花生根瘤菌诱变株感染大豆结瘤和固氮

花生根瘤菌诱变株感染大豆结瘤和固氮龙敏南，许良树，张凤章，曾定（生物学系）根瘤菌在共生固氮过程中会放Ｈ２，放Ｈ２是种能量浪费．Ｅｖａｎｓ等［１］研究表明：利用具有吸氢酶活性的根瘤菌［ＨＵｐ＋］接种豆科作物能提高固氮效率、增加作物产量．豆科植物根瘤菌的...     

大豆应用根瘤菌试验总结

为了“绿色”环保需要，降低氨忆施用量，节约成本，提高大豆的结瘤固氮能力，进而提高大豆产量和品质，通过试验根瘤菌对不同大豆品种的结瘤固氮效果，为生产大面积应用提供依据。     

快中子辐照大豆根瘤菌阶段研究

利用快中子对大豆根瘤菌(Rhizobium japonicum)进行辐射诱变。结果表明被诱变菌的菌落形态、颜色、结瘤数和共生固氮活性都发生变化。结瘤数高达对照的15倍左右,共生固氮活性最高提高8倍左右。诱变菌感染的大豆植株特性和结实量都发生了明显变化。     

大豆和花生根瘤菌氢酶的研究

根瘤菌在共生固氮过程中因放H2所消耗的能量约占固所氮总能量的40%-6%。吸氢酶则能回收和利用固氮过程所放的H2,养活能量损失,从而提高共生固氮效率。在厌氧条件下,加入防止酶蛋白聚合的试剂,利用DEAE-纤维素和Sephacry S-200柱层析,从自养性大豆根瘤菌和花生根瘤菌类菌体中分离并提纯膜结合态氢酶。纯化的两种氢酶表现相近的分子特征：均含有大（60kD,65kD)、小（30kD,35kD)两个亚基,均为NiFe-氢酶,并且有较高的吸H2活性。大豆根瘤菌氢酶的纯酶组分不含Cyt b599。花生根瘤菌L8-3菌株能进行化能自养生长,诱导出高吸H2活性。根瘤菌的吸H2能明显提高固氮活性。从具有高吸H2活性的花生根瘤菌中分离并克隆吸氢基因,采用PCR和探针杂交技术,获得含有吸氢基因的质粒pZ-55。利用多种限制性内切酶构建了质粒pZ-55的物理图谱。通过三亲本杂交,将含吸氢基因的重组质粒转移到不吸H2的花生和毛豆根瘤菌中,所获得的结合株在自生和共生条件下均表达吸H2活性。以结合株接种大田花生,获得的共生根瘤的吸H2活性比接种受体株提高4倍,花生叶片和种子的含N量、产量分别提高1.7%、8.9%和9.6%。     

大豆种植的科学原理

经过多年的研究，对于水热作用状况，活性磷的供给，土壤耕作方法，秸秆还田及微量元素钼的应用对作物的生长发育，大豆光合作用器官及共生器官的形成及工作，产量的高低和生物氮在参与氮形成中所占的比重等的影响得出了评价。大豆光合作用器官的发育水平及生物氮的供给决定了作物产量的高低，由于各年度的水热条件不同，磷营养的改善能提高大豆单产２．０～３．６公担／公顷。利用综合农业技术措施促使大豆单产提高１．５～２．６公担／公顷众所周知，在俄罗斯所进行的经济改革导致了在农业生产总体方面，特别是在大豆种植业方面的退步。大…     

细菌——植物叶际联合固氮研究进展

从生态学观点来看,自然界存在着三种生物固氨体系:自生固氮体系,如自生固氮菌在土壤中或培养基中独立生活时固定分子态氮;共生固氮体系,如根瘤菌和豆科植物共生时才能固定分子态氮,或者只在共生条件下才表现旺盛的固氮作用;联合固氮体系,是自生固氮体系与共生固氮体系的中间类型,固氮菌与相应联合的植物之间有较密切的相互影响,但既不同于典型的共生固氮体系,因为它不形成根瘤一样的特殊形态结构——根瘤,也不同於自生固氮体系,因为它有较大的寄主专一性,并且固氮作用比     

