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双语推荐：Agrobacterium

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近年来,根癌农杆菌（Agrobacterium tumefaciens）介导的食用菌遗传转化研究取得了一定的进展。为此,笔者对农杆菌介导转化的原理、转化的食用菌种类以及影响转化效率的相关因素进行了系统阐述。并分析了农杆菌介导食用菌转化中存在的问题,以及对该技术在食用菌中的应用前景进行了展望。
Molecular mechanisms operative in Agrobacterium tumefaciens-mediated transformation (ATMT), species of edible fungi transformed using A. tumefaciens, and factors affecting the efficiency of the transformation process,have been reviewed.Disadvantages associated with the use of ATMT in the context of edible fungi,and the future prospects of this methodology,have been discussed.

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采用16SrDNA测序方法对从内蒙古锡林郭勒天然草原四种主要禾本科牧草根际分离获得的18个固氮菌株进行属水平的鉴定,结果表明:其中7株属于土壤杆菌属(Agrobacterium)、3株属于产碱菌属(Alcali-genes)、1株属于固氮菌属(Azotobacter)、1株属于芽孢杆菌属(Bacillus)、3株属于假单胞菌属(Pseudomonas)、2株属于鞘氨醇单胞菌属(Sphingomonas),1株经16SrDNA序列分析和Biolog试验尚未找到与之相匹配的菌株,可能是一个新种。
Eighteen strains of N Fixation bacteria were identified by using 16sDNA sequence analysis at species level,which were isolated from rhizosphere of 4 species of gramineous grasses in Xilingol Grassland of Inner Mongolia.The results showed that 7strains of them belong to Agrobacterium,and there were 3 strains in Alcaligenes,3 strains in Pseudomonas,2 strains in Sphingomonas,1 strain each in Azotobacter and stran in Bacillus.One strain of N fixation bacterium was not identified because there was not similiar16sDNA sequence in 16sDNA bank which could fit it.Futher biolog analysis for the strain suggested it might be new species which need to test by using whole DNA sequence.

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太岁是自然界中一种组成及分类尚不明确的生物体。采用分子生物学方法对2种不同来源地的太岁样品进行菌种分离培养和分子鉴定。方法:太岁表面经75%乙醇消毒处理后,用麦芽汁培养基分离可培养菌株,提取太岁及分离菌株基因组DNA,以之为模板,用16S rDNA、18S rDNA和ITS通用引物进行PCR扩增,扩增片段连入pMD18T载体并测序,通过序列比对对太岁菌种组成进行初步鉴定。结果:从太岁1号样品中分离到假丝酵母(Can?dida)和粘质红酵母(Rhodotorula mucilaginosa),从太岁2号样品中分离到根癌农杆菌(Agrobacterium tumefaciens)和粘质红酵母。结论:2种来源不同的太岁样品中均存在可培养的粘质红酵母。
Objective: Taisui is an organism with undefined classification existed in nature. The object of this study was to isolate and molecular identity strains in two Taisui samples obtained from different source by molecu- lar method. Methods: After washing with 75% ethanol for about 10 s, the Taisui samples were inoculated into malt wort medium to obtain the cultured strains. Genomic DNA of Taisui samples were isolated and used as the template to amplify 16S rDNA, 18S rDNA and ITS by PCR. The PCR fragments were then ligated into pMD18T and sequenced respectively. Strain identification was performed by sequence alignment against NCBI GenBank. Re- sults: Candida and Rhodotorula mucilaginosa were found in No.1 sample while Agrobacterium tumefaciens and R. mucilaginosa were found in No.2 sample. Conclusion: Rhodotorula mucilaginosa was found in these two Taisui samples from different source.

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双孢蘑菇2个差异表达基因热休克蛋白基因(Hsp20)和4-氨基-4-脱氧分支酸合成酶基因(Adcs)通过根癌农杆菌(Agrobacterium)介导的花序浸渍法转化拟南芥(Arabidopsis thaliana),经草铵磷筛选、聚合酶链式反应(PCR)鉴定和蛋白质印迹(Western blot)鉴定表明,Hsp20基因和Adcs基因已整合到拟南芥基因组中并过表达.对转基因植株和野生型对照进行高温胁迫,比较其耐热性差异.结果显示:经过45℃热激1h处理,Hsp20基因转化拟南芥下胚轴恢复生长,部分幼苗存活;Adcs基因转化拟南芥与野生型一样下胚轴未恢复生长,幼苗基本未存活.实验表明,Hsp20基因对蘑菇的耐热性有直接作用,而Adcs基因对蘑菇的耐热性可能无直接作用.
As two differentiated expressing genes,heat shock protein 20 (Hsp20 )and 4-amino-4-deoxychorismate synthase (Adcs ) were transformed into wild type Arabidopsis plants by Agrobacterium tumefaciens-mediated floral dip method,after Basta screening,PCR and Western blot detection indicated Hsp20 and Adcs gene had been integrated into the genome of Arabidopsis and overexpressed.The performance of the transformed plants and the wild type under heat stress was analyzed.The results showed that, after 1 hour stress of 45 ℃,the hypocotyl of Hsp20 transgenic plants can recover extending,what''s more,some seedlings of trans-genic plants can survive;however,like wild type,the hypocotyl of Adcs transgenic plants can''t recover extending and their seedlings can''t survive.Consequently,the experimental results displayed that Hsp20 gene contributes thermotolerance to Agaricus bisporus di-rectly whereas Adcs gene probably not.

