甘薯EST资源的SSR信息分析

从NCBI公共数据库下载获得22371条甘薯EST序列,去除低质量的和冗余的序列后,得到总长为5．09×10^3kb的9204条唯一序列。从这些序列中搜索到总共436个SSR位点,平均相距11．68kb出现一个SSR。这些SSR的出现频率和平均长度分别为4．4％和24．28bp。在2-6bp的重复基元中,六核苷酸重复基元出现频率最（30．96％）,其次是三核苷酸重复基元（29．59％）和二核苷酸重复基元（24．54％）。出现最多的重复基元是AG／CT（16．28％）,其次是AAG／CTT（11．01％）。     

绿粘帚霉菌株发酵液抗真菌活性及稳定性测定

采用杯碟法测定绿粘帚霉（RCEF4099）菌株的发酵液对8种植物病原真菌的抗菌活性。结果表明RCEF4099菌株发酵液对5种供试病原真菌的抑菌圈直径在18mm以上。对菌株发酵液的稳定性测定结果表明菌株转接6代之前,活性稳定,从第7代开始其活性缓慢降低,第10代的抑菌圈直径仅比出发菌株减少了1．7mm。RCEF4099菌株发酵液有较好的热稳定性,发酵液加热到60％仍有较高活性。该菌株的发酵液对酸的稳定性较好,抑菌活性最强的pH值为4。     

链霉菌UDP-葡萄糖脱氢酸编码基因Ste6的克隆表达和性质研究

链霉菌139能够产生一种全新的胞外多糖——依博素（139A）,该多糖体内具有显著抗类风湿性关节炎活性。其生物合成基因簇（GenBank Accession Number：AYl31229）已被鉴定约31．3kb,包含22个开放阅读框（ste1—ste22）。以pET-30a为载体,克隆并在大肠杆菌BL21（DE3）中进行了ste6基因的表达,对该基因的克隆、表达与性质进行了研究。亲和层析法证实,纯化后重组蛋白具有催化UDP-葡萄糖脱氢变成UDP-葡萄糖醛酸的活性。这表明ste6编码产物是葡萄糖脱氢酶。为了证实ste6基因与依博素生物合成的关系,采用单交换基因破坏策略构建了ste6基因阻断突变株。结果初步显示ste6和依博素生物合成相关。     

黄单胞菌多糖20吨罐发酵

野油菜黄单胞菌（Xanthomonascampestris）L4在20t发酵罐中,以蔗糖为底物发酵72h,发酵液粘度达到7000～9500cp,产物对底物的转化率平均达到61．6％,为投产奠定了良好的基础。     

CYP3A4基因原核表达质粒构建与诱导表达

以质粒pCWNF14为模板,采用PCR技术扩增出CYP3A4基因,并将其接入pET-22b（＋）、pET-28b（＋）和pET-32a（＋）载体中,经PCR测序鉴定后,转化Rosetta（DE3）2pLysS细菌,并用IPTG诱导表达．采用考马斯亮蓝染色的方法检测重组蛋白表达,3个表达重组质粒中,只有pET-32a（＋）-CYP3A4可以表达目的蛋白;在低浓度IPTG（50μmol／L）诱导6h,所表达的CYP3A4蛋白约占细菌总蛋白的40％。且该蛋白不溶于8mol／L的尿素,而溶于去污剂10mmol／L 3-（（3-Cholamidopropyl）dimethvlammonio propanesulfonic acid（CHAPS））和0．3％ lauroyl sarcosine（SKL）．成功地使CYP3A4基因在原核系统中高效表达．     

海洋真菌Xylaria sp.(No.2508)生产xyloketal B的发酵优化

对海洋真菌Xylariasp．（No．2508）生产xyloketa]B的摇瓶发酵过程进行了初步优化。结果表明,Xylariasp．（No．2508）在培养120h时xyloketalB产量最高,当培养168h后菌体进入衰亡期且xyloketalB迅速降解,因此Xylariasp．（No．2508）生产xyloketalB的最适发酵周期为120h;Xylariasp．（No．2508）合成xyloketalB的过程及用乙酸乙酯萃取xyloketalB的过程对pH非常敏感,其中最适xyloketalB生产的发酵液初始pH为5．7,利用乙酸乙酯萃取xyloketalB时的发酵液最适pH为3。0;最适接种量为10％（v／v）;接种前发酵液中加入10g／L的大孔树脂XAD-16可以降低某些代谢产物对xyloketalB合成的抑制作用,从而使产量提高2倍左右,而菌体进入稳定期以后加入大孔树脂XAD-16对产物产量没有影响。     