野大豆快生型根瘤菌共生固氮及其生理生化特性研究

实验围绕野大豆——快生型根瘤菌共生固氮和抗药、耐盐、代谢产物等生理生化特性方面进行检测和研讨，旨在为筛选抗逆性强、高固氮活性菌株和农业应用提供理论依据。     

花生固氮根瘤菌应用研究

应用花生固氮根瘤菌，通过拌种方法在花生种子中接种，种植在未种过花生的花岗岩发育的壤土中，进行固氮效果测定。结果表明，施用的花生固氮根瘤菌明显增加了花生的结瘤量，植株生长健壮，群体生长旺盛，增加植株生物量，结荚率提高11%，植株含氮量提高52.9%，籽仁含氮量提高52.5%，产量提高40%，增产效果显著。     

增加田间二氧化碳的浓度使大豆根瘤菌固定更多氮素

每蒲式耳(35.238市升)大豆的籽实含有4磅氮素,另外还有2磅氮素含在根茎叶中。如果每英亩(6.070市亩)大豆平均产量为50蒲式耳(经折算,约为每亩430市斤),则总的含氮量为每英亩300磅。如果要把大豆产量翻一番,达到每英亩100蒲式耳,则总的氮素需要量约为每英亩600磅。明显地,要增加大豆的产量,主要要增加大豆对氮素的吸收量。大豆,一方面同其他作物一样,利用土壤肥料中的氮素;另一方面大豆还同根瘤菌形成共生关系,在根部形成根瘤,在根瘤中进行着共生性的氮素固定。这就使大豆既能利用土壤中的氮素,又能利用空气中丰富的氮素。     

山西省土著大豆根瘤菌共生结瘤固氮特性研究

在山西土壤气候条件下，大豆主栽品种自然结瘤率达９４．６％，单株结瘤量为３７．１±１５．２个，日固氮量０．４４～１，５６ｍｇ，每公顷约固氮３０ｋｇ。单株固氮总活性高峰期随品种和栽培条件的不同有所变化，而单瘤固氮活性的高峰均在花期。土著根瘤茵的固氮强度与结瘤量无相关性，固氮量与单瘤固氮酶活则成显著正相关（ｒ＝０．７１Ｐ＜０．０５）。覆膜栽培和秸秆还田均具有良好的共生固氮生态效应。     

Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium symbiosis

The biological reduction of atmospheric N2 to ammonium (nitrogen fixation) provides about 65% of the biosphere's available nitrogen. Most of this ammonium is contributed by legume-rhizobia symbioses, which are initiated by the infection of legume hosts by bacteria (rhizobia), resulting in formation of root nodules. Within the nodules, rhizobia are found as bacteroids, which perform the nitrogen fixation: to do this, they obtain sources of carbon and energy from the plant, in the form of dicarboxylic acids. It has been thought that, in return, bacteroids simply provide the plant with ammonium. But here we show that a more complex amino-acid cycle is essential for symbiotic nitrogen fixation by Rhizobium in pea nodules. The plant provides amino acids to the bacteroids, enabling them to shut down their ammonium assimilation. In return, bacteroids act like plant organelles to cycle amino acids back to the plant for asparagine synthesis. The mutual dependence of this exchange prevents the symbiosis being dominated by the plant, and provides a selective pressure for the evolution of mutualism.     

Extra-copy nifA enhances the nodulation efficiency of Sinorhizobium fredii

Previous investigations have shown that nifA gene is involved in nodulation and symbiotic nitrogen fixation regulation of Rhizobium. We study the role of nifA on nodulation of leguminous plants. We found that Sinorhizobium fredii harboring multi-copy plasmid carrying the constitutively expressed Klebsiella pneumoniae nifA exhibited an increase of noduiation activity and nodulation competitiveness on soybean plants. The Nod-factor secreted by the rhizobia cells containing the multi-copied nifA was assayed,and preliminary results showed that S. fredii containing the multi-copy plasmid carrying nifA produced higher strength of Nod-factor than the rhizobia containing the same plasmid carrying the vector did.     

