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。     

根瘤菌中的nodD基因及其与弗兰克氏菌（Frankia）中的类nodD基因的功能互补

根瘤菌可在几种豆科植物上成瘤,结瘤基因（ｎｏｄ基因）赋予这些细菌以寄主专一性的方式诱导瘤的形成。目前,已被鉴定的三类主要结瘤基因是：＂公共＂结瘤基因,如ｎｏｄＡＢＣ,这类基因是结瘤不可缺少的,并具有高度的结构和功能上的保守性;另一类是寄主专一性基因,这类基因具有种属或菌株的专一性,它们决定了细菌的寄主范围;最后一类是结瘤基因Ｄ（ｎｏｄＤ）,这类基因编码一类调节蛋白,并和植物分泌的酚类化合物一道起动其他结瘤操纵子的转录。一些根瘤菌种属只有一个拷贝的ｎｏｄＤ基因,而另一些则可拥有三个拷贝之多。苜蓿根瘤菌（Ｒ．ｍｅｌｉｌｏｔｉ）中存在三个拷贝的ｎｏｄＤ基因,其中的任何一个发生突变都以依赖于寄主的方式影响结瘤效果。在慢生型大豆根瘤菌（Ｂ．ｊａｐｏｎｉｃｕｍ）中,ｎｏｄＤ１突变菌株还可在大豆或其他豆科植物寄主上结瘤,只是稍往后推迟而已,ｎｏｄＤ２基因对ｎｏｄＹＡＢＣＳＵＩＪ操纵子诱导的影响却很小,两个ｎｏｄＤ拷贝突变后尚表现出结瘤活性。在只有一个ｎｏｄＤ基因拷贝的根瘤菌种属中,如豌豆根瘤菌（Ｒ．ｌｅｇｕｍｉｎｏｓａｒｕｍ）和茎瘤固氮菌（Ａ．ｃａｕｌｉｎｏｄａｎｓ）中,ｎｏｄＤ突变后会导致菌株丧失结瘤能力。一般说来,不     

慢生型大豆根瘤菌nfe基因的分子克隆

ｎｆｅＣ基因是从慢生型大豆根瘤菌中克隆到的,与竞争结瘤有关的基因。本研究从慢生型大豆根瘤菌菌株ＧＸ２０１的ｐＬＡＦＲ３为载体的基因文库中,筛选出与ｎｆｅ同源基因克隆。以转座子Ｔｎ５ｇｕｓＡ５诱变获得了ｇｕｓ基因表达的Ｔｎ５ｇｕｓＡ５插入突变质粒。     

豆科植物结瘤自调控分子机制研究进展

豆科植物通过与土壤中的根瘤菌共生发育形成根瘤,在根瘤中根瘤菌可以将空气中氮气转化成植物可以直接利用的铵态氮.共生过程中为了平衡氮素的摄取和能量的损耗,豆科植物形成了地上与地下信号交互的结瘤自调控机制(Auto-regulation of nodulation,AON),进而调节结瘤的数量.本文综述了AON分子调控机制近...     

慢生型大豆根瘤菌putA基因的功能分析

重组质粒pGX300上的putA基因是从慢生型大豆根瘤菌GX201中克隆到的编码脯氨酸脱氢酶的基因。通过Tn5gusA5定位诱变方法构建了putA基因的突变株GX20120，实验表明，GX20120的结瘤时间推迟，竞争结瘤能力降低。     

GUS基因标记法对慢生花生根瘤菌竞争结瘤和接种效果的研究

应用GUS基因标记技术,可简便、快速、准确、原位、直观地确定标记花生根瘤菌株形成的根瘤,从而方便地研究标记菌株与土著根瘤菌的竞争结瘤能力。无氮水培试验表明,标记菌株gusA4-5、gusA2-9分别与土著菌混和接种占瘤率为71.4%、77.0%。盆栽试验表明,接种供试菌株Spr4-5、Spr2-9占瘤率分别为57.9%、63.0%,比对照极显著增产52.5%、22.7%;接种Spr4-5比Spr2-9极显著增产24.2%。初步说明两个供试菌株的竞争结瘤力比土著根瘤菌强,菌株Spr2-9强于Spr4-5;Spr4-5比Spr2-9有效性高,是结瘤适量,竞争结瘤能力强的高效菌株。     