麦迪霉素产生菌酮基还原酶基因在大肠杆菌中的表达

用PCR法扩增麦迪霉素产生菌酮基还原酶（MKR）基因,得到约0．8kb的DNA片段,扩增片段重组到利用依赖T7RNA聚合酶的高效表达载体pT7－7中,在大肠杆菌中表达出28．9kD的蛋白质。表达的蛋白质具有生物活性。     

用Genome shuffling技术选育紫杉醇高产菌株

以树状多节孢（Nodulisporium sylviforme）紫杉醇产生菌为研究对象,探索了紫杉醇产生菌的基因组重排育种的基本规律,重点研究了紫杉醇产生菌的原生质体融合和基因组重排育种的方法．采用薄层层析（TLC）、高效液相色谱（HPLC）和质谱（MS）分析筛选重组子,通过四轮基因组重排成功选育出了3株遗传稳定的高产紫杉醇菌株,其中一株重排菌株F4-26的发酵液中紫杉醇含量达到516-37μg几,比原始出发菌株NCEU-1紫杉醇产量提高了64．41％,比亲本菌株紫杉醇产量提高了31．52％-44．72％．     

链霉菌基因克隆载体及基因文库的构建

利用麦迪霉素产生菌中分离的质粒pSMY1(10．8kb),将pIJ30的硫链丝菌素抗性基因(tsr)克隆到psMY1上,获得了具有硫链丝菌素抗性选择标记质粒pSJ10。通过DNA体外缺失,片段播入、λ-COS片段的插入及与大肠杆菌质粒的重组等基因技术,获得了一系列psMY1衍生质粒,包括双标记质粒pSM3,大肠杆菌／链霉菌穿梭质粒pBMJ2和穿梭粘粒pNMJ1。通过对这些质粒的分析,确定了质粒psMY1的复制必需区为3．06kd的EcoRI—sphI片段。质粒pSJ10、pSM3、pNMJ1具有可选择标记,多个单一确切的可克降位点,有一定范围的宿主并能在链霉菌中稳定存在等特点,故可作为链霉菌基因克隆的载体。其中pNMJl(11．15kb)是大肠杆菌／链霉菌穿梭粘粒,能有效地运载28—38kb的外源DNA片段,并能在体外包装λ噬菌体外壳,转导大肠杆菌。利用载体pNMJl,通过λ-噬菌体蛋白体外包装,在大肠杆菌中建立了螺旋霉素产生菌的基因文库,其基因覆盖率可达90％。重组DNA分子可通过转化转移到链霉菌中。     

硫霉素环化酶基因在变铅青链霉菌TK24中的表达及基因定位

将含有硫霉素环化酶基因的重组质粒p6BCl2转化变铅青链霉菌(Streptomyceslividans)TK24,含有p6BCl2的转化子细胞抽提液分别与琉霉素生物合成阻断变株Y,发酵液以及纯化的Y。中间产物经过体外共培养可产生活性物质．化学分析表明与Y,发酵液混合后产生的是硫霉素,与纯化的Y。中间产物混合产生的是一种不稳定的活性物质。说明硫霉素环化酶基因在S.lividans TK24中得到了表达,其产物以Y。中间产物为底物并弥补了Y,中的缺陷。对p6Bcl2中4．5kb外源片段进行了限制酶酶切分析,建立了酶切图谱．利用含硫霉素环化酶基因的S．Lividans TK24转化子体外转化Y,的应用体系,将硫霉素环化酶基因定位在0．9kb Hinc I—Pst I片段上,并证明了硫霉紊环化酶的活性与IPNS同源片段无关。以上实验为进一步研究琉霉素环化酶基因的结构打下了基础。     

野油菜黄单胞菌野油菜致病变种8004菌株wxcA基因与EPS的产量有关

在以前的工作中,采用转座子Tn5 gusA5对野油菜黄单胞菌野油菜致病变种(Xcc)8004菌株进行诱变,获得一批胞外多糖(EPS)合成减少的突变体,对这些突变体的Tn5 gusA5的插入位点进行分析后,发现有两株突变体是wxcA基因不同插入位点的突变体。以前认为wxcA基因与脂多糖(LPS)的O-抗原合成有关而与EPS的合成无关。为明确wxc4基因的功能,对8004菌株的wxcA基因进行缺失,获得的△wxcA突变体的EPS产量与野生型菌株相比,减少了50％,并且一段PCR合成的包含wxcA基因的DNA片段能反式互补△wxcA突变体,恢复突变体的EPS产量。这证实了8004菌株的wxcA基因与EPS的合成产量有关。     