Isolation and application of effective nitrogen fixation rhizobial strains on low-phosphorus acid soils in South China

Soybean (Glycine max L.) is a very important food and oil crop in China. Legume-rhizobium symbiotic nitrogen (N) fixation is an important biological character and also the base of improving soil fertility of soybean. However, soybean production and development is severely limited in tropical and subtropical areas in China due to a lack of effective rhizobial inoculants adapting to low-phosphorus (P) acid soils. In the present study, 12 soybean rhizobial strains were isolated and purified from the nodules of two soybean genotypes contrasting in P efficiency, which were grown on different low-P acid soils with different soybean cultivation histories. Results from 16S rDNA sequence analysis showed that these 12 rhizobial strains belonged to the genus of Bradyrhizobium, which had higher nitrogenase activities compared to the control strain, Bradyrhizboium japonicum USDA110. A field experiment was carried out by applying rhizobial inoculants, a mixture of three rhizobial strains that showed the highest nitrogenase activity, on a typical low-P acid soil in South China. The results showed that, without inoculation, no nodules were formed in the three soybean genotypes tested; with inoculation, the nodulation rates in all were 100%. Inoculation with rhizobial inoculants not only made many nodules formed, but also increased soybean shoot biomass and yield, and improved nitrogen (N) and P nutrient status. Among which, shoot dry weight, N and P content of a soybean genotype, Huachun 3, inoculated with rhizobium were increased 154.3%, 152.4% and 163.2% compared to that without inoculation, respectively. We concluded that: (i) The effective indigenous rhizobial strains isolated in this study from soybeans on low-P acid soils in South China have the characters of broad host range, high nodulation efficiency, efficient N fixation, great low pH and low P tolerance. (ii) Soil environment and host types are the key factors to screen the effective rhizobial strains. Considering soil pH values and P efficiency of the host genotypes might increase the screening efficiency. (iii) Improving N status and facilitating root growth might be the mechanisms of increasing the P uptake in soybean plants inoculated with the effective rhizobial strains on low-P acid soils. (iv) Inoculation with the effective rhizobial inoculants could significantly improve growth, N and P content of soybean on low-P acid soils, which might be an effective approach to enhance soybean cultivation and development in these areas. Therefore, application and extension of inoculation techniques with effective rhizobial inoculants in legumes would result in great economical, environmental and ecological benefits. The authors contributed equally to this work Supported by National Key Basic Research and Development of China (Grant No. 2005CB120902), McKnight Foundation Collaborative Crop Research Program (USA) (Grant No. 05-780) and National Natural Science Foundation of China (Grant No. 30571111)     

豆科植物-根瘤菌共生固氮的分子机理

与豆科植物－根瘤菌共生固氮有关的基因涉及根瘤菌基因和宿主基因，根瘤菌基因有结瘤基因（nodD,nodAB-CIJ和hsn基因），根瘤菌细胞表面结构基因（exs,lps和ndv基因）和固氮基因（nif和fix基因）；宿主基因主要是结瘤素基因（ENOD和NOD基因）。根瘤菌结瘤基因表达后诱导产生结瘤因子。在根瘤发育过程中，这些基因在根瘤菌与植物之间进行着信息交换，并且具有不同的表达水平。结瘤因子和植物激素对它们进行着调节。     

富硒大豆低聚肽口服液的制备及其主要成分分析

富硒大豆蛋白经过酶解、超滤、调配、杀菌等工艺,制备成富硒大豆低聚肽口服液,分析了产品中的硒含量、大豆低聚肽含量及氨基酸组成,其中硒含量2.45 mg/100 ml,大豆低聚肽含量(分子量1000 D以下)达到638.25 mg/100 ml,含有Se-Cys在内的19种氨基酸,研究结果为富硒大豆低聚肽产品的质量标准制定提供了一定的理论依据.     

