苜蓿根瘤菌聚羟丁酸解聚酶基因(phbD )突变体的构建及其特性

摘要: 根据苜蓿根瘤菌(Sinorhizobium meliloti ) Rm1021基因组中与Ralstonia eutropha phaZ基因同源部分序列设计1对引物，从S. meliloti基因组中用PCR扩增出835 bp phbD 基因片段并克隆到载体pGEM?襆-T Easy上；通过在phbD基因内插入ΩSmSp和基因置换构建了phbD突变体。该突变体可积累比野生型菌株多1.0~2.6倍的聚羟丁酶（PHB）。在YMA和TY平板上形成非粘液型菌落，而在以乙酰乙酸或3-羟丁酸为唯一碳源的M9基本培养基（M9-AA，M9-HB）上形成粘液型菌落。碱性磷酸酶测定结果表明，通过接合引入phbD突变体菌株中的exoF-phoA融合基因在YMB培养基中低量表达，而在M9-AA和M9-HB中高量表达。     

接种根瘤菌对鸡骨草幼苗生长、结瘤和固氮能力的影响

用来自不同产地的鸡骨草根瘤菌接种盆栽幼苗,室内培养70 d后,测定植株生长和结瘤情况及植株含氮量。结果表明,接种根瘤菌对鸡骨草的株高、植株生物量、氮素相对积累量和固氮效率有明显的促进作用;不同来源的根瘤菌对鸡骨草生长和固氮的影响不同,来自玉林的根瘤菌菌株有较高的固氮效率。接种根瘤菌能促进鸡骨草生长,增加氮素累积,从而可以减少氮肥的使用,有利于充分发挥生物固氮的作用来提高原料药材的质量。     

胶质芽孢杆菌与苜蓿根瘤菌双接种对排土场紫花苜蓿生长和土壤性质的影响

为了明确胶质芽孢杆菌和苜蓿根瘤菌在矿区排土场复垦中的协同作用,以矿区排土场土壤作为基质进行盆栽试验,设置两菌单接种处理、双接种处理及对照,其中胶质芽孢杆菌和苜蓿根瘤菌用量分别为1.50%和0.25%,测定紫花苜蓿产量指标、叶片生理指标、土壤速效养分含量和土壤微生物指标。结果表明,接种胶质芽孢杆菌和苜蓿根瘤菌的各处理均能显著提高紫花苜蓿的产量指标,其中以双接种处理效果最优,将生物量和结瘤量分别比对照显著提高110.27%和124.32%;在生理指标方面,双接种处理效果低于单接种处理,未表现出协同作用;在土壤养分和微生物性质方面,单接种胶质芽孢杆菌仅显著提高土壤速效钾含量,单接种苜蓿根瘤菌对大部分指标均有显著效果,而双接种处理效果最佳,土壤碱解氮、速效钾含量比对照分别显著提高94.14%和84.55%,土壤微生物指标MBC含量、呼吸强度、蔗糖酶、脲酶、磷酸酶、过氧化氢酶活性比对照分别显著提高204.02%,65.86%,212.32%,91.87%,30.57%,51.87%。双接种处理对紫花苜蓿产量指标、养分和微生物指标表现出显著的协同作用。因此,1.50%用量胶质芽孢杆菌和0.25%用量苜蓿根瘤菌双接种能够有效提高排土场土壤的复垦效果,具有协同作用,可以作为矿区排土场复垦的高效方法。     

接种耐酸根瘤菌和施钙对酸性土上紫花苜蓿生长的影响

采用裂区设计法研究了重庆缙云山酸性土上施钙与接种耐酸根瘤菌对紫花苜蓿生长和品质的影响。结果发现:接种耐酸苜蓿根瘤菌对苜蓿植株瘤重、根鲜重、株高、植株上部鲜重、全氮含量和酸性土壤中根瘤菌数量的提高影响显著;施Ca2+ 5 mmol/L与10 mmol/L处理与不施Ca2+处理相比,能显著增加植株根鲜重、株高和地上部鲜重,也能显著增加瘤重;瘤重与植株地上部鲜重、根鲜重和株高呈极显著相关关系,与植株全氮含量呈显著相关关系,说明良好的结瘤性能能够提高酸性土上紫花苜蓿的产量和品质。     

土壤中大豆根瘤菌之间竞争结瘤的研究——Ⅲ.接种菌量对大豆生长的影响

本文研究了大豆根瘸菌PRC005的接菌量对大豆生长的影响.田间试验结果表明:接菌量在播种后40天和60天没有显著增加根瘤数、根瘤干重、地上部植株干重和植株含氮量.施氮肥处理和较高接菌量处理之间的大豆种子产量差异不显著,与不接菌对照处理相比,施氮肥和较高接菌量两个处理的种子产量显著增加.施氮肥处理没有使植株含氮量增加,并且还妨碍了大豆的结瘸作用.室内盆栽试验结果表明:只有当接菌量高于土著菌数1200倍时,才能显著地提高大豆的结瘤数和植株干重.     