用荧光酶基因(cfp)标记法研究菜豆根瘤菌的竞争性和有效性

试验采用三亲本杂交法将cfp基因导入一株有效性较高的菜豆根瘤菌SC18,获得了一株带cfp基因标记的菌株SC18-cfp。对SC18-cfp进行性状、标记基因的遗传稳定性检测,结果表明,cfp基因不仅能有效表达,而且性状稳定。随机挑取SC18-cfp的10个转移结合子在无氮砂培条件下进行标记菌株与亲本菌株在3个菜豆品种上的竞争结瘤试验。结果证实,10个转移结合子在供试植物根系上的占瘤率均接近50%,其有效性和竞争结瘤能力与亲本菌株间的差异不显著,品种间的差异性不显著;在控制田间试验中SC18-cfp比土著根瘤菌的竞争结瘤能力强,在品种A、B、C上的占瘤率分别达到了57.8%、60.0%、55.6%。接种SC18的植株干重、全氮量、叶绿素含量和豆荚产量均显著高于对照。     

抗药性根瘤菌基因工程菌的构建与应用

用三亲本杂交的方法将外源抗药性质粒导入到高效固氮的根瘤菌 Tal377中,外源质粒在 Tal377中能正常表达,但不影响其原有的固氮结瘤能力。利用此抗药性根瘤菌所携带的抗药性,在接种此根瘤菌的同时结合施加一定浓度的抗菌素,抗药性根瘤菌与出发菌株相比表现出一定程度地提高其结瘤固氮能力和占瘤率。     

百脉根根瘤菌自体诱导物合成酶基因mrlI2的初步研究

许多细菌均能产生一类叫做自体诱导物的信号分子,用于感知自身密度的变化,从而调控其生理行为,这种现象称为群体感应[1]。越来越多的研究表明,在根瘤菌中普遍存在着群体感应调控系统[2],并且它可能在根瘤菌与豆科植物共生结瘤固氮过程中起着重要作用。目前,在豌豆根瘤菌中共发现有4种群体感应系统(rai,rhi,cin和tra),其中cin系统位于上游,起总调控作用,tra系统控制着质粒的转移,rhi系统与结瘤数量有关,rai系统的功能还不清楚,它们共同组成了一个复杂的群体感应调控系统[3]。在苜蓿中华根瘤菌中,也发现群体感应系统与根瘤菌的生长和结瘤效率有关[4]。对中慢生型华癸根瘤菌和天山根瘤菌的研究     

活性氧调控豆科植物早期结瘤的研究进展

活性氧（reactive oxygen species,ROS）是一类具有高反应活性的氧衍生物,包括超氧阴离子（·O₂^—）、羟自由基（·OH）、过氧羟自由基（·HO₂）以及过氧化氢（H₂O₂）等。植物在进行有氧代谢或遭遇生物与非生物胁迫时会产生ROS,它不仅仅是有氧代谢的有毒副产物,同时能作为信号分子调节体内代谢过程,对抗外界环境。豆科植物形成根瘤时同样会产生ROS,这种ROS的变化区别于病原体入侵,而是作为一种信号物质参与结瘤过程。结瘤因子（nod factor,NF）诱导下ROS的产生参与了浸染线形成时细胞壁的重建、植物基质糖蛋白（matrix glycoprotein,MGP）的交联和肌动蛋白微丝的成核和延长过程。细胞质膜NADPH氧化酶（respiratory burst oxidase homologue,RBOHs）是共生过程中ROS产生的主要途径,Rboh基因的过表达会促进根瘤菌浸染和根瘤形成,同时根瘤中的共生微粒体数量增加,固氮效率提高,而表达受抑制后会减少ROS的产生,同时下调结瘤相关基因RIPs、NIN、ENOD2的表达,抑制固氮酶活性。此外,ROS时空上的变化与Ca²⁺相关联,协同调控根系结瘤。ROS的产生是植物与微生物早期的识别信号,通过认识ROS在早期结瘤过程中的作用有助于我们进一步理解共生关系建立的特异性。本文就ROS在早期结瘤过程中的产生及其发挥的作用做了综述,指出ROS通过直接或间接作用诱导结瘤基因的表达,是豆科植物根瘤形成以及功能固氮的重要信号分子。     