Nitrogen fixation. Endocrine disrupters and flavonoid signalling

Nitrogen fixation is a symbiotic process initiated by chemical signals from legumes that are recognized by soil bacteria. Here we show that some endocrine-disrupting chemicals (EDCs), so called because of their effect on hormone-signalling pathways in animal cells, also interfere with the symbiotic signalling that leads to nitrogen fixation. Our results raise the possibility that these phytochemically activated pathways may have features in common with hormonal signalling in vertebrates, thereby extending the biological and ecological impact of EDCs.     

有机氮源对柠檬酸发酵的影响

对柠檬酸发酵使用的薯干原料中的总氮、可溶性氮及非蛋白氮的含量进行了测定 ,并通过发酵实验考察了培养液中含氮量与柠檬酸产量的关系。在薯干培养液中添加某些氨基酸或玉米蛋白的酶解液进行发酵实验 ,结果证明 ,添加适量的有机氮源对柠檬酸产量有促进作用 ,特别是对质量低劣的薯干原料促进效果更加明显 ,柠檬酸产量可比对照提高27 %     

黄腐酸对大豆内源激素的调控及结瘤能力的影响

为探讨黄腐酸提高大豆结瘤能力的作用机理,以辽宁慢生根瘤菌(Bradyrhizobium liaoningense)CCBAU 05525和冀豆17为材料,采用高效液相色谱法(HPLC)探究黄腐酸对大豆结瘤初期内源激素含量、分布及结瘤的影响.结果表明:黄腐酸质量浓度为500 mg/L时大豆结瘤数量最多,比对照组显著提高73.1%,同时该条件下激素含量和分布变化最为明显,具体表现为大豆根、茎、叶中玉米素(ZT)、生长素(IAA)、赤霉素(GA3)含量增加,根中脱落酸(ABA)含量减少,叶中ABA含量增加.而黄腐酸质量浓度为1 000 mg/L时则表现为抑制大豆结瘤. 上述结果为解析黄腐酸提高大豆结瘤固氮的机理、增加大豆产量提供了理论依据.     

A unifying framework for dinitrogen fixation in the terrestrial biosphere

Dinitrogen (N(2)) fixation is widely recognized as an important process in controlling ecosystem responses to global environmental change, both today and in the past; however, significant discrepancies exist between theory and observations of patterns of N(2) fixation across major sectors of the land biosphere. A question remains as to why symbiotic N(2)-fixing plants are more abundant in vast areas of the tropics than in many of the mature forests that seem to be nitrogen-limited in the temperate and boreal zones. Here we present a unifying framework for terrestrial N(2) fixation that can explain the geographic occurrence of N(2) fixers across diverse biomes and at the global scale. By examining trade-offs inherent in plant carbon, nitrogen and phosphorus capture, we find a clear advantage to symbiotic N(2) fixers in phosphorus-limited tropical savannas and lowland tropical forests. The ability of N(2) fixers to invest nitrogen into phosphorus acquisition seems vital to sustained N(2) fixation in phosphorus-limited tropical ecosystems. In contrast, modern-day temperatures seem to constrain N(2) fixation rates and N(2)-fixing species from mature forests in the high latitudes. We propose that an analysis that couples biogeochemical cycling and biophysical mechanisms is sufficient to explain the principal geographical patterns of symbiotic N(2) fixation on land, thus providing a basis for predicting the response of nutrient-limited ecosystems to climate change and increasing atmospheric CO(2).     

A general method for site-directed mutagenesis in prokaryotes

The genetic analysis of genes from prokaryotic species for which experimental genetic systems have not yet been developed is often limited by the difficulty of producing mutations in those genes. We report here a general technique applicable to Gram-negative prokaryotes for site-directed mutagenesis of cloned DNA fragments which we have applied to the study of the symbiotic nitrogen fixation genes of Rhizobium meliloti. In particular, we mutagenized cloned R. meliloti restriction fragments in Escherichia coli with transposon Tn5 and then replaced the wild-type parental DNA sequences with the mutant DNA sequences in the R. meliloti genome. Using this method we show that an R. meliloti DNA restriction fragment, cloned previously on the basis of homology to Klebsiella pneumoniae nif genes, contains gene(s) essential for symbiotic nitrogen fixation. In addition, we use this method to construct a physical genetic map of a subset of the R. meliloti nif genes.