接种根瘤菌对白格、大叶合欢和银合欢生长与结瘤的影响

Effects of inoculation of Rhizobium suspension on nodulation and plant growth were examined with Albizzia procera,Albizzia lebbeck and Leucaena leucocephala seedlings grown on sterilized and non-sterilized soil media.Inoculation resulted in nodule number increases of 28.6,29.02and 23.9 times in sterilized soil and 3.4,3.6and 3.27 times in non-sterilized soil for A.procera,A.lebbeck and L.leucocephala seedlings respectively.Total dry mass increased by 127.6%,66.7%and 60.7% in sterilized soil and 100%,95.5%and 52.65% in non-sterilized soil for these three legume trees,respectively,after a period of two months.Significantly high inoculation responses of oot length,root diameter,collar diameter,shoot length,and dry mass of root,shoot,leaves and nodules were also observed in both steilized and non-sterilized soil media as compared to respective control treatments,The response to inoculation was strong in sterilized and modest in non-sterilized soils.The significantly higher response to Rhizobium inoculation over control in all the species tested suggested that application of Rhizobium greatly enhanced plant growth ,nodulation,biomass production and nitrogen-fixing activity of the nodules.     

外源甘氨酸对镉胁迫下苜蓿幼苗生长和氧化损伤的影响

以苜蓿为试验材料,分析了甘氨酸处理对幼苗镉(Cd)毒害缓解的生理机制。Cd毒害降低了苜蓿幼苗超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性,导致幼苗出现氧化伤害,如丙二醛(MDA)含量升高,伊文斯蓝(Evans blue)染色加深。甘氨酸处理提高了苜蓿幼苗SOD和CAT活性,降低了MDA的累积,减少了氧化伤害。Cd毒害还降低了幼苗硝酸还原酶(NR)活性,外源甘氨酸处理则显著提高其NR活性。施用NR抑制剂钨酸钠降低了苜蓿幼苗的NR活性,同时抵消了甘氨酸对Cd胁迫幼苗的生长缓解作用。表明甘氨酸通过提高苜蓿幼苗的抗氧化能力和NR活性,从而提高幼苗对Cd胁迫的耐受性。     

钙磷对酸铝土壤中苜蓿根瘤菌迁移定殖和群体感应的影响

采用分室(内、外室)培养法,以紫花苜蓿及其耐酸根瘤菌91522为材料,在酸性土壤(pH4.45)中额外补充Al3+至中度铝毒水平,从培养装置根箱外接种,探讨了补充Ca2+、P后,耐酸苜蓿根瘤菌在酸性土壤上的存活、迁移以及群体感应的变化动态。结果表明,补充5 mmol kg-1Ca2+处理的土壤根瘤菌的数量在97 d种植期内均显著高于对照(Ca0P0,即无Ca2+无P处理),Ca2+5 mmol kg-1+P 0μmol kg-1处理近根区(距苜蓿植株根1～2 cm)土壤最大根瘤菌数量为同期对照的6.15倍;在此基础上补充P后根瘤菌的数量进一步增加,"Ca2+5 mmol kg-1+P 4μmol kg-1"处理近根区最大根瘤菌数量为同期对照处理的9.40倍。因此推断Ca2+和P的上述作用存在交互效应。施加Ca2+、P能够显著提高土壤中根瘤菌群体感应物质N-酰基高丝氨酸内酯衍生物(N-acyl-homoserine actones,AHLs)的含量,且Ca2+5 mmol kg-1效果好于Ca2+10mmol kg-1处理。根瘤菌数量在远根区(距苜蓿植株根6～8 cm)与近根区变化规律一致,即接种1周后根瘤菌数量由远根区向近根区逐渐增加,接种后30 d内达到最高值,之后数量下降并趋于稳定。但近根区根瘤菌数量和AHLs含量均高于远根区,说明宿主根际微环境也能够影响根瘤菌数量和群体感应。初步认为,酸性有铝土壤上补充Ca2+5 mmol kg-1和P 30μmol kg-1对耐酸根瘤菌的存活、迁移和群体感应有良好的改善效果。该处理最终显著地增强了对酸性土壤极为敏感的苜蓿植株的耐酸铝胁迫能力,较对照显著(p0.05)改善了酸铝胁迫下苜蓿的植株根鲜重(为对照的4.67倍)、地上部鲜重(3.10倍)、含氮量(2.47倍)和根瘤数(14.74倍)等农艺性状。     

