大肠杆菌不耐热肠毒素(LT)突变体LTR72的表达与鉴定

将重组质粒PLTR72和PLT分别转化大肠杆菌JM101，DH5α和C600，并接种于LB和CAYE-2中培养，用GM1-ELISA法检测菌体裂解液中2者的表达情况，将表达产物纯化后用SDS-PAGE等方法鉴定。结果表明，3种大肠杆菌在2种培养基中都可表达出LT突变体和LT，并可将产物分泌到大肠杆菌胞周质中，成为具有GM1结合活性的突变体蛋白，鉴定纯化后的突变体的组成形式与野生型LT相同。均是1A：5B。     

Sensitivity to fluopicolide of wild type isolates and biological characteristics of fluopicolide-resistant mutants in Pseudoperonospora cubensis

Cucurbit downy mildew caused by the oomycete pathogen Pseudoperonospora cubensis is a devastating disease that is distributed worldwide and affects cucumber in open fields and greenhouses. Fluopicolide, which was a novel systemic fungicide and was released in 2008, it is very effective in controlling downy mildew on cucumber and grape, potato late blight and pepper Phythophthora blight and reduces the loss caused by the diseases, but so far the potential for P. cubensis to develop resistance to fluopicolide has not been investigated. Hence, a laboratory study was undertaken to assess the risk of P. cubensis developing resistance to fluopicolide. Baseline sensitivity to fluopicolide was determined by using 75 P. cubensis isolates collected from cucumber-growing greenhouses in Hebei province, where no fluopicolide had been used for control of cucumber downy mildew before. Values of effective concentrations for 50% inhibition (EC₅₀) of sporulation ranged from 0.02 to 0.40 μg ml⁻¹ and were distributed as a unimodal curve, indicating that all 75 isolates were sensitive to fluopicolide. Sporangia of nine sensitive isolates were ultraviolet (UV)-irradiated, and four fluopicolide-resistant mutants were acquired at a mutation frequency of 7.4 × 10⁻⁷. Seven mutants resistant to fluopicolide were obtained from seven isolates by sporangia adaptation on fluopicolide-treated leaves of cucumber. The EC₅₀ values for all eleven fluopicolide-resistant mutants ranged from 3.37 to 13.06 μg ml⁻¹ with mean resistance factors of 7.9–118.0. After 10 sporangia transfers on fungicide-free leaves of cucumber, all eleven resistant mutants remained resistant to fluopicolide with mean resistance factors of 8.2–81.3. Seven resistant mutants from the selection for resistance and one resistant mutant from UV mutagenesis exhibited stable resistance; however, the other three resistant mutants from UV irradiation became significantly less resistant. Compared to their respective sensitive parents, the eleven resistant mutants exhibited diversity in latent period, infection frequency, lesion extension and sporulation ability. Five out of the eleven resistant mutants exhibited prolonged latent period and three out of the eleven resistant mutants provided decreased infection frequency (IF) compared to their respective parents, indicating that in some cases, resistance mutation might affect the latent period and IF of P. cubensis. There were significant differences in pathogenicity and ability to produce sporangia, but this seemed not to be caused by resistance mutation. No cross-resistance was detected between fluopicolide and azoxystrobin, metalaxyl, dimethomorph, or cymoxanil. In all, there could be a moderate to high risk of field populations of P. cubensis developing resistance to fluopicolide, and populations of P. cubensis should be monitored regularly for their shift of sensitivity over years of application.     

一份新的水稻斑点叶突变体spl32的鉴定和基因定位

从F2(粤晶丝苗2号/H4)群体中,鉴定出一份显性斑点叶突变体spl32(spotted leaf 32)。其叶片褐色斑点受自然光诱导,在幼穗分化期从叶尖逐渐扩散至叶鞘,台盼蓝染色表明斑点并非由细胞死亡引起。以从F5杂合个体分离出的正常叶色植株为对照,斑点叶植株的穗粒数、结实率显著降低。斑点出现后,spl32的POD活性和MDA含量均显著高于对照;同时,spl32叶片光合色素含量降低,但荧光动力学参数并无显著变化。抽穗期人工接菌表明,spl32对水稻白叶枯病菌抗性较对照显著提高。遗传分析表明spl32斑点性状由一个显性基因Spl32(t)控制,利用F2(02428/spl32)群体将其定位在第11染色体Ind-c和RM206之间,推测该基因为一个新的水稻斑点叶基因。     

嗜酸乳杆菌突变株产共轭亚油酸异构酶的纯化研究

以嗜酸乳杆菌NCFM和Lakcid作为出发菌株,通过紫外诱变及化学诱变筛选得到突变株.代谢亚油酸生成共轭亚油酸(CLA),研究转化过程中的关键性酶-亚油酸异构酶的酶学性质,采用硫酸铵沉淀、透析等对该酶进行纯化,用SDS-PAGE测得该酶的亚基分子量为60.5 ku;并得出该酶的最适温度是44℃,最适pH是6.0左右,金属离子Fe2+、K+对异构酶的反应有促进作用,Zn2+等对反应都起抑制作用.     

