固氮相关的两个植物基因转化烟草及其表达

豆科植物凝集和血红蛋白分别在植物识别其相应的根瘤菌和在根瘤内降低氧分压保护固氮酶的共生固氮作用中起重要作用。将豌豆（Ｐｉｓｕｍ ｓａｔｉｖａ Ｌ．）凝集素基因（ｐｌ）和Ｐａｒａｑｓｐｏｎｉａ ａｎｄｅｒｓｏｎｉｉ血红蛋白基因（ｐｈｂ）构建到同一植物表达载体上,通过根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导法转化烟草（Ｎｉｃｓ     

豌豆凝集素和血红蛋白基因对水稻的转化和表达

为了扩大根瘤菌的突破产范围和试探根瘤菌在非豆科植物上的固所为作用,将豌豆凝集素基因（pl）和Parasponia andersonii血红蛋白基因 （phb）构建在同一个植物表达载体上,用基因枪法将其导入水稻（Oryza sativa L.ssp.japonica）。经PCR扩增和Southern杂匀分析,证明外源目的基因已整合到水稻基因组中。GUS组织化学染色及豌豆凝集素基因的Western印迹实验和表达产物的原位杂交,证实外源基因在转基因水稻中表达。在40个转化植株中18株有pl和phb基因的PCR产物,得率为45％。再用18株植物做pl基因的Western blot检测,有3株有翻译表达,占40株的7.5％,18株的17％。为水稻与根瘤菌的相互作用和固氮作用的可能性研究奠定了一定的基础。     

Transformation of Pea Lectin Gene and Parasponia Haemoglobin Gene into Rice and Their Expressions

Lectin and leghemoglobin in legumes play the important roles, respectively, in recognition of host plants to their rhizobial bacteria, and lowering the oxygen partial pressure around bacteroids and protecting nitrogenase from oxygen in symbiotic nitrogen-fixing nodules. In order to extend the host range of the rhizobial bacteria and to make them fix nitrogen in non-legumes, pea lectin gene ( pl ) and Parasponia hemoglobin gene ( phb ) have been constructed into a plant expression vector (pCBHUL) and the vector pCBHUL was introduced into rice calli from immature young embryos by particle bombardment. After the calli were regenerated into plantlets on the resistant-selecting media containing hygromycin, they were identified by PCR and Southern blot hybridization. It was indicated that the pl and phb genes were integrated into nucleic genome of the transformed rice plants. GUS activity and the product of the pl gene were determined by GUS staining, Western blot and in situ hybridization at translational level. Eighteen out of 40 plants resistant to hygromycin were positively identified by PCR analysis with the rate of 45%. The pl gene was expressed in 3 out of 18 plants with 17% and 7.5%in 40 plants. The results may provide a clue for exploring whether Rhizobium leguminosarum bv. viceae could extend its host range and make the transgenic rice plants have the possibility of being symbiotic, or associative to nitrogen fixation.     

Bioengineering of symbiotic systems: creation of new associative symbiosis with the use of lectins on the example of tobacco and colza

"Barbate roots" in tobacco and colza transgenic on lectin gene were obtained with the use of a wild strain of Agrobacterium rhizogenes 15834 transformed with pCAMBIA1305.1 plasmid containing the full-size lectin gene (psl) from the Pisum sativum. Influence of expression oflectin gene on colonization oftransgenic roots with symbiont of pea (Rhizobium leguminosarum) was investigated. The number of adhered bacteria onto the roots transformed with lectin gene was 14-fold and 37-fold higher in comparison with the control; this confirms the interaction of R. leguminosarum with pea lectin at the surface of the transformed roots of tobacco and colza. The developed experimental approach, based on the simulation of recognition processes and early symbiotic interactions with lectins of pea plants, may, in perspective, be used for obtaining stable associations of economically valuable, nonsymbiotrophic plant species with rhizobia.     

Bioengineering of symbiotic systems: Creation of new associative symbiosis with the use of lectins on the example of tobacco and oil seed rape

Hairy roots in tobacco and oil seed rape transgenic on lectin gene were obtained with the use of a wild strain of Agrobacterium rhizogenes 15834 transformed with pCAMBIA1305.1 plasmid containing the full-size lectin gene (psl) from the Pisum sativum. Influence of expression of lectin gene on colonization of transgenic roots with symbiont of pea (Rhizobium leguminosarum) was investigated. The number of adhered bacteria onto the roots transformed with lectin gene was 14-fold and 37-fold higher in comparison with the control; this confirms the interaction of R. leguminosarum with pea lectin at the surface of the transformed roots of tobacco and oil seed rape. The developed experimental approach, based on the simulation of recognition processes and early symbiotic interactions with lectins of pea plants, may, in perspective, be used for obtaining stable associations of economically valuable, nonsymbiotrophic plant species with rhizobia.     

