E.aero高产13-丙二醇菌株选育及发酵培养基研究(Ⅰ)

以淡水湖泊泥土中分离出的 30 0多株肠杆菌 (Enterobacter)为出发菌株 ,利用常规筛选方法选出 2株 1 3 丙二醇产生菌 (Enterobacteraerogenes)。经UV、DES、NTG、EMS、LiCl单独及复合诱变 ,选育出一株 (E aero N 56) 1 3 PD高产突变株。通过单因素实验 ,确定了E aero N 56菌株 1 3 PD发酵培养基为 :甘油 90g L ,NH4Cl₁ 50g L     

应用聚合酶链式反应快速特异鉴定假单胞菌和伯克霍尔德氏菌（R）-3-羟基酯酰载酯蛋白-辅酶A转酰基酶基因

聚羟基脂肪酸酯 (PHA)是一类具有广泛应用前景的可降解生物塑料。因其可以以葡萄糖等廉价底物直接发酵生产PHA而日益受到重视。目前的研究表明在积累中长链PHA的假单胞菌中 ,由phaG基因编码的（R）3 羟基酯酰载酯蛋白 辅酶A转酰基酶 (PhaG)起关键作用 ,但目前为止对该蛋白还知之甚少。通过聚合酶链式反应 (PCR)建立了一种快速、特异鉴定phaG基因的方法 ,应用该方法成功地从两株积累不同PHA的假单胞菌Pseudomonasstutzeri 1317和Pseudomonasnitroreducens 080 2中分别克隆得到phaG基因 ,并在phaG基因突变株PseudomonasputidaPHAG_N-21中表达成功。同时 ,还首次报道了从非假单胞菌菌株Burkholderiacaryophylli AS 1.274 1中鉴定得到phaG基因 ,提示PhaG介导的中长链PHA合成途径作为一种通用的代谢模式在细菌中广泛存在 ,为进一步实现从廉价的非相关底物合成中长链PHA提供了必要的分子生物学基础。     

分离自鸡眼草和木蓝的根瘤菌分类研究

采用数值分类方法对分离自鸡眼草(Kummerowia)和木蓝(Indigofera)的根瘤菌及已知参比菌株进行聚类分析,发现在83%的相似性水平上形成2个不同与已知菌种的新类群。以SDS全细胞蛋白电泳技术快速聚类分群扩大菌株数,在86%的相似性水平上,分离自鸡眼草的24株菌形成第1类群,分离自木蓝的20株菌形成第2类群。DNA同源性测定结果表明,这2个类群中心菌株SH713和SHL042与13个已知根瘤菌种的DNA同源性均小于61%。因此,分离自鸡眼草和木蓝的根瘤菌分别构成2个独立的根瘤菌新种群。     

水稻黄单胞细菌Harpin蛋白的遗传多样性及其诱导烟草过敏反应和抗病性功能

利用PCR方法从水稻黄单胞细菌(Xanthomonas oryzae)两个致病变种(pv. oryzae和pv. oryzicola)的12个菌株中, 扩增出了编码诱导植物过敏反应蛋白激发子Harpin的3类hrfA (hypersensitive response functioning factor A)同源基因hrf1, hrf2和hrf3. 同源性分析表明, 这些同源基因推测的编码产物均富含甘氨酸, 缺乏酪氨酸, 在序列的第45位附近的氨基酸都有一个半胱氨酸. Harpin_Xoo由来源于水稻白叶枯病菌的hrfA基因编码, 产物包括两类, 一类代表菌株为JxoⅢ, 其中GGG-GG基序的重复数为3个, 分子量为15.6 kD; 另一类代表菌株为Pxo112, 产物中GGG-GG基序的重复数为4个, 分子量为15.9 kD, 这两个基因分别命名为hrf1和hrf3. Harpin_Xooc由来源于水稻条斑病细菌的hrfA基因编码, 代表菌株为RS105, 其中GGG-GG重复数为2个, 分子量为15.3 kD, 水稻黄单胞菌Harpin分子中的一个半胱氨酸残基是其特有的. 将这3类基因与已报道的hpa1和xop1基因编码的氨基酸序列进行聚类分析, 结果可以将其分为4类, 来源于水稻黄单胞菌的Harpin_Xoo和Harpin_Xooc被分在相邻的两类中. 对3种产物进行比较研究结果, 在相同条件下3个代表菌株JxoⅢ, RS105和Pxo112的hrfA基因(hrf1, hrf2和hrf3)表达蛋白的相对浓度分别为: 0.389, 0.530, 0.083 mg/mL. hrf1, hrf2和hrf3在大肠杆菌(Bl21)中的表达产物(Harpins)在烟草上均能激发过敏反应和诱导抗病性, 其生物活性依次为Hrf2, Hrf1和Hrf3.     

