含发光酶基因的转座子质粒载体的改造及向根瘤菌HN01的转座

将从自杀性重组质粒pDB30上分离的3．6kb含卡那霉素抗性基因和发光酶基因（luxAB）的BglⅡ酶切片段，克隆到小Mr的高拷贝质粒pIJ2925上，构成新的质粒，pHNLX1011；然后将pHNLX1011经BglⅡ部分酶切，酶连在含蔗糖酶基因（sacB）的自杀性重组质粒载体pMH1701上，载体分别采用BglⅡ酶切和BamHⅠ酶切．连接转化后共得到4种重组质粒类型的菌株，它们具有不同发光活性；将它们分别与快生型大豆根瘤菌HN01进行三亲本杂交，发现它们的转座频率均较pDB30高．对经转座标记后的根瘤菌进行发光活性检测，只有pHNC3质粒所转座的根瘤菌浇具有较强发光活性．所得发光根瘤菌衍生菌株能在野大豆和栽培大豆黑农39根际形成正常根瘤，能用X光片记录下根瘤的发光活性．     

干旱地区几种豆科植物根瘤菌的数值分类和SDS-PAGE全细胞蛋白电泳分析

从新疆、内蒙干旱地区苜蓿属、草木樨属、锦鸡儿属植物的根瘤中分离出５４株根瘤菌，对其中４８株根瘤菌和２２株参比菌一起进行了数值分类研究，在８５％的相似性水平上，未知菌分为３个不同于已知种的新群．另加入６株分离自锦鸡儿属植物的根瘤菌，共７６株菌进行了全细胞蛋白ＳＤＳＰＡＧＥ分析，在８０％的相似性水平上，已知菌相应成群，未知菌除ＸＪ９６３４２在６８％的相似性水平上和群５、６、７、８、Ｒ．ｇａｌｅｇａ聚群外，其余未知菌则分布在４、５、６、７、８、９、１０七个群中．群４中所有菌株属于数值分类的群１；群６中有３株菌属于数值分类的群３；群９中有８株菌为数值分类群２的菌株．反映了两种方法在分类上得到的结果比较近似．     

我国蚕豆根瘤菌的多样性和系统发育研究

采用数值分类、16S rDNA PCR-RFLP、IGS PCR-RFLP等方法对分离自我国11个省的50株蚕豆根瘤菌及11株参比菌株进行了表型测定和遗传型研究,同时对5株蚕豆根瘤菌的代表菌株进行了16S rDNA全序列测定.表型测定的结果表明,在80%的相似水平上供试菌株分为4个群,各群间存在地区交叉;16S rDNA PCR-RFLP的聚类结果与数值分类的聚类结果有很好的一致性;IGSRFLP反映的多样性更明显,形成的遗传群较多,可用于菌株间的鉴别.实验结果表明我国蚕豆根瘤菌具有极大的表型多样性和遗传多样性.系统发育研究结果表明,蚕豆根瘤菌的代表菌株均位于快生根瘤菌属(Rhizobium)系统发育分支,与R.leguminosarum USDA2370的全序列相似性达99.9%,说明蚕豆根瘤菌属于Rhizobium,系豌豆根瘤菌的一个生物型.图4表3参12     

中国根瘤菌

陈文新著科学出版社出版(2011年1月)ISBN 978-7-03-029789-1$108.00 16开本圆脊精装内容简介本书分为四篇:第一篇为概论,主要包括根瘤菌的特点、功能、起源与进化,分类历史与分类系统,根瘤菌与豆科植物共生分子机制,基因组学及后基因组学研究;第二篇为根瘤菌多样性及系统学的现代研究技术与方法;     

根瘤菌全细胞组分的红外光谱特征

利用红外光谱仪记录并分析了36株根瘤菌全细胞组分的红外光谱图，所得图谱分辨率高、重现性好，菌株之间的光谱特征和吸收带的位置、强度各不相同。尤其在指纹区（1700-650cm^-1），不同菌株的图谱有较为明显的差别，利用红外光谱技术可以进行根瘤菌的鉴别，菌株水平的差异能充分表现出来，红外光谱分析在一定程度上可反映根瘤菌的分类地位，但实际应用起来，仍有一定的困难。     

