菜豆根瘤菌Rhizobium etli CFN42全基因组含信号肽分泌蛋白的鉴定与分析

利用菜豆根瘤菌Rhizobium etli CFN42全基因组测序结果,结合计算机技术和生物信息学分析方法,对菜豆根瘤菌蛋白质组中的分泌蛋白进行预测和分析,以便更深入地了解共生固氮的分子机制。经过分析发现,在该根瘤菌5 963个蛋白质中,具有信号肽的分泌蛋白332个,占总数的5.4%主要涉及底物转运、物质代谢和信号转导等方面。在有功能描述的186个分泌蛋白中,识别了Ⅰ、Ⅱ、Ⅲ、Ⅳ型分泌系统和丝氨酸蛋白酶,这些蛋白质可能参与了根瘤菌对宿主植物的侵染,是两者分子对话的关键参与者;在假定蛋白中,通过结构域分析,发现了SH3b、BA14K和EFh结构域,含有这些结构域的分泌蛋白可能参与了根瘤信号转导途径。在根瘤菌共生结瘤过程中,分泌蛋白发挥了重要作用。     

Analysis of Sec-type Signal Peptides in the Ti and AT Plasmids of Agrobacterium tumefaciens C58 Cereon

利用Signal P3.0和PrediSi软件结合L值和TMHMM筛选，对根癌农杆菌(Agrobacterium tumefaciens )C58 Cereon菌株的Ti和AT质粒所编码的745个ORF编码蛋白中的信号肽进行了预测，发现35条为分泌型蛋白，其中6条属于RR-motif型信号肽，2条蛋白的信号肽为细菌素-信息素型信号肽，新注释了24条分泌型信号肽，其中1条来自Ti质粒，23条来自AT质粒的蛋白。这些信号肽N结构域的长度变化为2~19 aa,平均为6.8 aa，H结构域的长度变化为8~34 aa，平均为17.1 aa。     

十字花科黑腐病菌分泌蛋白表达谱分析

本文旨在分析Xcc8004菌株分泌蛋白的蛋白表达谱,建立Xcc8004分泌谱分析方法。首先将诱导的Xcc8004分泌蛋白进行液体酶解,通过HPLC阳离子交换柱分离多肽,采用EASY-nLC结合Orbitrap质谱鉴定分泌谱。经过过滤筛选后,共精确鉴定蛋白1532个,SignalP分析其中包含286个含信号肽且不含跨膜区,其中118个为假定蛋白。对286个可能的分泌蛋白进行基因本体评注分析,其中,主要为行驶水解酶类,受体蛋白,信号传导等重要的微生物-植物互作介导蛋白。本研究通过液相色谱-质谱联用能够高效鉴定Xcc8004的分泌蛋白,对这些鉴定的分泌蛋白的信息学分析有利于我们进一步研究Xcc8004的致病机制,为研究微生物植物相互作用鉴定了方法与理论基础。     

巴斯德毕赤酵母新型分泌表达载体构建

巴斯德毕赤酵母分泌表达载体pPICINU是利用来源于克鲁维酵母（Kluyveromyces marxianus)的菊粉酶基因信号肽DNA序列（ISP）构建的。表达实验结果表明，带有分泌表达载体pPICINU的β-1，3－1，4葡聚糖酶基因重组菌，具有与带有表达载体pPIC9K(带有α因子信号肽）的β-1，3－1，4葡聚糖酶基因重组菌相同的分泌效率。     

根癌农杆菌Ti和AT质粒基因组中的分泌型信号肽分析

利用SignalP3.0和PrediSi软件结合L值和TMHMM筛选,对根癌农杆菌(Agrobacteriumtumefaciens)C58Cereon菌株的Ti和AT质粒所编码的745个ORF编码蛋白中的信号肽进行了预测,发现35条为分泌型蛋白,其中6条属于RR-motif型信号肽,2条蛋白的信号肽为细菌素-信息素型信号肽,新注释了24条分泌型信号肽,其中1条来自Ti质粒,23条来自AT质粒的蛋白。这些信号肽N结构域的长度变化为2￣19aa,平均为6.8aa,H结构域的长度变化为8￣34aa,平均为17.1aa。     