根癌农杆菌介导的真菌遗传转化研究进展

 农杆菌介导遗传转化成为真菌实现菌种改良、新基因标记及目的基因的筛选、分离和克隆的重要方法之一。该方法操作简单、受体范围广、转化效率高、单拷贝率高及突变体遗传稳定,解决了传统转化方法的瓶颈。利用该方法成功实现了多种真菌大容量高质量突变体库的构建,且在各类真菌突变体库构建的基础上,相关基因的筛选和功能基因的验证工作成为下一步的研究热点,进而为各类病原菌致病机理的深入研究提供依据,为抗病育种等亟待解决的问题奠定基础。     

一株灰葡萄孢细胞壁完整性缺陷突变株的分子鉴定和表型分析

为探明灰葡萄孢细胞壁相关基因功能,用荧光增白剂Calcofluor White（CFW）从灰葡萄孢T-DNA插入突变体库中筛选细胞壁完整性缺陷突变株,发现一株突变株D-59对CFW的敏感性与野生型相比提高了2.8倍。通过TAIL-PCR扩增突变株的T-DNA插入位点的侧翼序列并对其进行序列分析表明,T-DNA插入于突变株BC1G₀0770.1基因的外显子部位。RT-PCR的结果显示,D-59的BC1G₀0770.1基因不表达。突变株D-59的表型分析表明,突变株的菌丝稀疏,菌落呈土黄色,产孢量明显下降,孢子萌发异常,致病能力减弱,细胞壁的几丁质含量下降了48%。     

Flower color chimera and abnormal leaf mutants induced by C heavy ions in Salvia splendens Ker-Gawl.

The effect of ¹²C⁶⁺ heavy ions bombardment on mutagenesis in Salvia splendens Ker-Gawl. was studied. Dose–response studies indicated that there was a peak of malformation frequency of S. splendens at 200 Gy. Abnormal leaf mutants of the bileaf, trileaf and tetraleaf conglutination were selected. Meanwhile, a bicolor flower chimera with dark red and fresh red flower was isolated in M1 generation of S. splendens. Random amplified polymorphic DNA (RAPD) analysis demonstrated that DNA variations existed among the wild-type, fresh and dark red flower shoots of the chimera. The dark red flower shoots of the chimera were conserved and cultivated at a large-scale through micropropagation. MS supplemented with 2.0 mg/L BA and 0.3 mg/L NAA was the optimal medium in which the maximum proliferation ratio (5.2-fold) and rooting rate (88%) were achieved after 6 weeks. Our findings provide an important method to improve the ornamental quality of S. splendens.     

Participation of Xanthomonas axonopodis pv. citri hrp cluster in citrus canker and nonhost plant responses

The participation of the Xanthomonas axonopodis pv. citri hypersensitive response and pathogenicity ( hrp ) cluster in interactions with host and nonhost plants was characterized in pathogenicity and avirulence models. The hrp cluster encodes the type III secretion system indispensable for trafficking of proteins to the plant cell. Mutations in operons hrpB and hrpD and the hrpF gene failed to produce canker in citrus plants or hypersensitive response in cotton plants. The interaction of the phytopathogen with various nonhost plants has been characterized . The results showed that the hypersensitive response is activated in leaves of cotton, bean, tobacco, tomato, pepper and Nicotiana benthamiana , and that genes present in operons hrpB and hrpD and the hrpF gene are required for pathogenicity in hosts and induction of the hypersensitive response in nonhost plants.     

Mating Behaviour and Vegetative Compatibility in Spanish Populations of Botryotinia fuckeliana

Mating behaviour and vegetative compatibility were studied in Spanish populations of Botryotinia fuckeliana. Fifty-seven isolates out of the 61 tested were sexually fertile with one or more of the reference strains of known mating type (MAT1-1 or MAT1-2). Thirty-nine isolates were heterothallic, giving fertile crosses when mated with the MAT1-1 (24 isolates) or the MAT1-2 (15 isolates) reference strain. Eighteen isolates crossed successfully with both MAT1-1 and MAT1-2 reference strains, and were referred to as homothallic or MAT1-1/2. Both mating types were widespread, being represented in isolates from two regions, from the same and different greenhouses, from different hosts, and from different years of isolation. Isolates were paired on Malt Extract Agar + NaCl to evaluate vegetative compatibility. Most of the paired isolates were unable to fuse and showed a different reaction of incompatibility. Nitrate-non-utilising (nit) mutants were selected by growth on a medium amended with 30–50gl^–1 potassium chlorate. Over 600 chlorate-resistant sectors were recovered from 40 isolates at a mean frequency of 0.15–2.39 sectors per colony, but only 11% were identified as nit mutants by their thin growth with no aerial mycelium on minimal medium. However, most of these nit mutants reverted to wild type during six months of storage on chlorate-amended medium. Genetic complementation between nit mutants occurred only in two cases between mutants from the same isolate.