花生根瘤菌的生长特性和脂肪酸组成研究(英文)

用73株慢生根瘤菌B．Japonicum、B．elkanii和B．"intermedium"为对照．对22株花生根瘤菌的生长速度、产碱能力和细胞脂肪酸组成进行了测定．结果表明：花生根瘤菌属于慢生根瘤菌属（Bradyrhizobiumsp．arachis）；除花生根瘤菌含有比大豆根瘤菌（B．japonicum）较高的脂肪酸16：1w5c（16个碳原子1个双键的脂肪酸）而外，花生根瘤菌与大豆根疚菌相似，表明了他们在系统发育及进化方向上的一致性．细胞脂肪酸分析法是根瘤菌系统发育研究中一种简便而可信的方法．     

内蒙古中东部地区几种豆科牧草根瘤菌的表型多样性及水分梯度对其分布的影响

选用101株分离自扁蓿豆、胡枝子、野豌豆、锦鸡儿属植物的根瘤菌菌株，进行了营养利用、抗生素抗性、耐逆性和酶活性测定等84个表型性状分析，发现分离自同种寄主植物的根瘤菌由于地理来源的不相同而存在着较大的多样性．通过数值分类，各已知种分别聚群，101株未知菌在84％的相似水平上分为4个各不同的群．根据1、2、3、4群菌的聚类情况，结合在不同水分梯度下采集的豆科牧草根瘤菌，结果发现除个别菌外，绝大部分菌株呈现随着水分梯度的不同，各菌以类群形式分布．证明土壤中水分含量对根瘤菌的分布有很大影响．     

花生根瘤菌的生长特性和脂肪酯组成研究

用７３株慢生根瘤菌Ｂ．Ｊａｐｏｎｉｃｕｍ，Ｂ．ｅｌｋａｎｉｉ和Ｂ．“ｉｎｔｅｒｍｅｄｉｕｍ”为对照，对２２株花生根瘤菌的生长速度，产碱能力和细胞脂肪酸组成进行了测定，结果表明，花生根瘤菌慢生根瘤菌属（Ｂｒａｄｙｒｈｉｚｏｂｉｕｍｓｐ．ａｒａｃｈｉｓ），除花生根瘤菌含有比大豆根瘤菌（Ｂ．ｊａｐｏｎｉｃｕｍ）较高的脂肪酸，１６：１ｗ５ｃ（１６个碳原子１个双键的脂肪酸）而外，花生根瘤菌与大豆根瘤菌相似，     

四川攀西干热河谷区根瘤菌的多样性研究

采用数值分类、BOXAIR-PCR和16S rDNA PCR-RFLP方法,对分离自四川攀西干热河谷区11种豆科植物上的43株根瘤菌的表型和遗传多样性进行了研究.数值分类结果表明,攀西地区根瘤菌具有极大的表型性状多样性,在80%相似性水平上绝大多数供试菌株单独成群,能耐60℃(10min处理)的高温,在低温(10℃)或者低pH(4.0)条件下生长很差.与数值分类结果相比较,BOXAIR-PCR的分群结果更分散,说明供试菌株在遗传性状上也具有多样性.在数值分群基础上,选取15个代表菌株进行16S rDNA PCR-RFLP分析.结果表明,供试的15株代表菌株遗传差异明显,仅CCBAU61224与Rhizobium leguminosarum USDA2370T,CCBAU61303与Agrobacterium tumerfacienseIAM13129T具有相同的遗传图谱类型.数值分类和16S rDNA PCR-RFLP分析具有较好的一致性,BOXAIR-PCR适合于对根瘤菌种内菌株间的遗传多样性研究.图4表2参21     