L-乳酸脱氢酶基因克隆及功能分析

构建了一株产D ,L_乳酸的乳杆菌 (Lactobacillussp .)MD_1的基因文库。利用乳酸脱氢酶和丙酮酸裂解酶缺陷的EscherichiacoliFMJ14 4作为宿主 ,通过互补筛选分离克隆到乳酸脱氢酶基因 (ldhL)。核酸序列分析表明 ,该基因以ATG为起始密码子编码 316个氨基酸残基组成的蛋白质 ,预测的分子量为 33 84kD ;5′端存在典型的启动子结构 ,3′端的终止子是不依赖于 ρ因子的转录终止子。ldhL编码的蛋白质有 3个保守区域 ,其中Gly13～Asp50保守区域是NADH的结合位点 ,Asp73～Ile10 0和Asn12.3～Arg15.4保守区是酶的活性部位。该ldhL和其他乳杆菌的ldhL基因和编码的氨基酸序列相似性较低 ,核苷酸序列相似性最高仅为 64.1% ,氨基酸序列相似性最高仅为 68.9% ,是新的L_乳酸脱氢酶基因     

转化5a-娠烷-3β，17a-二羟基一20一酮-21-醋酸酯为氢化可的松诺卡氏菌62-1菌株和蓝色梨头霉AS3．65的协同转化

本文研究了诺卡氏菌(Noeardia) 62-1 菌株和蓝色梨头霉(Absidia coerulea) AS3.65在一起,转化5a-娠烷-3β,17a-二羟基-20-酮-21-醋酸酯一步生成氢化可的松的协同转化作用。由于诺卡氏菌62一l菌株的水解酶活性弱,A1一脱氢酶的活性强,所以在单独转化中形成RSA,P．S和ALRS等多种产物,但蓝色梨头霉有很强的水解酶活性并具11β一羟化酶,所以在和诺卡氏菌的协同转化中,只测到少量RSA和微量不欲得的△1-RS,生成的主要中间产物是RS,它紧接着进一步转化成氢化可的松,这是一种理想的转化方法。     

芒果生长素反应因子类蛋白的cDNA克隆和表达

通过SSH法获得了一个与不定根形成相关的差异表达的cDNA片段,其推导的氨基酸序列与拟南芥的生长素反应因子(ARF)类蛋白具有较大的同源性,因此将它命名为MiARF。用所设计的基因特异引物进行3′RACE扩增获得包含完整读码框架(ORF)的MiARF1（GenBank登录号为AY255705）和MiARF2（GenBank登录号为AY300808）。MiARF1全长为3272bp,其中,ORF含2523bp,5′非翻译区（5′UTR）含285bp, 3′非翻译区(3′UTR)含464bp。由该序列所推导的氨基酸序列与拟南芥ARF2（BAB10162）的ID值为64%, E值为0,在DNA结合区域（DBD）、III和IV区域的同源性更高,ID值均大于80%。MiARF2cDNA全长为1474bp,其中ORF含981bp,5′非翻译区含285bp, 3′非翻译区含208bp,由该序列所推导的氨基酸序列与拟南芥ARF2（BAB10162）的ID值为84%,E值为e-151。 MiARF2仅具有DBD保守区并与MiARF1的基本相同,但缺乏III和IV区域。Virtural Northern 杂交表明：MiARF2在生根的组织中表达水平高, 而在非生根的组织中未见表达;MiARF1在生根及非生根的组织中均有表达。     

类诺卡氏菌属的两个新种

根据71-N46等5株菌的基丝与气丝都形成横隔,并断裂为杆状、椭圆或球状,无鞭毛不运动的节段;细胞壁组份I型：DNA中GC克分子百分比为66．3％等,认为这5株菌应属于类诺卡氏菌属,除71-N46为这属的典型种白色类诺卡氏菌(Nocardioides albus Prauser)外,本文报道两个新种：黄色类诺卡氏菌新种(Nocardioidea flavus n. sp.)和情黄类诺卡氏菌新种(Nocardioides fulvus n. sp. )。     