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通过分离湖南省不同地区辣椒的内生固氮菌,探索其多样性,为内生固氮菌资源应用于现实生产提供理论依据.利用改良无氮培养基对辣椒的根、茎、叶中内生固氮菌进行分离和纯化,并进行菌落计数比较其菌群密度,再对分离出的菌株进行16S rDNA序列扩增,分析其多样性,最后通过乙炔还原法结合气相色谱法测其固氮酶活性.结果筛选到内生固氮细菌30株,经16S rDNA序列测定和分析,结果显示土壤杆菌属(Agrobacterium sp.)、根瘤菌属(Rhizobium sp.)、泛菌属(Pantoeasp.)、假单胞菌属(Pseudomonas sp.)相对频率较高,是辣椒内生固氮菌的优势种.经乙炔还原法测定固氮酶活性表明,该30株菌株均具有较弱固氮酶活性,范围为4 ～ 183nmol/(mL·h),且不同地区同种菌的固氮酶活性存在一定差异.
To separate different areas of Hunan province pepper endogenous azotobacter,to explore its diversity as endogenous azotobacter resources applied to provide theoretical basis for practical production.Improved nitrogen free culture medium was used for separation and purification of pepper root,stem and leaf of endogenous azotobacter.Then the bacteria colony density counts,16S rDNA sequences with strains isolated amplification were analized by the acetylene reduction method in combination with gas chromatography to measure the nitrogenase activity.30 strains endogenous azotobacter were screened ; 16S rDNA sequence was determined and analized.The relative activities of Agrobacterium sp.,Rhizobium sp.,Pantoea sp.,Pseudomonas sp.,were higher which meant better N fixing endogenous azotobacter dominant species in pepper.By using the acetylene reduction method to determine the nitrogenase activity,results showed that all 30 strains fell in a weaker nitrogenase activity scope of 4-183n

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羟氨氧化酶(Hydroxylamine oxidase,HAO)的作用方式直接决定了异养硝化好氧反硝化细菌的代谢途径,分离得到纯度较高的HAO也就成为研究这类细菌脱氮机制的重要环节。以异养硝化好氧反硝化细菌Agrobacterium tumefaciens LAD9为代表,建立了该菌株HAO的分离纯化方法:首先采用渗透压休克法提取细胞周质液,然后采用DEAE Sepharose CL-6B离子交换层析和Sephacryl S-100凝胶过滤层析对细胞周质液进行分离纯化。结果表明,经过离子交换层析可得到分子量分别为55.3、35.7和19.2kD的杂蛋白,进一步经过凝胶过滤层析即可得到电泳纯的HAO,纯化倍数为5.79,产率为39.71%。对其酶学性质的初步研究表明,该菌株HAO的分子量为18.8 kD,能够将羟胺氧化为亚硝酸盐氮,且Fe2+的加入可显著增强其酶活。
The metabolic pathway of heterotrophic nitrification-aerobic denitrification bacteria was directly determined by the actions of their hydroxylamine oxidase(HAO). Isolating high-purity HAO from this kind of bacteria has become particularly important to explain the mechanism of nitrogen removal. In this study, the separation and purification technic of HAO from a novel heterotrophic nitrification-aerobic denitrification strain Agrobacterium tumefaciens LAD9 was established. Electrophoretic purity of HAO could be sucessfully purified through DEAE Sepharose CL-6B ion-exchange chromatography and Sephacryl S-100 gel filtration from its periplasm. The final purification fold was 5.79 and the yield was 39.71%. SDS-PAGE eclectrophoresis results revealed that the molecular weight of HAO in the strain LAD9 was 18.8 kD. Studies on enzymatic properties showed that the purified enzyme could oxidize hadroxylamine to nitrite and its activity could be enhanced by the addition of Fe2+.