慢生型大豆根瘤菌的遗传多样性研究

采用选择性扩增片断长度多态性 (简称AFLP)DNA指纹技术对来自泰国北部的 2 45株慢生型大豆根瘤菌进行遗传多样性的研究 .通过AFLP图谱揭示出该地区的慢生型大豆根瘤菌有较显著的遗传多样性 .从 2 45株中选择出 92个代表株用计算机进行的树状图分析结果表明 ,所分析的菌株在 82 %的相似性水平上聚类成 8个群 .对这 92个代表株的部分菌株和慢生型大豆根瘤菌的参比菌株进行多聚酶链反应 (PCR)扩增的 16SrDNA的 4种限制性内切酶长度多态 (简称 16SrDNAPCR RFLP)分析 ,得出 2个不同的 16SrDNAPCR RFLP类型的菌株 ,分别与慢生型大豆根瘤菌的参比菌株Bradyrhizobiumjaponicum和Bradyrhizobiumelkanii相同 .图 1表 2参 14     

根瘤菌菌体及胞外多糖红外光谱特征

记录并分析了根瘤菌４个新类群菌株的菌体与胞外多糖的红外光谱,表明它们在特定光谱区显示特征吸收峰．根瘤菌菌体光谱特征在鉴定上具有一定意义;胞外多糖的结构为β－配糖键连接,其光谱特征与数值分类结果关系不明显     

根瘤菌的遗传多样性与系统发育研究进展

根瘤菌 (ｒｈｉｚｏｂｉａ)是一类广泛分布于土壤中的革兰氏阴性细菌。它可以侵染豆科植物根部或茎部形成根瘤和少数茎瘤 ,固定空气中的分子态氮形成氨 ,为植物提供氮素营养 .据估计 ,全球每年生物固氮量达 1.75× 10 8ｔ,为世界工业氮肥产量的4 .37倍[1] .根瘤菌与豆科植物的共生固氮作用是生物固氮中固氮效率最高的体系 .因此 ,根瘤菌与豆科植物的共生固氮作用是实现农业可持续发展的重大研究课题之一 .研究根瘤菌的遗传多样性 ,对于发掘新的根瘤菌种质资源 ,保藏并合理利用根瘤菌基因资源库 ,选育高效菌株直接应用于农业生产有重要意义 .…     

兰坪铅锌尾矿区豆科植物根瘤菌的多样性及抗性研究

对兰坪铅锌尾矿区自然发生的植物进行调查,共发现18种植物,分属于10科。对野豇豆（Vigna vexiauata（Linn.）Bench.）、长波叶山蚂蝗（Desmodium sequax Wall.）、三叶草（Trifolium pratense Linn.）、白刺花（Sophora davii（franch.）Skeels）、密花序黄芪（Astragalus forrestii Simpson.）、美丽胡枝子（Lespedeza Formosa（Vog.）Koehne.）6种8株豆科植物根瘤进行根瘤菌的分离纯化,共得到419株细菌,经镜检初步确定有94株为根瘤菌。对其中31株根瘤菌的淀粉水解、糖发酵等12项生理生化指标进行测定,并以平均连锁法进行聚类分析。结果表明,兰坪铅锌尾矿区31株根瘤菌在50%的相似水平上可分为6个类群,其表型特性存在明显差异,同一种植物中可能存在不同代谢类型的根瘤菌,同一种代谢类型的根瘤菌也可与不同植物共生。抗逆性测定结果表明,菌株（4G.7、5.4.3、7.4.3）对温度、（4A.12、4G.7、4G.10、5.4.3）对pH、（3.7.9、5.4.3）对盐、（3.7.19、4A.12、6.1.2、6.1.13）对抗生素有较强耐受性或抗性,大多数菌株能在较宽的温度、盐度和pH范围内生长。     

