学染色检测到了GUS瞬间表达活性;并对影响CUS基因瞬间表达的因素进行了分析．以期为提高基因枪法遗传转化效率提供参考。     

根癌农杆菌介导的高羊茅遗传转化研究

采用携带卡那霉素抗性基因nptⅡ和Na^ ／H^ 反向运输AtNHX1基因的表达载体pROK2／AtNHX1(带有35S启动子)和pROK2U／AtNHX1(带有ubi1启动子)的根癌农杆菌AGL1和GV3101,对4个品种高羊茅下胚轴来源的胚性愈伤组织进行了遗传转化。胚性愈伤组织经根癌农杆菌感染和共培养后,用50～150mg／L巴龙霉素筛选抗性愈伤组织,获得1126棵再生植株,用10～20mg／L卡那霉素进一步筛选再生植株,总共得到525棵绿色抗性植株。抗性植株的总DNA用AtNHX1基因的特异引物进行PCR检测,其中21棵为PCR阳性,最高转化频率为1．77％。Southern杂交结果证实,外源基因以低拷贝整合到高羊茅的基因组中,实验发现,在不同品种之间转化效率有所差异。     

施肥对高羊茅草坪越夏的影响

施用N、K、Ca和杀真菌剂对高羊茅（Festuca arundineces Schreb.cv.Pixie)越夏影响的研究结果表明,施氮肥可以打破高羊茅的夏季休眼,促进植株生长,促进植株对N、K的吸收,增加植株叶绿素含量,提高草坪质量,而不会明显降低草坪草的抗热性。但施氮肥后褐斑病等病害加重,通过喷洒杀真菌剂可控制病害的发生。     

Comparative analysis of transgenic tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) plants obtained by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and particle bombardment

Agrobacterium-mediated transformation and particle bombardment are the two most widely used methods for genetically modifying grasses. Here, these two systems are compared for transformation efficiency, transgene integration and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The bar gene was used as a selectable marker and selection during tissue culture was performed using 2 mg/l bialaphos in both callus induction and regeneration media. Average transformation efficiency across the four callus lines used in the experiments was 10.5% for Agrobacterium-mediated transformation and 11.5% for particle bombardment. Similar transgene integration patterns and co-integration frequencies of bar and uidA were observed in both gene transfer systems. However, while GUS activity was detected in leaves of 53% of the Agrobacterium transformed lines, only 20% of the bombarded lines showed GUS activity. Thus, Agrobacterium-mediated transformation appears to be the preferred method for producing transgenic tall fescue plants.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of meadow fescue (<Emphasis Type="Italic">Festuca pratensis</Emphasis> Huds.)

Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved. The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines, the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated one or two copies of the uidA gene. C. Gao and J. Liu contributed equally to the work.     

A linkage map of meadow fescue (<Emphasis Type="Italic">Festuca pratensis</Emphasis> Huds.) and comparative mapping with other Poaceae species

A genetic linkage map has been constructed for meadow fescue (Festuca pratensis Huds.) (2n=2x=14) using a full-sib family of a cross between a genotype from a Norwegian population (HF2) and a genotype from a Yugoslavian cultivar (B14). The two-way pseudo-testcross procedure has been used to develop separate maps for each parent, as well as a combined map. A total number of 550 loci have been mapped using homologous and heterologous RFLPs, AFLPs, isozymes and SSRs. The combined map consists of 466 markers, has a total length of 658.8 cM with an average marker density of 1.4 cM/marker. A high degree of orthology and colinearity was observed between meadow fescue and the Triticeae genome(s) for all linkage groups, and the individual linkage groups were designated 1F–7F in accordance with the orthologous Triticeae chromosomes. As expected, the meadow fescue linkage groups were highly orthologous and co-linear with Lolium, and with oat, maize and sorghum, generally in the same manner as the Triticeae chromosomes. It was shown that the evolutionary 4AL/5AL translocation, which characterises some of the Triticeae species, is not present in the meadow fescue genome. A putative insertion of a segment orthologous to Triticeae 2 at the top of 6F, similar to the rearrangement found in the wheat B and the rye R genome, was also observed. In addition, chromosome 4F is completely orthologous to rice chromosome 3 in contrast to the Triticeae where this rice chromosome is distributed over homoeologous group 4 and 5 chromosomes. The meadow fescue genome thus has a more ancestral configuration than any of the Triticeae genomes. The extended meadow fescue map reported here provides the opportunity for beneficial cross-species transfer of genetic knowledge, particularly from the complete genome sequence of rice.Communicated by P. Langridge     

Low allelopathic potential of an invasive forage grass on native grassland plants: a cause for encouragement?

