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1.
用pSUP1011载体系统,Tn5诱变慢生大豆根瘤菌110、123,其KmR菌落出现频率为5x10-8一5x 10-7。Tn5的SmR。基因能在慢生大豆根瘤菌中表达。用卡那霉素加链霉素来筛选插入变株,可完全排除自然突变的干扰。从4450个KmR变株中检出营养缺陷型7株,其中His-(4)、Glu-(1)和Trp-(2),吸氢功能缺陷型10株,其中2株His-同时是吸氢缺陷(Hup-)和共生固氮缺陷(Nif-),其原养型回变体(回变频率为0.5×10-8)都同时恢复了吸氢与固氮功能,又失去了对卡那霉素的抗性。4株对氧特别敏感的Hup-缺陷型,在培养状态下无吸氢活性,但结瘤固氮时不放氢。  相似文献   

2.
花生接种根瘤菌共生固氮酶活性的研究   总被引:2,自引:0,他引:2       下载免费PDF全文
1983—1984年,分别利用生茬土和重茬土进行了盆栽花生接种根瘤菌共生固氮酶活性的研究,试验结果表明。花生接种8B6,97-1两株根瘤菌,显著提高结瘤数量,瘤重和固氮酶活性,提高植株干物重和含氮量。共生固氮酶活性高  相似文献   

3.
接种不同大豆根瘤菌株的根瘤放氢和吸氢的研究   总被引:2,自引:0,他引:2       下载免费PDF全文
本实验测定和比较了七株大豆根瘤菌与三个大豆品种共生时的放氢和固氮效率。证明了大多数菌株接种不同寄主,其根瘤的放氢、吸氢和固氮效率的差异均明显。但USDA110在三个品种上所结根瘤不放氢或极少放氢;它的固氮量最高。根瘤的能量利用率与植株的氮积累及产量的相关性尚需进一步验证。  相似文献   

4.
介绍了影响根瘤菌共生固氮效率的主要因素及遗传改造 :包括土壤因素、宿主植物、四碳二羧酸转移酶基因dct、固氮调节基因nifA、吸氢酶基因hup、共生质粒 (基因 )、缺陷型回复突变等。  相似文献   

5.
青海蚕豆根瘤菌共生固氮效应的研究   总被引:4,自引:0,他引:4  
刁治民 《微生物学杂志》2000,20(3):20-22,32
蚕豆根瘤菌属于快生型根瘤菌 ,2 4h形成菌落。在盆栽试验中 ,蚕豆根瘤菌Qx -2与蚕豆具有良好共生效应 ,其株高、茎叶干重及含氮量 ,株瘤数 ,根瘤重量以及固氮酶活性等 ,均明显高于不接种对照 ,固氮率提高 69.4 9%。接种根瘤菌是提高蚕豆共生固氮效应的技术措施。  相似文献   

6.
巴西固氮螺菌中吸氢酶基因同源性的分子检测   总被引:2,自引:0,他引:2  
罗永华  郭俊 《微生物学杂志》2002,22(3):12-13,34
通过TTC(2 ,3,5 氯化三苯基氮唑 )实验筛选出 12株能产生吸氢酶蛋白 (Hup+ )的巴西固氮螺菌 (Azospir rilumbrasilense)菌株。用Qiagen柱分离提纯含豌豆根瘤菌的hup基因片段的质粒 pHVT10 9和pHVT115 ,并用地高辛标记法标记pHVT115 ,与 12株Hup+ 巴西固氮螺菌的总DNA进行斑点杂交 ,结果显示pHVT115所含的吸氢酶基因 (hup基因 )片段与巴西固氮螺菌的大部分菌株的hup基因同源性不强。这一结果表明hup基因在固氮生物中存在着遗传多样性 ,异源hup基因探针不一定都适宜于探测hup基因。  相似文献   