一株水稻内生菌久留里副伯克霍尔德(Paraburkholderia kururiensis)的分离鉴定及促生功能的评价

从水稻根中分离出的内生细菌 HMC50通过 16S rRNA基因和全基因组序列分析鉴定为久留里副伯克霍尔德菌( Paraburkholderia kururiensiss)。在基因组水平预测到该菌株具有与促进植物生长、鞭毛介导的运动性和解毒作用相关的主要特征,发现与固氮相关的 18个基因中包括关键基因 nifH以及色氨酸合酶α链(EC4.2.1.20)基因和色氨酸合酶β链(EC4.2.1.20)基因,因此推测该菌株具有固定 N 2和分泌生长素(IAA)的能力;通过植物促生特性试验的验证,发现 HMC50的确具有分泌 IAA的能力(29.57 mg/L)并且具有较高的固氮酶活性 11.9nmol/(mL·h),除此之外,该菌株同时具有产铁载体的能力,定量检测 A/Ar是 0.29,1-氨基环丙烷羧酸脱氨酶活性为 0.35 U/mg,溶解无机磷的能力较强(菌落溶磷透明圈直径HD与菌落直径 CD的比值为 3.97);盆栽试验发现该菌株对水稻有促进生长的潜能,接菌后水稻幼苗的根长、茎长、根重以及总鲜重上都优于空白对照组     

苹果甘露糖结合蛋白2基因(MdMBP2)的克隆及其重组蛋白的活性研究

甘露糖结合蛋白(MBP)是一类包含B-lectin结构域的植物凝集素,通过与糖的结合参与多种生理过程,包括细胞和细胞之间的相互作用以及寄主和病原之间的相互作用等.为了探讨苹果甘露糖结合蛋白在抗轮纹病防御反应中的作用,本研究从苹果(Malus demestica)枝条韧皮部cDNA鉴定了一个甘露糖结合蛋白的编码基因,命名为MdMBP2(GenBank accession No.JX126857).该基因全长1 553 bp,开放阅读框为1 368 bp,编码一个由455个氨基酸残基组成的蛋白质,分子量和等电点分别为50.4 kD和8.68.该蛋白的氨基酸序列包含保守的B-lectin结构域,N端有27个氨基酸残基组成的信号肽,C端有一个PAN-apple结构域.同源性和系统演化分析显示,MdMBP2蛋白与葡萄(Vitis vinifera)和大豆(Glycinemax)的表皮分泌蛋白(EP)及拟南芥(Arab idopsis thaliana)的甘露糖结合蛋白具有较高的序列相似性.包含B-lectin结构域的蛋白可以分成两个群,MdMBP2属于Class Ⅰ,与ClassⅡ蛋白相比,Class Ⅰ缺少SLP结构域和蛋白激酶结构域.利用荧光定量PCR技术检测MdMBP2基因的表达,发现MdMBP2基因在被检测的几种组织中均有不同程度的表达,但在皮和叶中的表达量较高,这种表达模式表明,MdMBP2可能参与多种生理途径.另外,轮纹病病原菌(Botryosphaeria dothidea)侵染能诱导MdMBP2转录本的积累,且其时序表达变化与轮纹病发病过程相关,暗示该基因参与了苹果抗轮纹病的防御反应.通过体外重组表达MdMBP2基因的重组蛋白,并用重组蛋白进行糖结合实验,发现MdMBP2蛋白能与真菌细胞壁组分高甘露糖型聚糖和甘露寡糖有较高的结合活性,表明MdMBP2蛋白可能参与病原真菌的识别.本研究分离鉴定了一个新的苹果甘露糖结合蛋白的编码基因,认为该基因参与苹果对轮纹病病原的防御反应,可能在寄主对病原的识别过程中发挥作用.     