紫花苜蓿“Vector”品种高效根瘤菌筛选及其田间竞争结瘤能力研究

Seventeen Sinorhizobium meliloti strains from seven provinces in China were used to screen highly effective strains for alfalfa cultivar in a greenhouse study and their symbiotic relationship and competitive ability were studied in the field. CCBAU30138 was the most effective strain, as evidenced by increase in dry weights. A field experiment showed that the inoculation of alfalfa with CCBAU30138 resulted in increases of 11.9% and 19.6% of dry matter production and crude protein production, respectively, in forage of monocultured plants. The total dry matter yields of alfalfa and tall fescue in binary culture were increased by 16.3% by inoculation of alfalfa with this strain. These results showed that S. rneliloti strain CCBAU30138 was an effective inoculant both in the greenhouse and in the field. The analysis of randomly amplified polymorphic DNA （RAPD） by polymerase chain reaction （PCR） from nodule extracts showed that the strain CCBAU30138 had high competitiveness in the field. It occupied 47.5% of nodules in alfalfa monoculture and 44.4% of nodules in alfalfa-tall fescue binary culture after 20 weeks of growth. In conclusion, a simple system to select highly effective and competitive symbiotic strains specific to alfalfa was established. Using this system, a s.train suitable for the alfalfa cultivar ‘Vector＇ grown in Wuqiao County of Hebei Province was obtained.     

Survival and persistence of genetically modified Sinorhizobium meliloti in soil

Studies were conducted to evaluate the survival and persistence of Sinorhizobium meliloti 104A14 and two acid phosphatase-negative mutants in Kirkland (fine, mixed, thermic Udertic Paleustolls) silt loam soils with various fertility levels, and to assess the impact of inoculation on nodule occupancy and soil microbial community structure in the inoculated alfalfa (Medicago sativa L.) rhizosphere. Recovery of the inoculated strains was 100% (in the order of 10⁸ cells g⁻¹ soil) immediately following inoculation to soils, but decreased from 10⁸ cells g⁻¹ soil to undetectable levels in a nutrient-poor soil within 32 days. In a nutrient-rich soil, approximately 2–3% (4.7–7.43×10⁶ cells g⁻¹ soil) of the mutants and 23% (5.84×10⁷ cells g⁻¹ soil) of the wild-type inocula persisted for more than 64 days. Survivability and persistence of the wild-type S. meliloti were significantly greater than that of the genetically modified acid phosphatase negative mutants in all the soils tested. The persistence and nodule occupancy of the introduced S. meliloti in sterile and non-sterile soils were also tested for two repeated alfalfa growth periods in the same plant growth units, with a 1 month interval in between and no additional inoculation for the second period. Nodule occupancy of the introduced S. meliloti in non-sterile soils ranged from 30 to 60% for the first period and 85 to 100% for the second period. Our results suggest that survival and persistence of S. meliloti was enhanced by alfalfa cultivation and increased soil fertility, but impaired by mutation of acid phosphatase genes regardless of phosphorus nutritional levels. Moreover, inoculation with genetically modified S. meliloti strain 104A14 promoted indigenous bacterial growth in soil (increased bacterial population from 1.4×10⁶ to 4.3×10⁶ cells g⁻¹ soil), but not the growth of fungi and yeast. However, inoculation of the wild-type S. meliloti or genetically modified mutants did not result in significant changes in microbial community structure as indicated by EP indices and ratios of r/K strategists.     

根瘤菌结瘤基因与竞争结瘤基因的研究进展

根瘤菌的结瘤基因与竞争结瘤基因在根瘤菌与结瘤的过程中起着关键的作用。随着研究的深入,已经发现,分离和定位了数十个结瘤基因和几个与竞争结瘤有关的基因,本文将简要地介绍了这方面的研究成果。     

抗春雷霉素大豆根瘤菌突变株的诱变及其生物固氮特性的分析

根瘤菌与豆科植物共生可以固定大量的氮。根瘤菌剂接种豆科作物是一项普遍推广应用、有效的农业技术。但由于大量土著根瘤菌的存在，产生竞争障碍，降低了接种菌剂的占瘤率。大多数的土著根瘤菌对春雷霉素敏感，因此接种抗春雷霉素的高效结瘤固氮根瘤菌，并用春雷霉素处理种子，可抑制土著根瘤菌，提高接种菌剂的占瘤率，从而达到高结瘤、高固氮和提高产量的目的。本文将探讨诱变对根瘤菌抗春雷霉素突变的作用，并对获得的抗性突变株的生物固氮特性进行分析。     