7.
如东田菁根瘤菌遗传多样性及高效促生菌株筛选   总被引:1,自引:0,他引:1  
摘要:【目的】研究江苏如东沿海滩涂田菁根瘤菌的遗传多样性和系统发育,筛选高效促生田菁根瘤菌。【方法】采用16S rRNA基因、持家基因(recA、atpD、glnⅡ)、共生基因(nodA、nifH)的系统发育分析,并进行温室盆栽接种试验筛选高效菌株。【结果】分离到的32株田菁根瘤菌分布于Ensifer、Neorhizobium、Rhizobium,并与E. meliloti、N. huautlense、E. sesbaniae、R. pusense 亲缘关系最近。共生基因nodA、nifH的系统进化分析结果基本一致,均与E. saheli 最为相近。7株代表菌株均能耐受5%的NaCl(W/V)浓度,其中YIC5082耐盐性最强,能够耐受6%的NaCl。温室盆栽试验表明7 株代表菌株均能与田菁有效结瘤固氮,其中6株菌显著提高了植株的株高和鲜重。【结论】江苏如东沿海滩涂田菁根瘤菌具有较丰富的遗传多样性,其中N.huautlense、E.meliloti为优势种。该地区的田菁根瘤菌多数具有较好的共生有效性及耐盐性,YIC5077促生效果最为突出,并表现出较强的固氮及结瘤能力,具有良好的应用前景。  相似文献   

8.
通过三亲本杂交将携带豌豆根瘤菌吸氢基因的质粒pAL_(618)转移到台湾毛豆根瘤菌292-C中,经抗性筛选、质粒检测和吸氢活性测定,得到能稳定遗传的结合株292-C2和292-C3。在自生条件下结合株均表现较高的吸氢活性,而受体株292-C不吸氢,在共生条件下结合株表现为高吸氢,而受体株表现为放氢。  相似文献   

9.
【背景】对根瘤菌多样性的研究有助于推进根瘤菌种质资源的利用。【目的】研究紫花苜蓿内生和非内生根瘤菌的表型和遗传多样性,比较菌株在5个苜蓿品种上的共生效应,验证根瘤菌群体共生效应由苜蓿品种决定的假设。【方法】从甘肃省白银会宁旱作区、兰州安宁灌区、武威凉州灌区3个栽培区域的陇中、清水、WL168HQ、甘农3号、甘农9号等紫花苜蓿品种中分离内生(植株种子、花、叶、茎、根表皮、根中柱和根瘤)和非内生(根际土壤和田间土壤)根瘤菌菌株,通过表型数值分类、 16SrRNA基因限制性片段长度多态性(Restrictionfragmentlengthpolymorphism,RFLP)、16S rRNA基因测序、持家基因多位点序列分型(Multilocus sequence typing,MLST),以及结瘤基因nodC和固氮基因nifH片段序列测定,研究紫花苜蓿根瘤菌的表型和遗传多样性,并采用主成分分析研究根瘤菌菌株在5个紫花苜蓿品种上的共生效应差异。【结果】共分离得到43株内生根瘤菌和10株非内生根瘤菌,叶片和花中没有分离到根瘤菌菌株。53株根瘤菌以及对照菌株R.GN5和S.12531表型特征数值分类聚为8个群,菌株表型多样性丰富。经16SrRNA-RFLP分析共形成22种RFLP分型组合,基因型Ⅰ分布最广泛(24),其次为基因型Ⅻ(5)、ⅩⅤ(5)和ⅩⅨ(3),其余16株菌各代表1种基因型,菌株遗传多样性丰富。16S rRNA基因测序和MLST分析将所有菌株划分为Rhizobiumradiobacter、R.rosettiformans和Ensifermeliloti。仅从7株E.meliloti代表菌株和对照菌株S.12531中扩增到nodC和nifH基因,说明E.meliloti菌株均能结瘤固氮。E.meliloti菌株G3L3接种甘农3号,LP3、LL1和LL2接种陇中,QL2接种清水,LL1、LL2和WLP2接种WL169HQ苜蓿均能显著促进植株的单株结瘤数、地上干重和粗蛋白含量。E.meliloti菌株接种甘农3号、甘农9号和清水苜蓿品种后所有参数值在PC1轴上分别聚在-1-1之间,在PC3轴上聚在-1.5-1.5之间;接种陇中和WL168HQ苜蓿的参数值较分散,PC1轴上分散在-1.5-4之间,PC3轴上分散在-3-4之间。【结论】紫花苜蓿内生和非内生根瘤菌菌株多样性丰富,表型和遗传多样性与其来源没有直接关系。菌株G3L3与甘农3号,LP3、LL1、LL2与陇中,QL2与清水、LL1、LL2、WLP2与WL169HQ苜蓿品种共生匹配和适应能力强。在甘农3号、甘农9号和清水紫花苜蓿品种上群体共生效应相似,在陇中和WL168HQ紫花苜蓿上共生效应差异明显。本研究内生和非内生根瘤菌菌株的群体共生效应根据苜蓿品种而定,根瘤菌菌株与苜蓿品种间的信号识别程度存在差异。  相似文献   