NAC1上游调控区表达特征及其与侧根激素诱导的关系

从拟南芥(Arabidopsis thaliana）中克隆到与侧根原基发生相关的转录因子基因NAC1上游调控区序列,构建由该序列驱动β-葡聚糖苷酶基因(GUS)的植物表达载体并转化烟草(Nicotiana Tabaccum),经筛选获得了在根组织高GUS活性而地上部痕量表达的转基因烟草植株。对转基因植株进行GUS活性和染色分析,结果表明NAC1上游调控区驱动的GUS基因表达具有根部组织特异性,在侧根顶端分生组织区、侧根原基基部和幼嫩侧根基部表达。用IBA,GA₃,GA₄₊₇处理转基因植株根部,NAC1上游调控区驱动的GUS表达均增强,表明生长素、赤霉素可显著诱导NAC1上游调控区的表达,并参与侧根发生的调控。     

利用反向PCR方法扩增细菌热激蛋白HSP60基因

利用PCR简并引物扩增出HSP6 0基因中一段约 6 0 0bp的核心片段 ,将该核心片段标记为探针 ,与基因组DNA进行Southern杂交 ,选择出适宜的限制性内切酶 ,以便消化基因组DNA得到大小合适的、含有HSP6 0基因的酶切片段。将酶切片段自身环化后作为模板进行反向PCR ,引物的延伸方向自核心片段出发延环化分子向未知序列区进行 ,可扩增出核心区上下游的序列。应用该方法 ,扩增并测定了寓齿双歧杆菌 (Bifidobacteriumdenticolens)DSM1 0 1 0 5 T、奇异双歧杆菌 (Bifidobacteriuminopinatum)DSM1 0 1 0 7T 和阴道加德纳氏菌 (Gard nerellavaginalis)ATCC1 40 1 8T 的HSP6 0全基因序列及青春双歧杆菌 (Bifidobacteriumadolescentis)JCM1 2 75 T98%以上的HSP6 0全基因序列。结果表明 ,反向PCR方法可有效的扩增细菌HSP6 0基因     

将云南原小单孢菌转入纤维化纤维菌的多相分类研究

对中国普通微生物菌种保藏中心(CGMCC)保藏的云南原小单孢菌(Promicromonospora yunnanensis)AS4.1333进行的多相分类研究表明,菌株AS4.1333与纤维化纤维菌(Cellulosimicrobium cellulans)DSM43879T关系密切,它们的16S rRNA基因序列相似性为99.6%,DNA同源性为89.3%。胞壁组分、枝菌酸、甲基萘醌、磷酸类脂和DNAG Cmol%测定等化学分类结果支持了上述结论。在形态和生理生化特性上,菌株AS4.1333与CellulosimicrobiumcellulansDSM43879T之间也表现出非常相似的性状。根据系统发育分析、化学分类、形态和生理生化特性、DNA同源性测定等研究结果,对菌株AS4.1333的分类地位做了修正,将其从原小单孢菌属中排除,认为菌株AS4.1333与Cellulosimicrobium cellulansDSM43879T是同一个种,建议将菌株AS4.1333由云南原小单孢菌(Promicromonosporayunnanensis)转入纤维化纤维菌(Cellulosimicrobium cellulans)。     

Rhodococcus cercidiphylli sp. nov., a new endophytic actinobacterium isolated from a Cercidiphyllum japonicum leaf

An endophytic actinobacterium, designated YIM 65003(T), was isolated from a surface sterilized leaf sample of Cercidiphyllum japonicum collected from Yunnan province, south-west China. The morphological and chemotaxonomic properties of the isolate were typical of members of the genus Rhodococcus. Analysis of the 16S rRNA gene sequence revealed that the isolate was most closely related to Rhodococcus fascians DSM 20669(T) (99.6%) and Rhodococcus yunnanensis YIM 70056(T) (99.0%). DNA-DNA hybridization with the above microorganisms (46.3% and 48.8%, respectively), in combination with differences in the biochemical and physiological properties, suggested that strain YIM 65003(T) should be classified within a novel species of the genus Rhodococcus, for which the name Rhodococcus cercidiphylli sp. nov. is proposed, with YIM 65003(T) (=CCTCC AB 207160(T)=DSM 45141(T)) as the type strain. The 16S rRNA gene sequence of strain YIM 65003(T) has been deposited in GenBank under the accession number EU325542.     