Insights into the Effect of a Natural Arsenate Dose on Growth, Nodulation and Redox Metabolism of Soybean Plants

<正>Dear Editor,Arsenic(As) is a harmful metalloid that occurs in soil and water; its concentration varies considerably among geographic regions, with groundwater being the principal source of human contamination(Smedley and Kinniburgh, 2002). Besides the direct contamination effect of drinking water that contains high As concentration, human poisoning may also occur after inges-     

外源赤霉素对富集植物繁缕生长及镉积累的影响

为了强化镉富集植物繁缕对镉污染土壤的修复效率,采用盆栽试验,研究了喷施不同浓度赤霉素(GA_3)对繁缕生长及镉积累的影响。结果表明,繁缕的生物量随GA_3浓度的增加而增加,但光合色素含量、抗氧化酶(SOD、POD和CAT)活性、可溶性蛋白含量、可溶性糖含量、镉含量及镉积累量均呈先增后降的趋势。在GA_3浓度为10 mg L~(-1)时,繁缕地上部分及整株镉积累量均达到最大值,分别为244.86、348.97μg plant~(-1),较各自对照分别增加了31.20%(P0.05)和25.51%(P0.05)。因此,采用繁缕修复镉污染土壤时,以10 mg L~(-1)的GA_3提高繁缕的修复效率最佳,可作为植物修复的强化措施。     

Survey of genes identified in Sinorhizobium meliloti spp., necessaryfor the development of an efficient symbiosis

The symbiosis between the soil bacteria Rhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium or Bradyrhizobium and leguminous plants is characterised by a specific multistep signal exchange. Only when a compatible rhizobial strain encounters its leguminous host, nodules will be formed on the roots of the host. During infection of this nodule, the microsymbiont evolves into a bacteroid form which, when provided with plant-derived carbon sources, is able to convert atmospheric nitrogen to ammonia that subsequently is supplied to the plant. The developmental programme underlying nodule organogenesis and functioning has been studied intensively for several decades. In this review, several observed plant phenotypes resulting from an ineffective symbiosis between plants and mutant rhizobial strains are represented. Besides the influence of the bacterial nodulation, nitrogen fixation and surface polysaccharide genes on symbiosis, the role of other genes important for the formation of effective nitrogen fixing nodules will be explained.     

外源激素对金花茶花蕾生长的影响(简报)

采用不同激素在不同时期和时间间隔处理金花茶花蕾的方法研究外源激素对花蕾生长的影响。结果表明，用１０００ｍｇ·Ｌ－１的赤霉素（ＧＡ３）处理直径在０．３２～１．１２ｃｍ范围内的金花茶花蕾，可以明显地促进花蕾的生长而无伤蕾现象。在抽梢前用ＧＡ３隔天处理，一个月后促进效果可达１００．７％，而用激动素（ＫＴ）处理则普遍抑制花蕾的生长。在抽梢后每周处理一次，则ＧＡ３的促进效果较差，一个月后促进效果只有４８．５％，而ＫＴ处理反而表现出一定的促进效应     

生物素缺乏及肉鸡和种鸡日粮中添加生物素效果的研究

５４０只１日龄红布罗商品代雏鸡随机分为９组，第１组饲喂以小麦为基础的诱发日粮，第２和第３组在１组基础上分别添加３００（μｇ／ｋｇ日粮）生物素和动物脂肪（前期２％，后期３％）。第４至９组饲以玉米－豆饼型日粮，前后期分别添加０，５０，９０，１２０，１５０，２００和０，５０，７０，９５，１２０，１６０（μｇ／ｋｇ日粮）的生物素。结果表明：生物素缺乏造成足底皮肤炎，肝脏脂肪酸成分发生变化：不饱和脂肪酸（棕榈油酸）显著增加（Ｐ＜０．０１），饱和脂肪酸（硬脂酸）减少（Ｐ＜０．０５）。２９日龄绝食２４ｈ，饲喂诱发日粮的鸡，血糖、肝糖原和肝脏总脂肪含量下降，血浆甘油三酯增加，但无显著差异（Ｐ＞０．０５）。玉米－豆饼型日粮中添加不同水平生物素，绝食后血糖、肝糖原和甘油三酯含量未发生显著变化，随生物素添加水平提高，对足部皮肤状况有一定的改善作用，前期添加１５０或２００（μｇ／ｋｇ日粮）生物素对增重和饲料报酬较好，而后期添加生物素影响不大。种鸡试验选用３３６只２０周龄育成种鸡随机分为３组，分别饲以添加有０，１００，１５０μｇ／ｋｇ生物素的日粮。初步结果表明：生物素对蛋重无影响，日粮添加１００和１５０μｇ／ｋｇ生物素可提高受精率０