用AFLP技术检测慢生型花生根瘤菌竞争结瘤的研究

以 5株慢生型花生根瘤菌和天府 3号花生为材料 ,用 AFLP技术研究了慢生型花生根瘤菌 Spr2 - 9、Spr3- 3、Spr3- 5、Spr4- 5和 Spr7- 1的遗传特性和竞争结瘤能力。结果显示 ,供试条件下 ,传代次数对菌株的遗传性状无明显影响 ,2 8℃培养条件下 ,花生根瘤菌连续传 96代 ,其 AFLP指纹未发生明显变化 ;37℃培养 ,仅 Spr3- 3和 Spr3- 5能够存活并正常生长 ,其 AFLP指纹也未发生明显改变 ,然而其它菌株不能生长。将供试慢生型花生根瘤菌分别接种天府 3号花生 ,光照培养 30 d后 ,随机各取 4个根瘤 ,从根瘤中提取类菌体 DNA进行 AFLP分析 ,各根瘤类菌体 DNA的 AFLP指纹图谱与该菌株纯培养物 AFLP指纹相同。将 5个菌株混合接种天府 3号花生 ,不同菌株的占瘤率存在差异 ,Spr3- 3和 Spr3- 5的竞争结瘤能力最强 ,两菌株的占瘤率之和为 85.4% ;Spr4- 5的占瘤率为 1 2 .2 % ;Spr7- 1为 2 .4% ;而 Spr2 - 9的竞争结瘤能力最差。本试验结果说明 ,AFLP技术用于根瘤菌生态和竞争结瘤能力研究 ,具有下列优点 :简易、快速、准确 ;直接取豆科植物的根瘤提取 DNA,进行原位研究 ;在不改变菌株遗传特性 ,即不使用突变株的前提下 ,可以直接测定已知菌株的竞争结瘤能力     

用AFLP技术检测慢生型花生根瘤茵竞争结瘤的研究

以5株慢生型花生根瘤菌和天府3号花生为材料,用AFLP技术研究了慢生型花生根瘤菌Spr2—9、Spr3—3、Spr3—5、Spr4—5和Spr7—1的遗传特性和竞争结瘤能力。结果显示,供试条件下,传代次数对菌株的遗传性状无明显影响,28C培养条件下,花生根瘤菌连续传96代,其AFLP指纹未发生明显变化;37C培养,仅Spr3—3和Spr3—5能够存活并正常生长,其AFLP指纹也未发生明显改变,然而其它菌株不能生长。将供试慢生型花生根瘤菌分别接种天府3号花生。光照培养30d后,随机各取4个根瘤,从根瘤中提取类菌体DNA进行AFLP分析,各根瘤类菌体DNA的AFLP指纹图谱与该菌株纯培养物AFLP指纹相同。将5个菌株混合接种天府3号花生,不同菌株的占瘤率存在差异,Spr3—3和Spr3—5的竞争结瘤能力最强,两菌株的占瘤率之和为85．4％;Spr4—5的占瘤率为12．2％;Spr7—1为2．4％;而Spr2—9的竞争结瘤能力最差。本试验结果说明,AFLP技术用于根瘤菌生态和竞争结瘤能力研究,具有下列优点：简易、快速、准确;直接取豆科植物的根瘤提取DNA,进行原位研究;在不改变菌株遗传特性,即不使用突变株的前提下,可以直接测定已知菌株的竞争结瘤能力。     

竹黄菌基因组RAPD分子标记体系的建立

目的：建立随机引物扩增多态性DNA（RAPD）分子标记体系,为研究华东地区不同地理居群竹黄菌Shiraia bambusicola的遗传多样性奠定基础。方法：应用SDS法提取竹黄菌基因组DNA,并优化影响RAPD反应的条件因素。结果：竹黄RAPD的最佳反应体系为：25μlPCR反应体积,10×PCR缓冲液2.5μl,1.0UTaq酶,50ng模板DNA,2mmol/LMg2＋,0.2mmol/LdNTPs,0.4μmol/L随机引物。PCR循环程序为：94℃预变性4min,然后,94℃变性1min,38℃退火70s,72℃延伸90s,40次循环,最后72℃延伸6min,12℃保存。结论：建立了竹黄菌Shiraia bambusicola的最佳RAPD分子标记体系,可获得良好的竹黄菌RAPD指纹图谱。     