野油菜黄单胞菌野油菜致病变种中一个与EPS合成有关的新基因的鉴定

用转座子Tn5gusA5对野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris,简称Xcc)野生型菌株8004进行诱变,分离到一批胞外多糖(EPS)合成减少的突变体。采用TAIL-PCR(thermal asymmetric interlaced PCR)分析突变体的Tn5gusA5插入位点,发现其中一株编号为151D09的突变体的插入位点位于Xcc 8004菌株的基因组编号为XC3695的ORF内,该ORF功能尚未见报道。序列分析表明,该ORF演绎的编码产物与Serratia marcescens的kdtX基因和Klebsiella pneumoniae的waaE基因演绎的编码产物分别具有52%和50%的相似性,并具有第2家族糖基转移酶的功能域, 因此暂将该ORF命名为waxE基因。用同源双交换方法构建了waxE基因的缺失突变体,并采用PCR和Southern杂交的方法对突变体进行了验证。waxE基因缺失突变体在营养丰富培养基的生长繁殖不受影响,但其EPS产量与野生型菌株8004相比,降低35%左右,并且一段PCR合成的包含waxE基因的DNA片段能反式互补waxE基因缺失突变体,恢复缺失突变体的EPS产量,表明Xcc waxE基因与EPS的生物合成有关。     

Qualitative and comparative proteomic analysis of Xanthomonas campestris pv. campestris 17

The bacterium Xanthomonas campestris pathovar campestris (XCC) 17 is a local isolate that causes crucifer black rot disease in Taiwan. In this study, its proteome was separated using 2-DE and the well-resolved proteins were excised, trypsin digested, and analyzed by MS. Over 400 protein spots were analyzed and 281 proteins were identified by searching the MS or MS/MS spectra against the proteome database of the closely related XCC ATCC 33913. Functional categorization of the identified proteins matched 141 (50%) proteins to 81 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, we performed a comparative proteome analysis of the pathogenic strain 17 and an avirulent strain 11A to reveal the virulence-related proteins. We detected 22 up-regulated proteins in strain 17 including the degrading enzymes EngXCA, HtrA, and PepA, which had been shown to have a role in pathogenesis in other bacteria, and an anti-host defense protein, Ohr. Thus, further functional studies of these up-regulated proteins with respect to their roles in XCC pathogenicity are suggested.     

野油菜黄单胞菌野油菜致病型胞外多糖合成有关的DNA序列的克隆

以从野油菜黄单胞菌野油菜致病型（Xanthomonascampestrispv.campestris）胞外多糖突变体T117中克隆到的含有突变序列的DNA片段作探针,从野生型菌株中鉴定和克隆了含有相应序列的9．4kbHindⅢ片段。该片段可反式互补突变体T117,完全恢复其胞外多糖的合成,说明该片段至少含有一个完整的与该菌胞外多糖合成有关的基因。     

Comprehensive analysis of the extracellular proteins from Xanthomonas campestris pv. campestris B100

The extracellular proteome of Xanthomonas campestris pv. campestris (Xcc) cultivated in minimal medium was isolated from the cell-free culture supernatant and separated by two-dimensional gel electrophoresis. This technique resolved 97 clearly visible protein spots, which were excised, digested with trypsin and identified on the basis of their peptide mass fingerprints generated by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry. Using this approach 87 different proteins could be distinguished. The Signal P software predicted putative signal peptides for 53% of the extracellular proteins. These proteins are probably transported over the inner membrane and are localized in the periplasm, the outer membrane or secreted into the extracellular space. Among the secreted proteins are 11 degradative enzymes, which are involved in pathogenesis of Xcc. The proteins without obvious secretion signals are known to serve functions in the cytosol. How the cytosolic proteins are delivered to the extracellular space remains unclear.     

High-quality mutant libraries of Xanthomonas oryzae pv. oryzae and X. campestris pv. campestris generated by an efficient transposon mutagenesis system

A novel transposon mutagenesis system for the phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and X. campestris pv. campestris (Xcc) was developed using a Tn5-based transposome. A highly efficient transformation up to 10(6) transformants per microg transposon DNA was obtained. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses of Tn5 insertion sites suggested a random mode of transposition. The transposition was stable in the transformants for 20 subcultures. Eighteen thousand and 17000 transformants for Xoo and Xcc, respectively, were generated, corresponding to 4X ORF coverage of the genomes. The libraries will facilitate the identification of pathogenicity-related genes as well as functional genomic analysis in Xoo and Xcc.     