某些生物因子对苕子根瘤菌菌剂和土著根瘤菌竞争结瘤的影响

早在本世纪三十年代,国外已发现根瘤菌竞争结瘤的现象,并对三叶草根瘤菌品系之间的竞争结瘤进行了系统的研究。认为同时接种两个不同的根瘤菌品系,它们的结瘤率是取决于各个品系入侵竞争能力的大小,并引出了竞争优势种的概念。之后,随着免疫学、遗传标记和抗药标记等方法的相继应用,对竞争结瘤问题进行了深入的研究。     

Symbiotic dinitrogen fixation - its dependence on plant nutrition and its ecophysiological impact

The cultivation of symbiotic legumes and the consequent small uptake of nitrate by the host plant depresses soil pH and impairs subsequent legume growth. The rate of soil pH decline depends on the soil H⁺ buffer power, climatic conditions, soil permeability, and on the kind of legume cropping. It is shown for some leguminous crop species from the temperate climate that in many cases the N₂ fixation performance of Rhizobium/Bradyrhizobium symbiosis is more dependent on the growth conditions of the host plant than on the nitrogenase potential of the bacteroid. This is true for the supply of the host plant with phosphate, potassium and water as well as for light intensity. Nitrogen deficiency of the host plant because of insufficient nitrogenase activity was observed at low soil pH where nodulation and the development of the nitrogenase activity were delayed. Nodulation and nitrogenase activity are suppressed by high levels of available nitrogen in the soil. There are indications that the nitrogenase activity is very flexible and adjusts to the demand of the host plant. The mechanism of this regulation is not yet understood.     

Effect of combined nitrogen supply and nodulation on nitrate reductase activity and growth of pea plants

Nitrate reductase activity (NRA; EC 1.6.6.1) was measured in leaves, stems and roots of Pisum sativum cv. Lincoln supplied with different nitrate concentrations and inoculated with selected Rhizobium leguminosarum strains. As a control, noninoculated plants were grown in the same nutrient medium. NRA was determined by an in vivo‐nitrate assay. Although differences in tissue NRA were mainly related to nitrate concentration in the growing medium, nodulation much affected the NRA in the different plant organs, especially in root. An increased proportion of total plant NRA occurred in the leaves and stems as nitrate concentration was increased. Nitrate accumulation in leaves, stems and roots was correlated with the concentration of this ion in the nutrient solution. Nodulation also affects the nitrate accumulation in the different plant organs.     

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。     

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.     

Cropping history affects nodulation and symbiotic efficiency of distinct hairy vetch (Vicia villosa Roth.) genotypes with resident soil rhizobia

Compatible rhizobia strains are essential for nodulation and biological nitrogen fixation (BNF) of hairy vetch (Vicia villosa Roth, HV). We evaluated how past HV cultivation affected nodulation and BNF across host genotypes. Five groups of similar HV genotypes were inoculated with soil dilutions from six paired fields, three with 10-year HV cultivation history (HV+) and three with no history (HV?), and used to determine efficiency of rhizobia nodulation and BNF. Nodulation was equated to nodule number and mass, BNF to plant N and Rhizobium leguminosarum biovar viceae (Rlv) soil cell counts using qPCR to generate an amplicon of targeted Rlv nodD genes. Both HV cultivation history and genotype affected BNF parameters. Plants inoculated with HV+ soil dilutions averaged 60 and 70 % greater nodule number and mass, respectively. Such plants also had greater biomass and tissue N than those inoculated with HV? soil. Plant biomass and tissue N were strongly correlated to nodule mass (r ²?=?0.80 and 0.50, respectively), while correlations to nodule number were low (r ²?=?0.50 and 0.31, respectively). Although hairy vetch rhizobia occur naturally in soils, past cultivation of HV was shown in this study to enhance nodulation gene-carrying Rlv population size and/or efficiency of rhizobia capable of nodulation and N fixation.     