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【目的】从目前已知的参与拟南芥Arabidopsis thaliana次生壁加厚生长的转录因子着手,分析这些次生壁相关的转录因子是否能够调控木糖合成关键酶基因FRA8、IRX9、IRX10、IRX14、F8H、IRX9-L、IRX10-L和IRX14-L的表达,并且观察KNAT7基因显性抑制植株的表型.【方法】通过Gateway技术构建效应器和报告器,进行瞬时转录激活试验,同样构建pCAMBIA1304-p35S∷KNAT7-SRDX重组质粒,用农杆菌Agrobacterium tumefaciens花序浸染法将此质粒转化到野生型拟南芥植株中.【结果和结论】瞬时转录激活试验表明,转录因子KNAT7、MYB46、ERF72、SND1、NST2能够激活多个拟南芥木聚糖合成关键酶基因的表达,其中KNAT7能促进基因FRA8、IRX9和IRX14-L的表达.KNAT7基因显性抑制能显著影响拟南芥的生长.试验结果表明KNAT7基因可能在木聚糖的合成中起着重要的调控作用.
Objective] To analyze whether some transcription factors in Arabidopsis thaliana, known for the secondary cell wall thicken , could regulate the expression of the key genes of xylosyltransferase , such as FRA8, IRX9, IRX10, IRX14, F8H, IRX9-L, IRX10-L and IRX14-L, and observe the phenotype of KNAT7 dominant repression plant .[Method] Effectors and reporters were constructed by Gateway Tech-nology and the transient transcriptional activation assay was conducted .Construct pCAMBIA1304-p35S∷KNAT7-SRDX recombinant plasmid by Gateway Technology and transform this plasmid into wild A.thali-ana via Agrobacterium tumefaciens-floral dip method .[Result and conclusion] The transient transcription-al activation assay revealed that transcription factors KNAT 7, MYB46, ERF72, SND1, NST2 could acti-vate the expression of a number of the key genes of xylosyltransferase .KNAT7 could activate the expres-sion of FRA8, IRX9 and IRX14-L.Furthermore, dominant repression of KNAT7 significantly affected the

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为阐明四川部分地区野生白三叶根瘤菌的遗传多样性及系统发育地位，对分离自四川雅安、康定、泸定、西昌、成都和乐山6个地区白三叶根瘤的69株菌进行系统研究。采用16 S rDNA 限制性片段长度多态性分析（re-striction fragment length polymorphism，RFLP）和16S rDNA 基因、持家基因（recA、atpD、glnII）、结瘤基因（nodC）、固氮基因（nifH）系统发育分析的方法进行了研究。结果表明，16 S rDNA PCR-RFLP中所有供试菌株产生了4种酶切图谱类型，表现出较为丰富的遗传多样性。持家基因与16 S rDNA基因系统发育分析结果基本一致，9株代表菌株主要分布在α-变形菌纲（Alpha-Proteobacteria）的根瘤菌属（Rhizobium），并与豌豆根瘤菌三叶草生物型（R．leguminosarum bv．trifolii）ATCC 14480T 的亲缘关系较近。PCR可扩增出nodC和nifH基因片段，但从属于土壤杆菌属（Agrobacterium）的菌株LS1105中则扩增不出这两个基因。所有供试菌株被鉴定到了种的水平，证实了68株为白三叶根瘤菌，并通过不同采样地点菌株之间的比较，发现白三叶与根瘤菌的共生关系因地理分布不同而具有多样性，对于丰富白三叶根瘤菌资源及其开发利用具有重要意义。
The obj ective of this study was to determine the genetic diversity and phylogeny of 6 9 isolates of sym-biotic bacteria associated with wild white clover obtained from six sites in Sichuan Province,China:Ya’an, Kangding,Luding,Xichang,Chengdu and Leshan.Polymerase chain reaction restriction fragment length poly-morphism (PCR-RFLP)and phylogenetic analyses of the 16S rDNA gene,housekeeping genes (recA,glnII andatpD),nodulation gene (nodC)and nitrogen-fixing gene (nifH)were conducted in the research.Four PCR-RFLP fingerprint patterns were identified among the 69 isolates from the 16S rDNA analysis,indicating relatively high genetic diversity.The results of phylogenetic analysis of 16S rDNA and housekeeping genes were similar;nine representative strains mainly belonged to the genusRhizobiuminAlpha-Proteobacteriawere identified.In addition,nodCandnifH genes from strains from the Rhizobium genus were successfully ampli-fied by PCR,but strains belonging to the Agrobacterium genus