Isolation and characterization of a generalized transducing phage for Xanthomonas campestris pv. campestris.

We have isolated and characterized a lytic double-stranded DNA Xanthomonas campestris pv. campestris bacteriophage (XTP1) capable of mediating generalized transduction. The phage transduces chromosomal markers at frequencies of 10(-5) to 10(-6) transductants per PFU. We demonstrated its genetic utility by the isolation and cotransduction of linked transposon insertions to a nonselectable locus, xgl, required for the cleavage of 5-bromo-3-chloro-indoyl-beta-D-galactoside and showed that rif and str alleles in X. campestris are 75% linked. One-step growth experiments showed that the latent and rise periods were each 2 h and the average burst size was 35. The DNA genome is approximately 180 kb, presumably modified in a sequence-specific manner, and may be covalently attached to protein(s). Electron micrographs show the phage particle to have an icosahedral head and contractile tail with tail fibers uniquely attached to a location 40 nm proximal from the end of the tail.     

外源二甲基砷对油菜生长及土壤中砷生物有效性的影响

通过盆栽试验研究了土壤中添加外源二甲基砷(DMA)对油菜(Brassica campestris)生长及土壤中砷生物有效性的影响.结果表明:随着外源DMA添加量的增加,油菜的出苗率和生物量均在一定程度上表现出了低浓度促进而高浓度抑制的现象.当外源DMA添加量达到90 mg·kg-1时,第2季盆栽油菜的出苗率和生物量与对照相比分别下降了9.5%和57.0%,表明DMA对油菜生长的影响具有长期性;随着外源DMA浓度的增加,土壤中有效态砷及油菜体内的砷含量均表现出增加的趋势,相关性分析表明,该三者间具有极显著相关关系;添加入土壤中的DMA主要发生去甲基化作用,产物主要为As(V)及少量的As(Ⅲ),且随着外源DMA添加量的增加,As(V)和As(Ⅲ)的浓度均表现出增加的趋势.     

Microspore Culture of Hybrids Between Brassica napus and B. campestris

Embryos and regenerated plants were produced by isolated microspore culture of inter-specific hybrids between Brassica napus and B. campestris. The NLN media with different sucrose concentrations and pH values were tested and a protocol for optimal microspore culture of B. carnpestris was identified. The reciprocal hybrids between UM921 (B. campestris) and 911186 (B. napus) had significant higher embryo yield than other cultured hybrids. Obvious improvement of embryo yield and quality was achieved when hybrid plants of reciprocal UM921 × 911186 were grown under 10 ℃/5 ℃ (day/night) condition. There was significant correlation between embryo yield and seeds per pod on hybrid plants but no correlation between pollen fertility and embryo yield was detected among cultured.hybrids. The majority of microspore-derived plants from the reciprocal B. napus × B. campestris hybrids are aneuploids and 22.8% of the plants observed originated from the microspores with parent′s chromosome numbers, almost all n = 19. The factors affecting the embryogenesis in microspore culture of interspecific hybrids and the possible applications of the technique are discussed.     

野油菜黄单胞菌8004甘天丝亮特征序列酯酶及其酯酶结构域在大肠杆菌中的表达，包涵体复性及性质

【目的】了解野油菜黄单胞菌（Xanthomonas campestris pv. campestris）8004 GDSL(蛋白序列中甘氨酸、天冬氨酸、丝氨酸和亮氨酸特征序列)酯酶的性质。【方法】利用PCR方法扩增Xcc_est及其不同结构域的基因,这些基因以组氨酸标签融合蛋白的形式在大肠杆菌中获得表达。融合蛋白通过镍亲和色谱纯化。【结果】部分纯化的Xcc_est 在催化对硝基苯丁酸酯时,最适pH值为8.0,最适温度为52 °C。 Xcc_est 对于对硝基苯丁酸酯的Km值和Vmax值分别是47.6 ± 4.6 μmol/L, 和67.6 ± 7.8 U/mg,Xcc_est的酯酶结构域(Xcc_estN1-334)对于同一底物的Km值和Vmax值分别是 469.4 ± 9.8 μmol/L和2.5 ±0.9 U/mg。Xcc_est的成熟结构域(Xcc_estN26-606)可以获得成功复性,但是成熟酯酶结构域(Xcc_estN26-334)不能获得复性。复性后的Xcc_estN26-606底物谱较广,在室温下具有较高稳定性。【结论】复性的成熟结构域蛋白(Xcc_estN26-606)具有一定的生物转化应用前景。