刺槐核糖体蛋白同源基因RpRPL22在共生结瘤过程中功能研究

目的: 核糖体蛋白(RPs)属于多功能蛋白,能够参与调控细胞生长和响应胁迫条件。RpRPL22是一个从豆科植物刺槐中分离得到的结瘤相关基因,通过序列比对发现其与核糖体大亚基蛋白RPL22高度同源。对其如何通过调控根瘤菌侵染而在共生结瘤过程中发挥重要作用进行了较为深入的探索。方法: 利用实时荧光定量PCR技术(qRT-PCR)分析RpRPL22在接菌后不同时间及不同植物组织的表达变化。利用cDNA末端快速扩增技术(RACE)获得目的基因cDNA全长。通过GFP报告基因进行RpRPL22亚细胞定位分析。通过Gateway BP重组技术构建RNA干扰(RNAi)重组载体,借助电转化法将重组载体转至农杆菌K599,利用农杆菌介导植物根部,接菌后观察和测量植株表型。首先从宏观水平统计观察目的基因是否对结瘤过程有影响,其次从分子水平揭示目的基因在共生结瘤过程的重要功能。结果: 不同接菌时期、不同植物组织目的基因qRT-PCR相对表达量结果显示,几乎在所有取样的接菌时间,目的基因RpRPL22在接菌根中的相对表达量都低于未接菌对照根,只有接菌后第25天除外。在成熟的根瘤中,接菌后第25天该基因的表达量也最高。洋葱表皮和毛状根亚细胞定位结果均显示在椰菜花叶病毒(CaMV)的35S启动子控制下,RpRPL22融合绿色荧光蛋白GFP的荧光信号在细胞核和细胞质有明显的表达。RNAi转化植株的表型统计观察结果,比如植株鲜重、植株的有效结瘤数目较对照组均有明显的降低;同时RNAi转化植株在根瘤菌侵染过程形成的侵染线数目和根瘤原基数目较对照均显著降低。根瘤切片实验用于观察根瘤显微超微结构,结果显示RNAi植株根瘤中固氮区的受菌侵染细胞数目与对照相比明显减少。电镜观察根瘤单个受菌侵染细胞中类菌体形态显示,RNAi根瘤中类菌体侵染细胞胞体多呈不规则形状,皱缩变形严重,环类菌体周间隙空间增大,多共生体融合,表现出细胞凋亡的迹象。对照根瘤中的受菌侵染细胞胞体多呈圆形椭圆形,胞质饱满丰富且分布均匀,细胞发育正常,表明RNAi植株根瘤发育过程明显受阻。结论: 核糖体蛋白(RP)能够参与调控豆科植物共生结瘤过程,相关同源基因RpRPL22可能在起始根瘤菌侵染植物和阻止类菌体降解过程中起重要作用。     

3种丛枝菌根真菌与3种寄主植物的共生关系

【目的】利用丛枝菌根(Arbuscular mycorrhiza,AM)真菌对寄主植物的偏好性和不同寄主植物的功能互补作用,建立AM真菌的高效繁殖体系。【方法】以玉米(Zea may L.)、高粱[Sorghum bicolor(L.)Moench]和白车轴草(Trifolium repens L.)为寄主植物,采用寄主植物单作和间作的盆栽培养法,研究不同栽培模式对光壁无梗囊霉(Acaulospora laevis)、单孢球囊霉(Glomus monosporum)和根内球囊霉(G.intraradices)3种AM真菌繁殖能力的影响,通过地上部分生物量的分配分析,探索C3和C4植物对AM真菌共生关系的"功能互补"效应及机制。【结果】间作模式下,寄主植物地上部分总生物量和3种AM真菌的孢子密度均显著高于单作(P0.05);单作和间作栽培模式下,3种AM真菌对玉米地上部分生物量响应无显著差异(P0.05),但单孢球囊霉和根内球囊霉对高粱地上部分生物量产生显著影响(P0.05);两种间作栽培模式下,根内球囊霉对白车轴草地上部分生物量也产生了显著影响(P0.05)。【结论】3种AM真菌对3种寄主植物的共生偏好性不同,且C3和C4植物对AM真菌共生关系存在一定的"功能互补"效应,利用AM真菌的寄主植物偏好性和不同植物间的功能互补关系,增加AM真菌的孢子产量,有利于AM真菌高效繁殖体系的建立。     