菌寄生真菌黄蓝状菌(Talaromyces flavus)几丁质酶基因的克隆与生物信息学分析

菌寄生真菌的几丁质酶有很强的降解几丁质能力,在控制植物病害方面起着重要的作用.为克隆和研究菌寄生真菌黄蓝状菌(Talaromyces flavus)几丁质酶基因(tfchi1),本研究根据真菌几丁质酶基因的保守序列设计扩增基因中间片段的简并引物,采用RT-PCR、3'-RACE及5+-TAIL的方法获得了该基因的DNA和mRNA序列(GenBank:GU361769,GU361770).tfchi1长为2 561 bp,具有6个内含子,长度分别为129、78、68、65、53和59 bp,包含1 194bp的ORF,编码一个由397个氨基酸组成的蛋白.推导的tfchi1氨基酸序列以及蛋白质结构生物信息学分析表明,该蛋白具有典型的几丁质酶催化区保守序列SXGGW和DGXDXDWE,属于糖苷水解酶18家族几丁质酶,与柄篮状菌(Talaromyces stipitatus ATCC 10500)几丁质酶(XP_002480365)氨基酸序列同源性为96％,分子量为43.47kD,等电点为4.97.该蛋白无信号肽序列,有15个潜在的N-糖基化位点,Tfchi1的二级结构以无规卷曲和α-螺旋为蛋白的主要结构元件,三级结构中有(α/β)8的圆桶形结构.tfchi1转化毕赤酵母(Pichia pastoris )GS 115,酵母工程菌可分泌具几丁质酶活性的表达产物,重组蛋白的分子量与理论值相符.结果说明,本研究已从T.flavus中正确克隆了1个糖苷水解酶18家族几丁质酶基因.     

甘薯茎线虫β-1,4内切葡聚糖酶基因(Dd-eng-1b)cDNA全长的克隆与序列分析

β-1,4内切葡聚糖酶是植物寄生线虫食道腺分泌的一类细胞壁降解酶,在植物线虫的侵染过程中具有重要的作用.以马铃薯腐烂茎线虫(Ditylenchus destructor)为材料,用RT-PCR和RACE方法,获得了β-1,4-内切葡聚糖酶基因的cDNA全长,并将该基因命名为Dd-eng-1b(GenBank登录号为FJ430142).此cDNA全长序列为1 640 bp,包括1个1 443 bp的完整ORF,编码1含481个氨基酸的蛋白,其理论分子量为50.89 kD,等电点pI为6.94.序列比对分析表明,含有糖基水解酶的保守结构域,属于纤维素酶第五家族成员,N端具有19个氨基酸残基组成的信号肽,C端含有细菌式样的纤维素结合域(CBD Ⅱ).cDNA与基因组DNA重叠分析表明,此基因包含4个内含子,长度分别为36、76、187和344bp,切割点符合5'-GT……AG-3'的规律.系统进化树分析表明,该基因与细菌Bacillus subtilis和Erwina carotovora分泌的纤维素酶属于同一支,推测该基因可能来源于细菌的水平基因转移.     

固氮根瘤菌的生理特性及对非豆科生物的拌种效果

固氮根瘤菌（Ａｚｏｒｈｉｚｏｂｉｕｍ ｃａｕｌｉｎｏｄａｎｓ ＯＲＳ ５７１）不但能在毛萼田菁（Ｓｅｓｂａｎｉａ ｒｏｓｔｒａｔａ）上结出根瘤和茎瘤进行共生固氮，有屏氧功能可进行自生固氮，该菌生长过程分泌一定的植物中刺激作物生长。将该功晟菌种剂拌种小麦、水稻、玉米、马铃薯进行小区对比试验和大田示范。三年的结果表明，供试作物的经济性状得以不同程度的改善，经对照获得明显的增产增收效果。     

Survey of genes identified in Sinorhizobium meliloti spp., necessaryfor the development of an efficient symbiosis

The symbiosis between the soil bacteria Rhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium or Bradyrhizobium and leguminous plants is characterised by a specific multistep signal exchange. Only when a compatible rhizobial strain encounters its leguminous host, nodules will be formed on the roots of the host. During infection of this nodule, the microsymbiont evolves into a bacteroid form which, when provided with plant-derived carbon sources, is able to convert atmospheric nitrogen to ammonia that subsequently is supplied to the plant. The developmental programme underlying nodule organogenesis and functioning has been studied intensively for several decades. In this review, several observed plant phenotypes resulting from an ineffective symbiosis between plants and mutant rhizobial strains are represented. Besides the influence of the bacterial nodulation, nitrogen fixation and surface polysaccharide genes on symbiosis, the role of other genes important for the formation of effective nitrogen fixing nodules will be explained.     