10.
检测了四株大豆根瘤菌在不同的大豆品种上形成根瘤的放氢、吸氢、固氮活性及豆血红蛋白的含量;同时测定了植株干物质的积累。结果表明,所有固氮根瘤都放氢,自生条件下Hup~-根瘤菌形成的根瘤仍不具吸氢活性,相对固氮率在0.75左右。而Hup~(?)菌株根瘤的相对固氮率在0.91~1之间。寄主植物对Hup~(?)菌株的吸氢活性有影响。相关分析表明,根瘤的豆血红蛋白与吸氢活性呈负相关。干物质积累与固氮酶活性关系最密切,氢酶活性的影响是次要的。  相似文献   

11.
Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated. An R. japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R. japonicum strain SR139. Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen. All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically. Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules. Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation. When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments. Six E. coli transformants containing the nif/hup cosmids were conjugated with strain SR139. All strain SR139 transconjugants were Hup+ Nif+. Moreover, one nif/hup cosmid was transferred to 15 other R. japonicum Hup- mutants. Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup-. These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.  相似文献   

12.
The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup+) and hydrogenase negative (Hup) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N2-dependent or grown in the presence of 2 m M KNO3. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup strains accumulated more nitrite in the incubation medium than did those with Hup phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO3 and inoculated with single Hup+ and Hup R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup strains in the R. leguminosarum -pea symbiosis.  相似文献   

13.
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.  相似文献   

14.
The role of uptake hydrogenase was studied in Rhizobium leguminosarum bacteroids from the nodules of Pisum sativum L. cv. Homesteader. Uptake hydrogenase activity, measured by the 3H2 uptake method, was dependent on O-consumption and was similar to H2 uptake measured by gas chromatography. Km for O2 of 0.0007 atm (0.0709 kPa) and a Km for H2 of 0.0074 atm (0.7498, kPa) were determined. H2 increased the rate of endogenous respiration by isolates with uptake hydrogenase (Hup+) but had no effect on an isolate lacking uptake hydrogenase (Hup-). A survey of 14 Hup+ isolates indicated a wide range of H2 uptake activities. Four of the isolates tested had activities similar to or higher than those found in two Hup+ Rhizobium japonicum strains. H2 uptake was strongly coupled to ATP formation in only 5 of the 14 isolates. H2 increased the optimal O2 level of C2H2 reduction by 0.01 atm and permitted enhanced C2H2 reduction at O2 levels above the optimum in both a coupled and an uncoupled isolate. At suboptimal O2 concentrations a small enhancement of C2H2 reduction by H2 was seen in two out of three isolates in which H2 oxidation was coupled to ATP formation. Thus, the main function of uptake hydrogenase in R. leguminosarum appears to be in the protection of nitrogenase from O2 damage.  相似文献   

15.
Revertible hydrogen uptake-deficient mutants of Rhizobium japonicum.   总被引:21,自引:7,他引:14       下载免费PDF全文
We have developed mutants of Rhizobium japonicum which are deficient in H2 uptake capacity (Hup-) and which spontaneously revert to the parent type at a frequency consistent with that of a single-point mutation (ca. 1.0 x 10(-09)). The mutagenesis by nitrous acid and the selection of the Hup- phenotype by using penicillin and chemolithotrophy as enrichment for chemolithotrophy-deficient strains are described. Two mutants retain low but reproducible levels of ribulose bisphosphate-dependent CO2 fixation when grown on a low-carbon medium under an atmosphere of 1% O2, 4% H2, 5% CO2, and 90% N2. Neither O2 nor the artificial electron acceptors phenazine methosulfate or methylene blue supported detectable H2 uptake by the free-living Hup- mutants or by their bacteroids. Plant growth experiments under bacteriologically controlled conditions were conducted to assess the mutants' performance as inocula for soybean plants. Plants inoculated with Hup- strains had lower dry weights and contained less total N than did plants inoculated with the parent Hup+ strain. Use of either the Hup- mutants or the Hup+ parent strain as inocula, however, did not significantly affect the acetylene-reducing activity or the fresh weight of nodules. These results, obtained with apparently isogenic lines of H2 uptake-deficient R. japonicum, provide strong support for a beneficial role of the H2 uptake phenotype in legume symbiosis.  相似文献   