Identification of the sym plasmid of Rhizobium leguminosarum strain 1001 and its transfer to and expression in other Rhizobia and Agrobacterium tumefaciens

A plasmid of 150 Mdal from Rhizobium leguminosarum RCC1001 was found to be a Sym plasmid (pSym1) carrying genes for root nodulation and nitrogen fixation on plants of the pea vetch cross-inoculation group. The plasmid was expressed not only in different R. leguminosarum and R. trifolii hosts, but also in Agrobacterium tumefaciens and R. meliloti, although in root nodules induced by A. tumefaciens and R. meliloti hosts no nitrogen was fixed. The host range for root nodule induction appeared to be determined by pSym1 and only included plants of the pea vetch cross-inoculation group; in contrast, the host range for the induction of root hair deformations, which was found also to be determined by pSym1 was less restricted and included besides plants of the pea vetch group in addition plants of the clover group. This corroborates previous findings that host specificity for nodulation and nitrogen fixation is exerted at a stage after the induction of root hair deformations.     

Symbiotic Nitrogen Fixation and Ammonium Nitrogen Assimilation in rrrbrb-, rrRbRb-, and RRrbrb-Pea Mutants

Wrinkled-seeded pea mutants (Pisum sativum L., genotypes rrrbrb-, rrRbRb-, and RRrbrb-) have seeds with reduced, but different, starch content and modified starch properties. Analysis of these mutants revealed an enhanced capacity of root nodules for symbiotic nitrogen fixation and of host plant organs for assimilation of ammonium nitrogen. This observation was confirmed by morphological data on organization of symbiotic system, by elevated nitrogenase activity, high protein accumulation in plants due to nitrogen fixation, and by enhanced activity of glutamine synthase in leaves and glutamate dehydrogenase in roots of mutants, as compared with the organs of wild-type pea. It is supposed that the aforementioned advantages of mutants are related to accumulation in seeds of elevated protein reserves that satisfy their demand for nitrogen during formation of symbiotic systems.     

Increase in alfalfa nodulation, nitrogen fixation, and plant growth by specific DNA amplification in Sinorhizobium meliloti.

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

生物固氮研究中的几个热点问题

氮素化肥在农业生产中一直发挥重要作用,为了发展持续生态农业,全世界的研究者都在进行着长期不懈的努力,不断优化和拓展生物固氮系统。介绍固氮研究中的4个热点问题：⑴联合固氮;⑵根际微生物量氮及微生物活度;⑶通过豆科植物凝集素基因转化扩大根瘤菌宿主范围;⑷结瘤固与“类根瘤”固氮。     

Artificial colonization of non-symbiotic plants roots with the use of lectins

Rhizobia have the ability to increase growth of non-legume plants due to the production of phytohormones and protection of plant from diseases and pathogens. However, the practical use of these beneficial bacteria sometimes fails because of their inability to effectively colonize rhizoplane and rhizosphere of inoculated plants. We chose the legume lectins as a factor that allows plants to form associative symbiosis with rhizobia. To test the fact that transgenic tobacco, tomato and rape roots with pea lectin gene may affect specific interaction with rhizobia, transgenic roots have been artificially inoculated by fluorescently-labeled pea rhizobia R. leguminosarum and east galega rhizobia Rhizobium galega. Microscopic and microbiological tests have shown that the number of adhered R. leguminosarum onto tobacco, rape and tomato roots which transformed with pea lectin gene is higher in comparison with the control, but no such effect through inoculation of these plants with R. galegae has been found. This confirms the interaction of R. leguminosarum with pea lectin at the surface of transformed roots. Undoubtedly, the improvement of recognition and attachment processes by using lectins can lead to the achievement of a stable associative relationship between non-symbiotic plants and rhizobia.     

Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment

Abstract An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II ( npt II) promotor of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.     

Complete sequence of a Rhizobium plasmid carrying genes necessary for symbiotic association with the plant host

The soil bacteria rhizobia have the capacity to establish nitrogen-fixing symbiosis with their leguminous host plants. In most Rhizobium species the genes for nodule development and nitrogen fixation have been localized on large indigenous plasmids that are transmissible, allowing lateral transfer of symbiotic functions. A recent paper reports on the complete sequencing of the symbiotic plasmid pNGR234a from Rhizobium species NGR234⁽¹⁾, revealing not only putative new symbiotic genes but also possible mechanisms for evolution and lateral dispersal of symbiotic nitrogen-fixing abilities among rhizobia.     

Gene centres,a source for genetic variants in symbiotic nitrogen fixation: Host-induced ineffectivity inPisum sativum ecotype fulvum

Summary Pisum sativum ecotype fulvum forms ineffective nodules with Rhizobium strains, isolated from effective nodules of the cultivated pea in Europe. Rhizobium strains isolated from nodules of fulvum peas in Israel are fully effective on this host plant, but in association with the cultivated pea they induce nodules of poor N₂-fixing activity. The distribution of these fulvum-specific Rhizobium strains is restricted to regions where the fulvum pea occurs naturally. Rhizobium strains from other geographical regions induce mainly ineffective, or partially effective nodules on fulvum plants.A wide genetic variation, with regard to symbiotic response to a standard set of Rhizobium strains, was demonstrated in the fulvum plants collected in Israel. Based on variation in N₂-fixation three groups of plants can be distinguished. These plants offer the possibility for the study of host-genetic control on symbiotic nitrogen fixation.     