对胞必佳生产菌株的重新鉴定

主要是从形态学观察、菌株脂肪酸成分和16S rRNA基因全序列3个方面出发,重新对胞必佳生产菌株红色诺卡氏菌(Nocardia rubra)进行鉴定。结果表明,该菌株并非诺卡氏菌属中的红色诺卡氏菌,而属于红球菌属。16S rRNA序列相似性比较和系统进化树进一步说明,该菌株与Rhodococcus ruber(AY114117.1)的同源性最高,是1株红色红球菌。     

Nocardia testaceus sp. nov. and Nocardia senatus sp. nov., isolated from patients in Japan

Two actinomycete strains isolated from sputum between 1999 and 2001 in Japan were provisionally assigned to the genus Nocardia based on morphological criteria. These isolates were further studied in order to determine their specific taxonomic status. Detailed chemotaxonomic characterization and 16S rDNA gene sequence analysis of these isolates also confirmed that they belong to the genus Nocardia. The 16S rDNA sequence data of the two strains showed that they are most similar to that of Nocardia carnea and Nocardia flavorosea. However, DNA-DNA relatedness data showed that the two strains could be distinguished from N. carnea and N. flavorosea and therefore represented two new species within the genus Nocardia. The designation of the two isolated strains are Nocardia testaceus for IFM 0937(T) (=JCM 12235(T), DSM 44765(T)) and Nocardia senatus for IFM 10088(T) (=JCM 12236(T), DSM 44766(T)).     

Nonomuraea maheshkhaliensis sp. nov., a novel actinomycete isolated from mangrove rhizosphere mud

A strain of Nonomuraea was isolated from Maheshkhali, Cox's Bazar, an unexplored region of Bangladesh. Strain 16-5-14(T) is a Gram-positive, aerobic, non-motile actinomycete that formed branched substrate and aerial mycelia. On the basis of 16S rRNA gene sequence similarity studies, strain 16-5-14(T) was shown to belong to the genus Nonomuraea, being most closely related to Nonomuraea kuesteri. Chemotaxonomic data supported allocation of the strain as a member of the genus Nonomuraea. The strain 16-5-14(T) contained MK-9(H(4)) as the major menaquinone, the polar lipid was phosphatidylethanolamine and major cellular fatty acids were observed as C(16 : 0 )(15.5%), iso-C(16 : 0) (13.8%) and 10-methyl C(17 : 0) (9.6%). Results of DNA-DNA hybridization and physiological tests allowed genotypic and phenotypic differentiation of strain 16-5-14(T) from closely related species N. kuesteri. Thus 16-5-14(T) represents a novel species of the genus Nonomuraea. On the basis of evaluation of the morphological, physiological and chemotaxonomic characteristics, 16S rRNA gene sequence comparisons and DNA-DNA hybridization, Nonomuraea maheshkhaliensis sp. nov. (type strain, 16-5-14(T)=JCM 13929(T)=MTCC 8545(T)) is proposed.     

Saccharothrix yanglingensis sp. nov., an antagonistic endophytic actinomycete isolated from cucumber plant

An endophytic actinomycete strain, designated Hhs.015^T, was isolated from roots of cucumber seedlings. The endophytic isolate was identified by means of a polyphasic taxonomic approach. On the basis of 16S rRNA gene sequence similarities, strain Hhs.015^T was closely related to members of the genus Saccharothrix. DNA–DNA hybridization with the four closest relatives, Saccharothrix longispora NRRL B-16116^T, Saccharothrix xinjiangensis NRRL B-24321^T, Saccharothrix autraliensis CGMCC 4.1355^T and Saccharothrix espanaensis CGMCC 4.1714^T, gave similarity values of 33.8, 28.2, 44.1 and 29.5%, respectively, which indicated that strain Hhs.015^T represents a novel species of the genus Saccharothrix. This is consistent with the morphological, physiological and chemotaxonomic data. As a whole, these results suggest that strain Hhs.015^T represents a novel Saccharothrix species. The name Saccharothrix yanglingensis sp. nov. is proposed, with the type strain Hhs.015^T (=CGMCC 4.5627^T = KCTC 19722^T).     