Ensifer fredii symbiovar vachelliae nodulates endemic Vachellia gummifera in semiarid Moroccan areas

The purpose of this work was to study the genetic diversity of the nodule-forming bacteria associated with native populations of Vachellia gummifera growing wild in Morocco. The nearly complete 16S rRNA gene sequences from three selected strains, following ARDRA and REP-PCR results, revealed they were members of the genus Ensifer and the sequencing of the housekeeping genes recA, gyrB, dnaK and rpoB, and their concatenated phylogenetic analysis, showed that the 3 strains belong to the species E. fredii. Based on the nodC and nodA phylogenies, the 3 strains clearly diverged from the type and other reference strains of E. fredii and formed a clearly separated cluster. The strains AGA1, AGA2 and AGB23 did not form nodules on Glycine max, Phaseolus vulgaris and Medicago truncatula, and effectively nodulated V. gummifera, Acacia cyanophylla, Prosopis chilensis and Leucaena leucocephala. Based on similarities of the nodC and nodA symbiotic genes and differences in the host range, the strains isolated from Moroccan endemic V. gummifera may form a different symbiovar within Ensifer species, for which the name “vachelliae” is proposed.     

Fatty acid variations in symbiotic dinoflagellates from Okinawan corals

The fatty acid composition of polar lipids and triacylglycerols was determined in different morphophysiological types of symbiotic dinoflagellates (SD) isolated from the hydrocoral Millepora intricata and the scleractinian corals Pocillopora damicornis, Seriatopora caliendrum, Seriatopora hystrix and Stylophora pistillata from a fringing reef of Sesoko Island, Okinawa, Japan. The distribution of the fatty acids among the morphophysiologically distinct types of SD reported in these corals makes it possible to readily distinguish one type of SD from the other. Moreover, differences were found both in polar lipids and triacylglycerols. The polar lipids of SD from M. intricata showed a very distinctive fatty acid profile. A combination of large proportions of 18:4 (n-3), 18:5 (n-3), 22:5 (n-6), and 22:6 (n-3) and negligible amounts of 20:4 (n-6), and 20:5 (n-3) in SD from M. intricata was particularly noteworthy. The fatty acid profiles of SD from P. damicornis and SD isolated from S. caliendrum and S. hystrix differed in the proportion of 18:4 (n-3) and 22:6 (n-3). It is suggested that fatty acids might provide useful information on possible taxonomic differences among symbiotic dinoflagellates. It is assumed that biochemical differences can reflect the genetic diversity of the morphophysiological types of SD associated with several species of hermatypic corals from this region.     

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco

In arbuscular mycorrhizas, H⁺-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526–S532). The proton-pumping H⁺-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H⁺-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H⁺-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of β-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H⁺-ATPase activity in the periarbuscular membrane results from selective induction of two H⁺-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface. Received: 23 October 1999 / Accepted: 7 February 2000     

O-Methylated mannogalactan from the microalga Coccomyxa mucigena, symbiotic partner of the lichenized fungus Peltigera aphthosa

A structural study of the carbohydrates from Coccomyxa mucigena, the symbiotic algal partner of the lichenized fungus Peltigera aphthosa, was carried out. It produced an O-methylated mannogalactan, with a (1 → 6)-linked β-galactopyranose main-chain partially substituted at O-3 by β-Galp, 3-OMe-α-Manp or α-Manp units. There were no similarities with polysaccharides previously found in the lichen thallus of P. aphthosa. Moreover, the influence of lichenization in polysaccharide production by symbiotic microalgae and the nature of the photobiont in carbohydrate production in lichen symbiosis are also discussed.     