玉米花粉粒直接PCR技术研究

利用成熟花粉粒制成悬液作为玉米基因组DNA模板直接进行PCR扩散,研究了不同花粉悬液浓度、花粉上清液对RAPD-PCR的影响,结果表明：花粉悬液浓度在4μg/50μl以上,用10碱基随机引物均能扩增出较清晰的PCR条带,且与叶片按改良Guidet法提取的DNA模板扩增的RAPD带型无明显差异,利用花粉悬液能有效地进行基因组DNA变异的RAPD分析和基因SCAR标记的检测。玉米单株花粉量可用于数百次以上的PCR反应,较叶片等植物组织用常规法提取DNA快速、简便、廉价,可有效地应用于以PCR为基础的植物基因组DNA变异,农艺性状的RAPD标记和分子标记辅助育种的研究。     

小麦随机扩增多态性DNA的反应体系探索

随机扩增多态性DNA(RAPD)是近年来发展起来的一种DNA多态性检测技术。通过以小麦基因组DNA为模板 ,对RAPD反应程序中的一些重要参数进行摸索和优化试验 ,建立了随机扩增多态性DNA分析应用反应体系。即反应体积为 2 5μ1,Mg2 浓度为 2 0mM ,dNTP浓度为 150 μM ,模板DNA量 5ng～2 5ng ,随机引物量 30ng～ 4 5ng ,Tap酶 1单位 ;反应过程为 90℃变性 30s ,38℃结合 1min ,72℃延伸 2min(最后一个循环 10min) ,共 4 0个循环。该体系可有效地应用在小麦遗传育种研究中 ,重复性好 ,可靠性高。     

杨梅RAPD-PCR体系的正交优化研究

以杨梅DNA为模板,对影响杨梅RAPD-PCR扩增的重要参数进行了优化试验,以期建立杨梅RAPD反应的最佳体系。通过采用正交试验设计的方法,对杨梅RAPD-PCR条件进行了优化,结果表明最佳的杨梅RAPD-PCR的反应体系(20μl)中含有1×buffer,1.0U TaqDNA聚合酶,3.0mmol/L MgCl2,0.30mmol/L dNTPs,1.5μmol/L引物和模板DNA 30-40ng。适宜的扩增条件为94℃预变性3min,再进入38个PCR循环(94℃变性30s,38℃退火30s,72℃延伸90s),72℃延伸7min,4℃保存。     

水稻幼苗单株DNA的提取及其PCR—RAPD反应体系的建立

以杂交稻Ⅱ代63幼苗为DNA提取材料,建立了以所提取单株DNA为模板进行PCR－RAPD分析的最佳条件,获得稳定而理想的PCR扩增效果。     

应用GUS基因研究弗氏中华根瘤菌的结瘤及效果

应用GUS(葡萄糖苷酶 )基因标记技术将标记基因GUS导入受体菌S .fredii8855,标记菌株形成的根瘤可被GUS染色缓冲液染成蓝色 ,而土著菌形成的根瘤不能着色 .由此即可十分简便地确定土著菌的影响程度 .盆栽试验表明 ,S .fredii 8855的结瘤抗酸碱能力高于土著菌 ,能在土壤中较大范围内迁移 .当它的根瘤占有率不小于 43%时 ,接种能显著提高大豆产量 ,大豆产量与根瘤占有率呈正相关 (r=0 .98) ,而与总瘤数关系不大 (r=0 .1 3) .土壤N素显著抑制其结瘤 ,补加P能缓解这种抑制作用 .     

大肠杆菌ubiA基因的克隆及序列测定

以E.coli JM83染色体DNA为模板PCR扩增ubiA的结构基因,将PCR产物克隆于pUCm-T Vector,对PCR产物进行测序鉴定。与已发表的大肠杆菌ubiA基因序列比较,同源性达99％。     

分离自杭子梢等宿主根瘤的30株菌的结瘤特性的研究

对分离自杭子梢、菜豆和决明等宿主根瘤、处于Agrobacterium系统发育分支、DNA-DNA杂交与A.rubi的相似性达到100%的30株土壤杆菌,分属于Agrobacterium、Bradyrhizobium、Mesorhizobium、Rhizobium和Sinorhizobium 5个属的12个参比菌株。nodA PCR的结果表明,30株供试菌中扩增不出nodA,即没有结瘤性。以Sinorhizobium meliloti USDA1002T的nodA做探针对所提取的细菌总DNA进行斑点杂交,在65℃-68℃严谨洗膜条件下,该探针只能与同种的根瘤菌进行杂交,不能与其它属的根瘤菌或土壤杆菌杂交,初步推测共同结瘤基因nodA探针只能对种内根瘤菌的结瘤性进行鉴定。     