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农杆菌是一种革兰氏阴性土壤病原细菌,携带具有天然转基因功能的Ti质粒或Ri质粒,能将一部分遗传物质插入到寄主植物的染色体上,使其稳定遗传和表达,赋予植物新的性状。所以农杆菌作为最有效的转化媒介,已被广泛应用于多数双子叶植物和部分单子叶植物转基因研究。虽然农杆菌介导的转化技术具有操作简单、成本低廉,转基因沉默几率小,插入基因拷贝数少等优点,但农杆菌介导植物遗传转化是一个复杂的生物学过程,需要一系列农杆菌蛋白和植物蛋白相互作用,共同完成外源基因的转入和整合。植物相关蛋白在转化过程中起着重要作用。其中,阿拉伯半乳聚糖蛋白(AGP)、植物根钙粘附蛋白和类玻连蛋白等参与农杆菌附着于植物细胞表面的过程;鸟苷三磷酸腺苷酶(GTPase)和BTI蛋白协助T-DNA和Vir效应蛋白进入植物细胞;actin、GIP、VIP等蛋白参与T-DNA复合体在细胞质中的运输的过程;与Vir效应蛋白互作的VIP1、VIP2、KAPa、PP2C、Roc等蛋白协助T-DNA定位于植物细胞核;组蛋白、VIP1和VIP2等引导T-DNA在植物基因组上的整合。由于植物种类间存在巨大差异,上述一些植物蛋白基因的过表达虽能提高农杆菌转化某些植物的转化效率,但不能提高另一些植物的转化效率。在容易被农杆菌遗传转化的植物如拟南芥、水稻中的研究表明,VIP1、VIP2、AGP、H2A等蛋白与农杆菌转化关系密切,但这些蛋白在利用农杆菌转化较难的作物如小麦、玉米中的功能还不明确,因而需要在不同植物中继续筛选和鉴定与T-DNA转化相关重要蛋白的编码基因。目前,农杆菌介导的植物遗传转化有2个显著特点,一是农杆菌介导转化烟草、拟南芥、水稻等模式植物的技术日渐成熟,二是农杆菌介导转化小麦、玉米、大豆等重要作物的技术仍然没有本质突破,植物相关蛋白在T-DNA转运、整合等过程中的作用还需要深入研究和进一步明确。文章主要对参与农杆菌介导遗传转化植物整个过程中相关植物蛋白的研究进展进行了综述,以期为提高农杆菌转化顽拗型作物的转化效率提供参考。
Agrobacterium tumefaciens is a kind of Gram-negative soil bacterium, which harbors tumor inducing (Ti) or root inducing (Ri) plasmid. The T-DNA on the plasmid can enter plant cells and integrate into the host genome, and further endows plants new characteristics by stable inheritance. Thus, Agrobacterium tumefaciens, as a most efficient transformation vehicle, has been widely used in genetic engineering study for most dicotyledonous and part of monocotyledonous plants. Agrobacterium-mediated transformation technology has several advantages including simple process, low cost, less gene silence, and few copies of target genes. However, the transferring of target genes into plant genome by Agrobacterium is a complicated biological process, during which a lot of Agrobacterium proteins and plant proteins interact with each other for the mission of importation, transportation and integration of T-DNA. Associated host proteins play important roles almost in each step of the transformation of

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以玉米(Zea mays L.)HiII的幼胚为外植体,β-葡萄糖苷酸酶基因(GUS)为报告基因,通过农杆菌介导转化法对影响遗传转化体系的外植体大小、农杆菌浓度、热预处理温度、侵染时间、共培养及恢复培养时间、抗生素、筛选剂等因素进行优化,以建立农杆菌介导玉米幼胚遗传转化体系。结果表明,幼胚大小为1.0~1.5 mm,农杆菌菌液的OD600 nm为0.5,40℃热处理3 min,侵染8 min,共培养3 d和恢复培养4 d,100 mg/L羧苄青霉素作为抑菌剂,双丙氨膦作为筛选剂时为最佳遗传转化条件。通过该优化体系已获得多种转基因玉米材料,表明该体系具有较高的可重复性和可靠性。
In order to establish genetic transformation system of maize immature embryos mediated by Agrobacterium tumefaciens, factors including size of explants, concentration of Agrobacterium tumefaciens, temperature of thermal pretreatment, infection time, cultivation and recovery time, antibiotics, screening agent affecting genetic transformation system were optimized using immature embryos of HiII as explants and GUS as reporter gene. The results showed that the size of the embryos was 1.0 to 1.5 mm in length. The OD600nm of Agrobacterium was adjusted to 0.5 before inoculation. Pretreatment was 3 min at 40℃. Infection time was 8 min. It was co-cultivated 3 days and rested 4 days. 100 mg/L carbenicillin was used to eliminate Agrobacterium and bialaphos was used as selective agent. Transgenic plants were obtained by the optimized system, which was a highly and reliable system.

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