Increase in Alfalfa Nodulation, Nitrogen Fixation, and Plant Growth by Specific DNA Amplification in Sinorhizobium meliloti

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

Synthesis and accumulation of pea plastocyanin in transgenic tobacco plants

The pea plastocyanin gene in a 3.5 kbp Eco RI fragment of pea nuclear DNA was introduced into tobacco by Agrobacterium-mediated transformation. Regenerated plants contained pea plastocyanin located within the chloroplast thylakoid membrane system. Analysis of seedlings from a self-pollinated transgenic plant containing a single copy of the pea plastocyanin gene indicated that seedlings homozygous for the pea gene contained almost twice as much pea plastocyanin as seedlings hemizygous for the pea gene. Homozygous seedlings contained approximately equal amounts of pea and tobacco plastocyanins. The amount of tobacco plastocyanin in leaves of transgenic plants was unaffected by the expression of the pea plastocyanin gene. The mRNA from the pea gene in tobacco was indistinguishable by northern blotting and S1 nuclease protection from the mRNA found in pea. In both pea and transgenic tobacco, expression of the pea plastocyanin gene was induced by light in leaves but was suppressed in roots. Pea plastocyanin free of contaminating tobacco plastocyanin was purified from transgenic tobacco plants and shown to be indistinguishable from natural pea plastocyanin by N-terminal protein sequencing and ¹H NMR spectroscopy.     

Two C4-dicarboxylate transport systems in Rhizobium sp. NGR234: rhizobial dicarboxylate transport is essential for nitrogen fixation in tropical legume symbioses.

To investigate the role of dicarboxylate transport in nitrogen-fixing symbioses between Rhizobium and tropical legumes, we made a molecular genetic analysis of the bacterial transport system in Rhizobium sp. NGR234. This braod host range strain fixes nitrogen in association with evolutionarily divergent legumes. Two dicarboxylate transport systems were cloned from Rhizobium NGR234. One locus was chromosomally located, whereas the other was carried on the symbiotic plasmid (pSym) and contained a dctA carrier protein gene, which was analyzed in detail. Although the DNA and derived amino acid sequences of the structural gene were substantially homologous to that of R. meliloti, its promoter sequences was quite distinct, and the upstream sequence also exhibited no homology to dctB, which is found at this position in R. meliloti. A site-directed internal deletion mutant in dctA of NGR234 exhibited a (unique) exclusively symbiotic phenotype that could grow on dicarboxylates ex planta, but could not fix nitrogen in planta. This phenotype was found for tested host plants of NGR234 with either determinate- or indeterminate-type nodules, confirming for the first time that symbiosis-specific uptake of dicarboxylates is a prerequisite for nitrogen fixation in tropical legume symbioses.     

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.     

Attenuation of Symbiotic Effectiveness by Rhizobium meliloti SAF22 Related to the Presence of a Cryptic Plasmid

Several wild-type strains of Rhizobium meliloti isolated from alfalfa nodules exhibited different plasmid profiles, yet did not differ in growth rate in yeast-mannitol medium, utilization of 43 different carbon sources, intrinsic resistance to 14 antibiotics, or detection of 16 enzyme activities. In contrast, three measures of effectiveness in symbiotic nitrogen fixation with alfalfa (shoot length, dry weight, and nitrogen content) indicated that R. meliloti SAF22, whose plasmid profile differs from those of the other strains tested, is significantly less effective than other wild-type strains in symbiotic nitrogen fixation. Light microscopy of nodules infected with strain SAF22 showed an abnormal center of nitrogen fixation zone III, with bacteria occupying a smaller portion of the infected host cells and vacuoles occupying a significantly larger portion of adjacent uninfected host cells. In contrast, the effective nodules infected with other wild types or plasmid pRmSAF22c-cured segregants of SAF22 did not display this cytological abnormality.     

一氧化氮对豆科植物结瘤及固氮的影响机制

豆科植物-根瘤菌共生过程受双方基因复杂且精细的调控, 能够产生特异的根瘤结构并可将大气中的惰性氮气(N₂)转化为可被植物直接利用的氨态氮。结瘤与固氮受多种因素影响, 其中, 一氧化氮(NO)作为一种自由基反应性气体信号分子, 可参与调节植物的许多生长发育过程, 如植物的呼吸、光形态建成、种子萌发、组织和器官发育、衰老以及响应各种生物及非生物胁迫。在豆科植物中, NO不仅影响寄主与菌共生关系的建立, 还参与调控根瘤菌对氮气的固定并提高植株氮素营养利用效率。该文主要从豆科植物及共生菌内NO的产生、降解及其对结瘤、共生固氮的影响和对环境胁迫的响应, 阐述了NO调控豆科植物共生体系中根瘤形成和共生固氮过程的作用机制, 展望了NO信号分子在豆科植物共生固氮体系中的研究前景。