16.
Eight strains of Rhizobium lacking hydrogenase uptake (Hup) activity and 17 transconjugant strains carrying the hup cosmids pHU1, pHU52, or pHU53 (G. R. Lambert, M. A. Cantrell, F. J. Hanus, S. A. Russell, K. R. Haddad, and H. J. Evans, Proc. Natl. Acad. Sci. USA, 82:3232-3236, 1985) were screened for Hup activity and the presence of immunologically detectable hydrogenase polypeptides. Crude extracts of these strains were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with affinity-purified antibodies against the two subunits of purified hydrogenase (Mr 60,000 and 30,000). Derepressed transconjugants carrying the cosmid pHU52 were Hup+ and contained detectable levels of both hydrogenase subunit polypeptides. Non-derepressed strains, Hup- parent strains, and strains carrying cosmids other than pHU52 did not express Hup activity and contained no immunologically detectable protein. These data provide further evidence for the essential involvement of the smaller (Mr 30,000) subunit in the expression of hydrogenase activity in Rhizobium japonicum and suggest that the determinants for hydrogenase subunit synthesis are present on pHU52.  相似文献   

17.
18.
Initial observations showed a 100% increase in H2-uptake (Hup) activity of Rhizobium leguminosarum strain 3855 in pea root nodules (Pisum sativum L. cv Alaska) on plants growing in a baked clay substrate relative to those growing in vermiculite, and an investigation of nutrient factors responsible for the phenomenon was initiated. Significantly greater Hup activity was first measured in the clay-grown plants 24 days after germination, and higher activity was maintained relative to the vermiculite treatment until experiments were terminated at day 32. The increase in Hup activity was associated with a decrease in H2 evolution for plants with comparable rates of acetylene reduction. Analyses of the clay showed that it contained more Na+ (29 versus 9 milligrams per kilogram) and less K+ (6 versus 74 milligrams per kilogram) than the vermiculite. Analyses of plants, however, showed a large increase in Na+ concentration of clay-grown plants with a much smaller reduction in K+ concentration. In tests with the same organisms in a hydroponic system with controlled pH, 40 millimolar NaCl increased Hup activity more than 100% over plants grown in solutions lacking NaCl. Plants with increased Hup activity, however, did not have greater net carbon or total nitrogen assimilation. KCl treatments from 5 to 80 millimolar produced slight increased in Hup activity at 10 millimolar KCl, and tests with other salts in the hydroponic system indicated that only Na+ strongly promoted Hup activity. Treating vermiculite with 50 millimolar NaCl increased Na+ concentration in pea plant tissue and greatly promoted Hup activity of root nodules in a manner analogous to the original observation with the clay rooting medium. A wider generality of the phenomenon was suggested by demonstrating that exogenous Na+ increased Hup activity of other R. leguminosarum strains and promoted Hup activity of R. meliloti strain B300 in alfalfa (Medicago sativa L.).  相似文献   

19.
Peas (Pisum sativum L.) were inoculated with strains of Rhizobium leguminosarum having different levels of uptake hydrogenase (Hup) activity and were grown in sterile Leonard jars under controlled conditions. Rates of H2 evolution and acetylene reduction were determined for intact nodulated roots at intervals after the onset of darkness or after removal of the shoots. Hup activity was estimated using treatment plants or equivalent plants from the growth chamber, by measuring the uptake of H2 or 3H2 in the presence of acetylene. In all cases, the rate of H2 evolution was a continuous function of the rate of acetylene reduction. In symbioses with no demonstrable Hup activity, H2 evolution increased in direct proportion to acetylene reduction and the slopes were similar with the Hup strains NA502 and 128C79. Hup activity was similar in strains 128C30 and 128C52 but significantly lower in strain 128C54. With these strains, the slopes of the H2 evolution versus acetylene reduction curves initially increased with acetylene reduction, but became constant and similar to those for the Hup strains at high rates of acetylene reduction. On these parallel portions of the curves, the decreases in H2 evolution by Hup+ strains were similar in magnitude to their H2-saturated rates of Hup activity. The curvilinear relationship between H2 evolution and acetylene reduction for a representative Hup+ strain (128C52) was the same, regardless of the experimental conditions used to vary the nitrogenase activity.  相似文献   

20.
Thirteen Rhizobium leguminosarum strains previously reported as H(2)-uptake hydrogenase positive (Hup) or negative (Hup) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H(2) oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H(2)-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H(2)-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup strains in this symbiotic species.  相似文献   

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