潍坊、花园口土样中土著大豆根瘤菌的遗传多样性研究

采用１６Ｓ－２３Ｓ ｒＤＮＡ间隔区段（ＩＧＳ）ＲＦＬＰ分析和ＰＡＰＤ分析技术，分别对分离自山东潍坊和河南郑州花园口的２４株土著大豆根瘤菌进行２多样性分析。结果一 ７０％的相似性水平上，依ＩＧＳ－ＲＦＬＰ分析可将供试菌株分为１０群，依ＲＡＰＤ分析可将供试菌株分为８群。综合两种分析技术在５０５的相似性水平上将供试菌株分为潍坊群和花园口群。 在系统发育和分子进行上有明显的地理分隔作用。     

由一株青霉菌产生的聚乙烯醇降解酶

从纺织污水活性污泥中筛选得到一株新型聚乙烯醇(PVA)降解酶产生菌,根据形态学特征鉴定该菌属于青霉属(Penicillium sp.),实验室编号WStt02-21.这是由霉菌产生PVA降解酶的首例报道.在考察了菌株WSH02-21基本生长和产酶特性的基础上,研究了营养条件对PVA降解酶合成的影响.通过对营养条件的单因素考察和正交试验,确定了最优培养条件为PVA 40 g L-1、葡萄糖3.0 g L-1、NH4Cl 8.0 g L-1、KH2PO4 2.0 g L-1、酵母膏1.0 g L-1、:MgSO40.5 g L-1。、CaCl2 1.0 g L-1、NaCl 0.02 g L-1、FeSO4·7H2O 0.02 g L-1,初始pH 6.4.其中PVA浓度是影响Penicilliumsp.WSH02-21合成PVA降解酶的最重要因素.采用最优化条件进行验证试验,PVA降解酶酶活(4.4 U mL-1)略高于正交试验中的的最高酶活(4.3 U mL-1).图7表2参11     

Hydrogen production by nitrogenase as a potential crop rotation benefit

Both climate change and the adverse effects of chemical use on human and environmental health are recognized as serious issues of global concern. Nowhere is this more apparent than in the agricultural sector where release of greenhouse gases such as carbon dioxide, nitrous oxide and methane continues to be problematic and where use of nitrogen fertilizer is responsible for negative impacts on both human populations and ecosystems. The manipulation of biological nitrogen fixation (BNF) could help alleviate part of the difficulty by decreasing the need for nitrogen fertilizers, which require huge quantities of fossil fuel to produce and contribute to the release of nitrous oxide from soil as well as being responsible for the contamination of drinking water systems and natural habitats. BNF is performed by a variety of microorganisms. One of the most studied examples is the BNF carried out by rhizobial bacteria in symbiosis with their plant hosts such as pea and soybean. Hydrogen gas is an energy-rich, obligate by-product of BNF. Legume symbioses with rhizobia lacking hydrogenase enzymes (which can recycle hydrogen) have traditionally been viewed as energetically inefficient. However, recent studies suggest hydrogen release to soil may be beneficial, increasing soil carbon sequestration and promoting growth of hydrogen-oxidizing bacteria beneficial to plant growth; the alleged superiority of symbiotic performance in rhizobia possessing functional hydrogenases (HUP⁺) over those rhizobia without functional hydrogenases (HUP⁻) has also not been conclusively shown. The structure of the iron-molybdenum cofactor or FeMo-co of nitrogenase (the active site of the enzyme) has been elucidated through X-ray crystallography but the mechanism of nitrogen fixation remains unknown. However, studies of effects of hydrogen production on BNF have revealed potential candidate intermediates involved in the nitrogenase reaction pathway and have also shown the role of hydrogen as a competitive inhibitor of N₂, with hydrogen now considered to be the primary regulator of the nitrogenase electron allocation coefficient. The regulation of oxygen levels within legume root nodules is also being investigated; nitrogen fixation is energetically expensive, requiring a plentiful oxygen supply but too high an oxygen concentration can irreversibly damage nitrogenase, so some regulation is needed. There is evidence from gas diffusion studies suggesting the presence of a diffusion barrier in nodules; leghaemoglobin is another potential O₂ regulator. Possible functions of hydrogenases include hydrogen recycling, protection of nitrogenase from damaging O₂ levels and prevention of inhibitory H₂ accumulation; there is evidence for H₂ recycling only in studies where H₂ uptake has been strongly coupled to ATP production and where this is not the case, it is believed that the hydrogenase acts as an O₂ scavenger, lowering O₂ concentrations. The distribution of hydrogenases in temperate legumes has been found to be narrow and root and shoot grafting experiments suggest the host plant may exert some influence on the expression of hydrogenase (HUP) genes in rhizobia that possess them. Many still believe that HUP⁺ rhizobia are superior in performance to HUP⁻ species; to this end, many attempts to increase the relative efficiency of nitrogenase through the introduction of HUP genes into the plasmids or chromosomes of HUP⁻ rhizobia have been carried out and some have met with success but many other studies have not revealed an increase in symbiotic performance after successful insertion of HUP genes so the role of HUP in increasing parameters such as N₂ fixation and plant yield is still unclear. One advantage of the hydrogen production innate to BNF is that the H₂ evolved can be used to measure N₂ fixation using new open-flow gas chamber techniques seen as superior to the traditional acetylene reduction assay (ARA) conducted in closed chambers, although H₂ cannot be used for field studies yet as the ARA can. However, the ARA is now believed to be unreliable in field studies and it is recommended that other measures such as dry weight, yield and total nitrogen content are more accurate, especially in determining real food production, particularly in the developing nations. Another potential benefit of H₂ release from root nodules is that it stays in the soil and has been found to be consumed by H₂-oxidizing bacteria, many of which show plant growth–promoting properties such as the inhibition of ethylene biosynthesis in the host plant, leading to root elongation and increased plant growth; they may well be promising as biofertilizers if they can be successfully developed into seed inoculants for non-leguminous crop species, decreasing the need for chemical fertilizers. It has been suggested that rhizobia can produce nitrous oxide through denitrification but this has never been shown; it is possible that hydrogen release may provide more ideal conditions for denitrifying, free-living bacteria and so increase production of nitrous oxide that way and this issue will require more study. However, it seems unlikely that a natural system would release nitrous oxide to the same degree that chemical fertilizers have been shown to do.     