Tall fescue (Festuca arundinacea Schreb.), a highly competitive European grass that invades US grasslands, is reportedly allelopathic to many agronomic plants, but its ability to inhibit the germination or growth of native grassland plants is unknown. In three factorial glasshouse experiments, we tested the potential allelopathic effects of endophyte-infected (E+) and uninfected (E−) tall fescue on native grasses and forbs from Midwestern tallgrass prairies. Relative to a water control, at least one extract made from ground seed, or ground whole plant tissue of E+ or E− tall fescue reduced the germination of 10 of 11 species in petri dishes. In addition, the emergence of two native grasses in potting soil was lower when sown with E+ and E− tall fescue seedlings than when sown with seeds of conspecifics or tall fescue. However, when seeds of 13 prairie species were sown in sterilized, field-collected soil and given water or one of the four tall fescue extracts daily, seedling emergence was lower in one extract relative to water for only one species, and subsequent height growth did not differ among treatments for any species. We conclude that if tall fescue is allelopathic, its inhibitory effects on the germination and seedling growth of native prairie plants are limited, irrespective of endophyte infection. On the other hand, the apparent inability of these plants to detect tall fescue in field soil could hinder prairie restoration efforts if germination near this strong competitor confers fitness consequences. We propose that lack of chemical recognition may be common among resident and recently introduced non-indigenous plants because of temporally limited ecological interactions, and offer a view that challenges the existing allelopathy paradigm. Lastly, we suggest that tall fescue removal will have immediate benefits to the establishment of native grassland plants.     

高羊茅和多年生黑麦草内生真菌的分子检测

根据GenBank上报道的内生真菌Neotyphodium coenophialum和N.lolii的Nc25基因序列,设计了2对特异性引物FM1/R1和F3/R652,建立了一套适合于从高羊茅和多年生黑麦草种子中检测内生真菌N.coenophialum和N.lolii的常规PCR和巢式PCR方法。     

Characterization of microsatellite markers for rough fescue species (Festuca spp.)

One major challenge in genetic diversity analysis of minor grass species is the lack of informative molecular markers. A set of 210 simple sequence repeat (SSR) markers developed from wheat and barley were evaluated for their transferability to three rough fescue species [Festuca altaica Trinius, F. campestris (Rydb.) and F. hallii (Vassey) Piper]. Twelve SSR primer pairs displayed scorable polymorphism among and within the species. The number of alleles per primer pair ranged from three to 17 with an average of 8.3 for all the species and greatly varied for each species. About 82% of SSR variation resided within the species. Festuca hallii was genetically most distinct among the three species.     

A Clearing Technique for Detecting the Fungal Endophyte Acremonium sp. in Grasses

Leaf tissue of tall fescue Festuca arundinacea Schreb., hard fescue Festuca ovina L., red fescue Festuca rubra L. and perennial ryegrass Lolium perenne L. was stained with rose Bengal or aniline blue to detect the presence of the fungal endophyte Acremonium sp., Specimens were cleared using methyl salicylate, an optical clearing agent, and viewed using bright field microscopy. Tissue was presenred as dried tissue or stored in 70% aqueous ethyl alcohol before staining and clearing. Tissue was observed at 2, 4 and 12 weeks following clearing to check for stain retention. Staining with rose Bengal was inferior to aniline blue when followed by the clearing agent methyl salicylate. Fungal mycelia stained lighter with rose Bengal and were more difficult to detect than mycelia stained with aniline blue. The results illustrate the usefulness of combining staining and methyl salicylate clearing for detecting fungal endophytes.