Mixed Inoculations with Effective and Ineffective Strains of Rhizobium leguminosarum

An ineffective Rhizobium leguminosarum strain capable of forming green nodules of similar size and number as normally effective strains was tested for its ability to compete with an effective strain in nodule formation on the pea. The ineffective strain was found to be more competitive and influenced the pattern of nodulation by the effective strain on the same root system. Nodules containing both strains were pink and able to reduce acetylene.     

Rhizobium cauense sp. nov., isolated from root nodules of the herbaceous legume Kummerowia stipulacea grown in campus lawn soil

Three bacterial isolates (CCBAU 101002^T, CCBAU 101000 and CCBAU 101001) originating from root nodules of the herbaceous legume Kummerowia stipulacea grown in the campus lawn of China Agricultural University were characterized with a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that the isolates shared 99.85–99.92% sequence similarities and had the highest similarities to the type strains of Rhizobium mesoamericanum (99.31%), R. endophyticum (98.54%), R. tibeticum (98.38%) and R. grahamii (98.23%). Sequence similarity of four concatenated housekeeping genes (atpD, glnII, recA and rpoB) between CCBAU 101002^T and its closest neighbor (R. grahamii) was 92.05%. DNA–DNA hybridization values between strain CCBAU 101002^T and the four type strains of the most closely related Rhizobium species were less than 28.4 ± 0.8%. The G + C mol% of the genomic DNA for strain CCBAU 101002^T was 58.5% (Tm). The major respiratory quinone was ubiquinone (Q-10). Summed feature 8 (18:1ω7cis/18:1ω6cis) and 16:0 were the predominant fatty acids. Strain CCBAU 101002^T contained phosphatidylcholine and phosphatidylethanolamine as major polar lipids, and phosphatidylglycerol and cardiolipin as minor ones. No glycolipid was detected. Unlike other strains, this novel species could utilize dulcite or sodium pyruvate as sole carbon sources and it was resistant to 2% (w/v) NaCl. On the basis of the polyphasic study, a new species Rhizobium cauense sp. nov. is proposed, with CCBAU 101002^T (=LMG 26832^T = HAMBI 3288^T) as the type strain.     

Endophytic Occupation of Root Nodules and Roots of Melilotus dentatus by Agrobacterium tumefaciens

Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129^T and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules.     

Competition studies with Rhizobium trifolii in a field experiment

M c L oughlin , T.J. B ordeleau L.M. & D unican , L.K. 1984. Competition studies with Rhizobium trifolii in a field experiment. Journal of Applied Bacteriology 56 , 131–135.
Competition studies were carried out in a field experiment by comparing the ability of antibiotic marked Rhizobium strains inoculated at three inoculum levels in forming nodules against an effective indigenous Rhizobium population of 1.5 times 10⁵/g. Three inoculum strains were assessed. It was seen that by increasing the level of inoculum there was a corresponding increase in the number of nodules formed by the introduced strains. G1067 formed a high proportion (85%) at the highest level, whereas the other two inoculum strains occupied less than 50% of the nodules at the same level. Persistence of the inoculum strain was measured into the second season; G1032 could not be detected, G1006 (another 'foreign' strain) formed 43% and G1067 formed 97% of the nodules sampled. The indigenous Rhizobium population isolated from nodules was shown to be heterogenous, and 15 different intrinsic antibiotic resistance patterns were found. Fifty per cent of the isolates fell in the same group which showed a similar pattern to that of the inoculum strain G1067.     

Danish Rhizobium leguminosarum strains nodulating 'Afghanistan' pea (Pisum sativum)

A wild pea ( Pisum sativum L.) native to Afghanistan normally known to be resistant to nodulation with European strains of Rhizobium leguminosarum was nodulated early and effectively in field soil in Denmark. Isolates from nodules formed effective nodules abundantly on 'Afghanistan' on reinfection under aseptic conditions. Five types differing in isoenzyme composition pattern were found among 15 isolates from 'Afghanistan' nodules. None were identical with the 'Tom' strain from Turkey, which also forms effective nodules with 'Afghanistan'. The five types were also different with respect to isoenzyme pattern from Rhizobium leguminosarum strains isolated from a modern pea variety cultivated in the same field.     