植物细菌天山根瘤菌调控基因mrtR在群体感应中的作用

在许多种类的细菌中均发现了luxR/luxI类型的群体感应调控系统,其中luxR是调节基因,对luxI合成自体诱导物(AI,autoinducer)起着正调控作用。通过建库筛选后,测序比对发现天山根瘤菌中存在mrtR/mrtI系统。通过基因敲除和转录水平基因表达对mrtR进行研究,发现mrtR严格调控着mrtI的表达,同时mrtR的表达也受到AI影响,这表明mrtR表达的调控是一种自调控,同时根毛吸附实验显示该基因对根瘤菌与宿主间相互作用密切相关。     

The “soil microbial loop” is not always needed to explain protozoan stimulation of plants

Protozoa stimulate plant growth, but we do not completely understand the underlying mechanisms, and different hypotheses seek to explain this phenomenon. To test these hypotheses, we grew the grass Yorkshire fog (Holcus lanatus) in pots with soil, which contained either (1) no organisms but bacteria – or (2) bacteria and protozoa. Half of the pots received a glucose treatment so as to mimic an additional root exudation. We measured plant growth and plant nitrogen uptake, along with various microbial pools and processes that support plant growth. Protozoan presence significantly enhanced soil nitrogen mineralization, plant nitrogen uptake from organic nitrogen sources, plant nitrogen content, and plant growth. By contrast, we found no evidence that glucose addition, mimicking root exudation, increased soil nitrogen availability and plant nitrogen uptake. Moreover, although protozoan presence affected bacterial community structure, it did not affect the proportion of IAA-producing bacteria in the community or plant root morphology. These results refute the “soil microbial loop” hypotheses, which suggest that protozoan stimulation of plant growth results from complex interactions between plant roots, bacteria and protozoa. Our experiment thus favours the simple explanation that increased nitrogen availability is the key factor behind the positive protozoan effect on plant growth. To exploit natural resources in an efficient and environmentally friendly way, we need to understand in detail the functioning of ecosystems. This study stresses that to achieve this, it is still urgent, besides investigating intricate food-web and signal compound interactions, also to focus on the basic stoichiometric and energetic aspects of organisms.     

Effects of co-inoculation of Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 on plant growth,nodulation, nitrogen fixation,nutrient uptake,and yield of soybean in a field condition

ABSTRACT

Co-inoculation of nitrogen-fixing bacteria with plant growth-promoting bacteria has become more popular than single inoculation of rhizobia or plant-growth-promoting bacteria because of the synergy of these bacteria in increasing soybean yield and nitrogen fixation. This study was conducted to investigate the effects of Bradyrhizobium japonicum SAY3-7 and Streptomyces griseoflavus P4 co-inoculation on plant growth, nodulation, nitrogen fixation, nutrient uptake, and seed yield of the ‘Yezin-6’ soybean cultivar. Nitrogen fixation was measured using the acetylene reduction assay and ureide methods. Uptake of major nutrients [nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and magnesium (Mg)] was also measured. This study showed that single inoculation of SAY3-7 significantly increased shoot biomass; nodulation; Relative Ureide Index (RUI %), percent nitrogen derived from N fixation (% Ndfa); N, P, K, Ca, and Mg uptakes; during the later growth stages (R3.5 and R5.5), compared with control. These observations indicate that SAY3-7 is an effective N-fixing bacterium for the plant growth, nodulation, and nitrogen fixation with an ability to compete with native bradyrhizobia. Co-inoculation of SAY3-7 and P4 significantly improved nodule number; nodule dry weight; shoot and root biomass; N fixation; N, P, K, Ca, and Mg uptake; at various growth stages and seed yield in ‘Yezin-6’ soybean cultivar compared with the control, but not the single inoculation treatments. Significant differences in plant growth, nodulation, N fixation, nutrient uptake, and yield between co-inoculation and control, not between single inoculation and control, suggest that there is a synergetic effect due to co-inoculation of SAY3-7 and P4. Therefore, we conclude that Myanmar Bradyrhizobium strain SAY3-7 and P4 will be useful as effective inoculants in biofertilizer production in the future.     