Saccharopolyspora endophytica sp. nov., an endophytic actinomycete isolated from the root of Maytenus austroyunnanensis

A Gram-positive, aerobic, non-motile, non-acid-alcohol-fast strain, designated YIM 61095(T), was isolated from the root of Maytenus austroyunnanensis collected from a tropical rainforest of Xishuangbanna in Yunnan Province, south-west China. Strain YIM 61095(T) exhibited chemotaxonomic and morphological characteristics that were consistent with members of the genus Saccharopolyspora. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 61095(T) was a member of the genus Saccharopolyspora and was most closely related to Saccharopolyspora flava AS 4.1520(T) (97.7% sequence similarity). The major fatty acids were iso-C(15:0), iso-C(16:0), iso-C(17:0) and anteiso-C(17:0). The predominant quinone detected was MK-9(H(4)). The DNA G+C content was 66.2 mol%. The phenotypic characteristics and DNA-DNA hybridization relatedness data indicated that strain YIM 61095(T) should be distinguished from Saccharopolyspora flava AS 4.1520(T). On the basis of the evidence presented in this study, strain YIM 61095(T) represents a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora endophytica sp. nov. is proposed. The type strain is YIM 61095(T) (=KCTC 19397(T)=CCTCC AA 208003(T)).     

Bullera hoabinhensis sp. nov., a new ballistoconidiogenous yeast isolated from a plant leaf collected in Vietnam

VY-68, a ballistoconidiogenous yeast strain, isolated from a plant leaf at Cuc Phuong National Park of Ninh Binh Province, Vietnam, was assigned to the genus Bullera based on morphological and chemotaxonomical characteristics. Based on the sequence analyses of 18S rDNA, D1/D2 region of 26S rDNA, and internal transcribed spacer regions (ITS), VY-68 was phylogenetically closely related to Bullera pseudoalba and Cryptococcus cellulolyticus. DNA-DNA reassociation experiments among VY-68, B. pseudoalba and C. cellulolyticus revealed that strain VY-68 is a distinct species, and the latter two are conspecific. Bullera hoabinhensis is proposed for VY-68.     

Emendation of Dioszegia with redescription of Dioszegia hungarica and two new combinations,Dioszegia aurantiaca and Dioszegia crocea

During phylogenetic analyses of hymenomycetous yeasts based on 18S rDNA sequences, we found that Bullera armeniaca showed an extremely close phylogenetic relationship to Cryptococcus hungaricus. The analyses of internal transcribed spacer (ITS) regions of the two yeasts and the phylogenetically related species, Bullera aurantiaca and Bullera crocea, showed that B. armeniaca and C. hungaricus had identical sequences, indicating that these were conspecific. B. aurantiaca and B. crocea also showed high sequence similarity, 97.1% for ITS1, 100% for ITS2, and 98.7% for overall ITS regions. A DNA-DNA reassociation experiment revealed that B. armeniaca and C. hungaricus were conspecific and B. aurantiaca and B. crocea were two distinct species. These species occurred at a phylogenetically different lineage from that of Bulleromyces albus (anamorph: Bullera alba, type species of Bullera) and Filobasidiella neoformans (anamorph: Cryptococcus neoformans, neotype species of Cryptococcus). Based on these results, we emend the genus Dioszegia to include both ballistoconidium-forming and non-ballistoconidium-forming yeasts and redescribe the species Dioszegia hungarica. B. aurantiaca and B. crocea are also transferred to Dioszegia as Dioszegia aurantiaca comb. nov. and Dioszegia crocea comb. nov.     

Micromonospora siamensis sp. nov., isolated from Thai peat swamp forest

Morphological and chemotaxonomic characterization of actinomycete strain TT2-4T isolated from peat swamp forest soil in Pattaloong Province, Thailand, clearly demonstrated that this strain belongs to the genus Micromonospora. 16S rDNA sequence analysis for the strain supported the assignment of the strain to the genus Micromonospora and the similarity value of sequences between this strain and the closely related species, Micromonospora mirobrigensis was 99.1%, and M. carbonacea and M. matsumotoense were 98.8%. The DNA-DNA hybridization result and some physiological and biochemical properties indicated that strain TT2-4T was distinguished from the phylogenetically closest relatives. Based on these genotypic and phenotypic data, strain TT2-4T merits a new species in the genus Micromonospora and the name Micromonospora siamensis sp. nov. is proposed for the strain. The type strain is strain TT2-4T (=JCM 12769T =PCU 266T =TISTR 1554T).