我国豇豆和绿豆根瘤菌的数值分类及16S rDNA PCR-RFLP研究

对分离自中国14个不同省(自治区)的79株豇豆和绿豆根瘤菌及12株参比菌株进行了唯一碳、氮源利用,抗生素抗性,抗逆性和酶活性等128个表型性状的测定,并用MINTS软件进行聚类分析。表型性状测定结果发现,所有菌株都有极其广泛的碳、氮源利用谱,大多数菌株可在较宽的pH(pH5·0~11·0)值范围内生长,大部分菌株能在37℃高温条件下生长,个别菌株能耐受60℃高温较长时间(20~45min)的热激。聚类分析结果表明,全部供试菌株在63·5%的相似性水平上分为两大群:一个群为慢生菌群,另一群为快生和中慢生菌群;在79%的相似性水平上分为7个亚群。在数值分类的基础上,又将参比菌株增加到22株,对79株待测菌株进行了16SrDNAPCR-RFLP分析,16SrDNAPCR产物经HaeⅢ、HinfⅠ、MspⅠ和AluⅠ4种内切酶酶切共产生34种遗传图谱类型,经GelComparⅡ软件聚类后,在79%的相似性水平上也可划分为7个亚群,与数值分类的结果有很好的一致性。     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

Transfer of the symbiotic plasmid from Rhizobium leguminosarum biovar trifolii to Agrobacterium tumefaciens

This study examined the symbiotic properties of Agrobacterium transconjugants isolated by transferring a Tn5-mob-marked derivative of the 315 kb megaplasmid pRt4Sa from Rhizobium leguminosarum bv. trifolii 4S (wild-type strain) to Agrobacterium tumefaciens A136 as the recipient. The genetic characteristics of the AT4S transconjugant strains were ascertained by random amplified polymorphic DNA (RAPD) analyses and Southern hybridization using Tn5-mob and nod genes as probes. Several of these AT4S transconjugants carrying pRt4Sa were able to nodulate roots of the normal legume host, white clover. In addition, some AT4S transconjugant strains were able to induce nodules on other leguminous plants, including alfalfa and hairy vetch. A characteristic bacteroid differentiation was observed in clover and alfalfa nodules induced by the AT4S-series strains, although nitrogen-fixing activity (acetylene reduction) was not found. Furthermore, strain H1R1, obtained by retracing transfer of the pRt4Sa::Tn5-mob from strain AT4Sa to strain H1 (pRt4Sa cured derivative of 4S), induced Fix(+) nodules on clover roots. These results indicate the evidence that only nod genes can be expressed in the Agrobacterium background.     

Gene centres,a source for genetic variants in symbiotic nitrogen fixation: The symbiotic response of the cultivated pea toRhizobium leguminosarum strains from Europe and the Middle East

Summary Soil samples from several European countries; Sweden, the Netherlands, Spain, Italy and Greece, contained rhizobial populations capable of forming an effective symbiosis with the cultivated pea cv. Rondo from the Netherlands. The range of variation among the European Rhizobium strains, as expressed on pea cv. Rondo, was not so large and almost the same variation could be found within the rhizobial population within each country. Superior Rhizobium strains for the Dutch pea were not restricted to soils from the Netherlands but were also found in those from Sweden and Italy.Soils from Turkey and Israel also contained Rhizobium strains capable of nodulating pea cv. Rondo. However, the genetic variation among these Middle East Rhizobium strains was much larger than that of the European strains. When tested on pea cv. Rondo the majority of the Middle East strains belonged to the medium or low effective classes and only a few strains were comparable with European Rhizobium strains.Dutch Rhizobium strains induced effective nodules on both the Dutch pea cv. Rondo and the Swedish cv. L 110. However, in association with a Turkish Rhizobium strain effective nodules were formed on pea cv. Rondo and ineffective nodules on cv. L 110.We suggest that the genetic uniformity of EuropeanR. leguminosarum strains is the result of selection and domestication of Rhizobium strains originally derived from the gene centres of the pea plant.