~(15)N同位素稀释法测定燕麦根际固氮菌固氮量的研究

用15N同位素稀释法对接种到燕麦上的不同固氮菌的固氮能力进行了测定,结果表明:固氮菌处理的地上植株15N原子百分超为1.0871%～1.3791%,其中Pseudomonas spN4最低(1.0871%);植株地下部分为1.0921%～1.2751%,Pseudomonas spN4也是最低(1.0921%)。Pseudomonas spN4、A.lipoferumO6和Zoogloea spW6固氮能力较好。植株全氮含量增加2.08%～39.58%,A.lipoferumO6处理的全氮含量最高(39.58%)。接种固氮菌能够提高燕麦的全氮含量、固氮百分率和固氮量。     

利用15N2直接标记法研究水稻种植对稻田固氮量和固氮活性的影响

稻田固氮对土壤维持肥力有着重要的作用,但水稻种植与固氮菌及其活性之间的关系尚不清楚。本试验利用~(15)N_2直接标记法测定了下位砂姜土发育的简育水耕人为土在种水稻和不种水稻条件下的生物固氮量,及其在土壤不同层次(0～1、1～5、5～15 cm)和水稻中的分配,并通过实时荧光定量PCR技术测定了土壤中固氮菌nifH DNA及RNA基因数量。结果表明:种水稻处理显著提高了土壤各层固氮量,尤其提高了1～5 cm和5～15 cm土层土壤固氮量对总固氮量的贡献;种水稻处理的总固氮量是不种水稻处理的10.3倍;水稻植株中生物固定的氮占总固氮量的31.48%;在0～1 cm土层,种水稻处理显著提高了nifH RNA基因数量,而对nifH DNA基因数量的增加不显著。可见,水稻种植没有增加固氮菌的数量,稻田固氮量的增加是因为水稻种植极大地促进了固氮菌nifH基因的表达,提高了固氮菌的固氮活性。     

A synopsis of symbiotic nitrogen fixation and other apparently similar host‐microorganism interactions

Physical, physiological and biochemical processes occurring during colonization, infection, and nodule development and maintenance phases of an effective legume‐rhizobia symtiotic nitrogen fixation system are discussed. Limited knowledge of host reactions to invasion by rhizobia which result in failure to establish a symbiotic nitrogen fixation system are related to more thoroughly researched reactions of resistant host plants to invastion by pathogenic organisms. The most common resistant host responses are an increase in the production of phenolic compounds and phenol oxidizing enzymes. Many of the pheolic compounds or their quinones produced by enzymatic oxidation inhibit the action cell wall degrading enzymes and phytohormones, are antibiotic toward pathogenic organisms, and are phytotoxic to host plant cells. It is postulated that similar host responses result with rhizobia invasion and that the magnitude of these responses determine specificity of legume‐rhizobia symbiosis.     

甘肃省白龙江流域豆科植物根瘤菌共生固氮研究

对甘肃省白龙江流域甘南、陇南的大部分地区豆科植物——根瘤菌资源进行了调查,共采集21属45种豆科植物69份根瘤样品。从69份样品中获63株根瘤菌,对其中44株根瘤以13种寄主进行了回接试验,31株结瘤,结瘤率70.5%。该区根瘤一般带有粉红色,形态比较规则。乙炔还原活力测定结果表明,88.5%为有效根瘤。不同种根瘤固氮活性相差不大,但高于甘肃省其它地区。对豆科植物在蓄水保土,改良土壤状况中的地位进行了讨论。