Effect of salinity on root-nodule conductance to the oxygen diffusion in the Cicer arietinum-Mesorhizobium ciceri symbiosis

Nodule conductance to O2 diffusion has been involved as a major factor of the inhibition of N2 fixation by soil salinity that severely reduces the production of grain legumes. In order to determine the effect of this constraint on the nodule conductance, oxygen uptake by the nodulated roots of Cicer arietinum was measured by recording the concentration of O2 as a function of pO2 in a gas-tight incubator. After germination and inoculation with the strain Mesorhizobium ciceri UPMCa7, the varieties Amdoun 1 and INRAT 93-1 were hydroponically grown in a glasshouse on 1L glass bottles filled with nutrient solution containing 25 mM NaCl. Salinity induced a marked decrease in shoot (30% versus 14%), root (43% versus 20%), and nodule biomass (100% versus 43%) for Amdoun 1 relative to INRAT 93-1. Although salinity completely prevented nodule formation in the sensitive variety Amdoun 1, nodule number and biomass were higher in the first than in the second variety in the absence of salt. This effect was associated with a significantly higher O2 uptake by nodulated root (510 versus 255 micromol O2 plant(-1)h(-1)) and nodule conductance (20 versus 5 microm s(-1)) in Amdoun 1 than in INRAT 93-1. Salinity did not significantly change the nodule conductance and nodule permeability for INRAT 93-1. Thus, the salt tolerance of this variety appears to be associated with stability in nodule conductance and the capacity to form nodules under salt constraint.     

Simultaneous sludge reduction and nutrient removal (SSRNR) with interaction between Tubificidae and microorganisms: a full-scale study

A symbiotic ecosystem between Tubificidae and microorganisms was established at a full-scale wastewater treatment plant (WWTP). In this ecosystem Tubificidae were inoculated, and then adhered to the outer layers of carrier materials in an oxidation tank. During the long-term treatment of sewage volumes of 20,000 m(3)d(-1), the excess sludge production rate was reduced from 0.21 to 0.051 kg m(-3) and sludge settleability was significantly improved. When the influent concentrations of COD, NH(4)(+)-N, PO(4)(-)-P, and SS were in the ranges of 130.0-459.0 mg L(-1), 14.2-27.5 mg L(-1), 1.6-7.0 mg L(-1), and 60.0-466.0 mg L(-1), respectively, the COD and SS removal efficiency was increased by 8.7% and 13.6% within the symbiotic system compared to the control without Tubificidae. In addition, NH(4)(+)-N and phosphorus removal efficiency can also be improved. The results showed that both sludge reduction and nutrient removal were enhanced simultaneously significantly within the system utilizing the symbiotic interactions of Tubificidae and microorganisms.     

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.     

Impairment of NtAQP1 gene expression in tobacco plants does not affect root colonisation pattern by arbuscular mycorrhizal fungi but decreases their symbiotic efficiency under drought

We investigated in two tobacco (Nicotiana tabacum) plant lines (wildtype or antisense mutant) whether impairment in expression of the plasma membrane aquaporin gene (NtAQP1) affects the arbuscular mycorrhizal (AM) fungal colonisation pattern or the symbiotic efficiency of AM fungi. These two objectives were investigated under well-watered and drought stress conditions. Both plant lines had a similar pattern of root colonisation under well-watered and drought stress conditions. In contrast, under drought stress, AM wildtype plants grew faster than mycorrhizal antisense plants. Plant gas exchange also appeared to depend on the expression of NtAQP1 and parallelled the determined growth increments. The implications of enhanced symplastic water transport via NtAQP1 for the efficiency of the AM symbiosis under drought stress conditions are further discussed.