The Effects of Selective Logging on Inbreeding in Shorea megistophylla (Dipterocarpaceae) from Sri Lanka

The mating system of Shorea megistophylla , an endemic canopy tree from Sri Lanka, was quantified by allozyme analysis of progeny arrays using a mixed-mating model. Two adjacent populations were compared: one in forest that was selectively logged about 20 years ago, and the other in undisturbed primary forest. The selectively logged population had a lower multilocus outcrossing rate (t_m, = 0.71) compared to that of the undisturbed forest (t_m = 0.87). Only the progeny from the logged population showed evidence of either biparental inbreeding or Wablund effect, and the genotypic frequencies violated the assumptions of the mixed-mating model. Apomixis was detected in one isolated tree in the Royal Botanical Garden in Peradeniya by a multilocus test of progeny genotypic frequencies relative to the maternal genotype. However, significant levels of apomixis were not discerned in the natural (logged and unlogged) populations. These findings indicate that a reduction in population density of S. megistophylla following selective logging can significantly elevate the proportion of seeds produced through inbreeding. Adventive embryony may also increase in isolated trees that lack the chance to outcross.     

铜暴露对赤子爱胜蚓(Eisenia fetida)抗氧化酶活力的影响

通过溶液接触法和人工土壤法,研究了不同暴露环境中Cu对赤子爱胜蚓(Eisenia fetida)SOD酶、CAT酶和GST酶活力的影响.结果显示:溶液接触试验中, Cu显著抑制蚯蚓SOD酶活力(P<0.05),人工土壤试验中Cu则诱导SOD酶活力增加,预示暴露条件影响蚯蚓SOD酶对Cu的生物响应.人工土壤试验中, Cu暴露浓度与蚯蚓CAT酶和GST酶活力的相关系数分别为0.814和0.933,均存在极显著的剂量效应关系(P<0.001).在这两种暴露环境下,赤子爱胜蚓的CAT酶和GST酶活力对Cu浓度响应敏感.