高效亲和的花生寄主植物-根瘤菌株组合

本文针对栽培花生，从大、小共生体双方研究了寄主植物－根瘤菌株组合的亲和性。结果指出：（１）在温室水培盆栽条件下，用菌株１４７－３接种的寄主植物结瘤、固氮能力育成品种〉普通型；（２）血清学鉴定出５个不同血清型菌株，其竞争力或回收率与寄主品种、根瘤菌株和土著菌数密切相关；（３）在温室与田间条件下，不同花生寄主一根瘤菌株组合存在着广谱或特异共生亲和性与非亲和性的差异，并鉴定出高效、广谱亲和的品种徐州６－     

Low root zone temperature effects on bean (Phaseolus vulgaris L.) plants inoculated with Rhizobium leguminosarum bv. phaseoli pre-incubated with methyl jasmonate and/or genistein

Abstract

Methyl jasmonate (MeJA) has recently been shown to act as a plant-to-bacteria signal. We tested the hypothesis that pre-induction of Rhizobium leguminosarum bv. phaseoli cells with genistein and/or MeJA would at least partially overcome the negative effects of low root zone temperature (RZT) on bean nodulation, nitrogen fixation and plant growth. Otebo bean plants were grown at constant air temperature (25^oC) and two RZT regimes (25 and 17^oC) and inoculated with R. leguminosarum bv. phaseoli pre-induced with MeJA and/or genistein. Our results indicate that low RZT inhibited nodulation, nitrogen fixation and plant growth. The plants growing at low RZT began fixing nitrogen seven days later compared to those at higher RZT. The low RZT plants had fewer nodules, lower nodule weight, less N fixation, slower plant growth, fewer leaves, smaller leaf area, and less dry matter accumulation comared to plants at a higher RZT. Rhzobium leguminosarum bv. phaseoli cells induced with genistein and/or MeJA enhanced bean nodulation, nitrogen fixation and growth at both optimum and suboptimum RZTs. The results of this study indicate that MeJA improves bean nitrogen fixation and growth at both optimum and suboptimum RZTs, and can be used alone or in combination with genistein to partially overcome the low RZT induced inhibitory effects on nodulation and nitrogen fixation.     

Changes in Phosphate Fractions Extracted From Different Organs of Phosphorus Starved Nitrogen Fixing Pea Plants

The present work investigates the impact of phosphorus (P) starvation on plant growth, symbiotic nitrogen fixation, and internal P status (determined as extracted P fractions) of leaves, roots, and nodules of 27-days–old pea (Pisum sativum L) plants inoculated with Rhizobium leguminosarum bv viciae strain D293. The procedure of separation of organic and inorganic P compounds in 10% perchloric acid (HCLO₄) and the absorption of nucleotides in active charcoal gave several fractions, containing different phosphorus compounds, which were extracted and determined as inorganic phosphate after combustion. These are acid soluble and insoluble P, sugar P, nucleotide P, and inorganic P. The P starvation of plants inhibited significantly plant dry mass accumulation, nodulation rate and specific nitrogenase activity of nodules. These results were accompanied with lower quantities of total P per plant, acid soluble and acid non-soluble P fractions in all plant organs. The inhibited accumulation of P in the acid soluble P fraction was associated with decrease of sugar, nucleotide and inorganic P in all plant organs. The most negatively affected were all P fractions extracted from nodules and leaves. The low content of inorganic P in the stressed plant tissues was regarded as primary reason for induced alterations in the content of analyzed P fractions.