Investigation of the biological roles of autophagy in appressorium morphogenesis in Magnaporthe oryzae

Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentiation in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.     

用抑制差减杂交技术分离烯丙异噻唑诱导水稻特异表达的基因

烯丙异噻唑(PBZ)处理水稻根能使其产生对稻瘟病的系统获得性抗性,因此在东南亚稻区被广泛用于防治稻瘟病,然而关于其作用的分子机理还知之甚少．运用抑制差减杂交技术,试图通过分离鉴定受PBZ诱导调控的关键基因,探索其作用的分子机理．以PBZ处理后的水稻叶片cDNA为目标群体(tester),以未处理水稻叶片cDNA为对照群体(driver),用经过对照cDNA差减的、烯丙异噻唑处理的cDNA群体构建了一个含260个重组子的差减文库．通过差示筛选鉴定出了26个。PBZ诱导水稻特异表达和增强表达的候选克隆．对26个cDNA克隆进行了双向测序和同源性比较,发现其中3个克隆：rJAB1,rTAB2和蛋白磷酸酯酶2Aδ调节亚基同型物基因,位于抗病相关信号转导途径上,它们与哺乳动物和人类免疫途径上的信号因子有明显相似之处,因此推断可能与诱导抗性有关．另外8个克隆与已知基因同源性为70％～99％．经Northern杂交分析,其中rJAB1(编码c-jun激活区结合蛋白1)受烯丙异噻唑和稻瘟菌诱导表达;膜糖蛋白同源基因及肌动蛋白(actin)α1受烯丙异噻唑诱导表达,部分克隆为低丰度转录本．     

抗稻瘟病水稻BAC文库的构建与鉴定

水稻是一种重要的粮食作物，同时也是一种重要的单子叶植物模式.稻瘟病是水稻生长中的一种严重病害，因此分离和克隆新的稻瘟病抗病基因具有重要的应用价值.以一个抗稻瘟病农家种水稻为材料，以plndigoBAC5为载体，构建了细菌人工染色体（BAC）文库.该文库共有90 000个转化子，插入频率99％，插入片段平均长度为105 kb，由此推测这个文库覆盖水稻基因组约20倍.利用其中的45 000个转化子建立了4维PCR筛选体系，并且通过4维PCR筛选体系，筛选获得了7个含水稻分蘖基因（MOC1）的阳性克隆.因此该文库是一个高质量、高覆盖率的水稻BAC文库，能有效用于目的基因的分离.     

A Gγ subunit promoter T-DNA insertion mutant――A1-412 of Magnaporthe grisea is defective in appressorium formation, penetration and pathogenicity

Heterotrimeric G-proteins consisting of α, β and γ-subunits are essential for the transduction of ex- tracellular signals to various downstream intracellular effectors in eukaryotes. Previous studies showed that Gα and Gβ were involved in regulating     

MoFLP1, encoding a novel fungal fasciclin-like protein, is involved in conidiation and pathogenicity in Magnaporthe oryzae

Fasciclin family proteins have been identified as cell adhesion molecules in various organisms. In this study, a novel Magnaporthe oryzae fasciclin-like protein encoding gene, named MoFLP1, was isolated from a subtractive suppressive cDNA library and functionally analyzed. Sequence analysis showed that the MoFLP1 gene contains an open reading frame (ORF) of 1050 nucleotides encoding 349 amino acids with a calculated molecular weight of 35.85 kDa and a pI of 7.76. The deduced MoFLP1 protein contains a 17-amino acid secretion signal sequence and an 18-amino acid sequence with the characteristics of a glycosylphosphotidylinositol (GPI) anchor additional signal at its N- and C-terminuses, respectively. Potential N-glycosylation sites and domains involving cell adhesion were also identified in MoFLP1. Sequence analysis and subcellular localization by the expression of MoFLP1-GFP fusion construct in M. oryzae indicated that the MoFLP1 protein is probably localized on the vacuole membrane. Two MoFLP1 null mutants generated by targeted gene disruption exhibited marked reduction of conidiation, conidial adhesion, appressorium turgor, and pathogenicity. Our results indicate that fasciclin proteins play important roles in fungal development and pathogenicity in M. oryzae.     

稻瘟菌致病性变异突变体的筛选及共分离分析

通过筛选稻瘟菌(Magnaporthe grisea)P131小种的REMI(Restriction Enzyme MediatedIntegration)转化体库获得对水稻品种梅雨明致病性变异的突变体,命名为PX1.与野生型菌株P131相比,该突变体对水稻品种梅雨明致病性丧失,在洋葱表皮上侵染钉形成率显著降低,而孢子萌发率和附着胞形成率差异不显著.遗传分析表明,该突变体的突变表型和潮霉素抗性标记共分离,说明突变是由于外源质粒插入引起的,因此,可以此为标记克隆控制该表型的基因.     

Expression, purification and characterization of a phyAm-phyCs fusion phytase

The phyA^m gene encoding acid phytase and optimized neutral phytase phyCs gene were inserted into expression vector pPIC9K in correct orientation and transformed into Pichiapastoris in order to expand the pH profile ofphytase and decrease the cost of production. The fusion phytase phyA^m-phyCs gene was successfully overexpressed in P. pastoris as an active and extracellular phytase. The yield of total extracellular fusion phytase activity is （25.4±0.53） U/ml at the flask scale and （159.1±2.92） U/ml for high cell-density fermentation, respectively. Purified fusion phytase exhibits an optimal temperature at 55 ℃ and an optimal pH at 5.5-6.0 and its relative activity remains at a relatively high level of above 70% in the range ofpH 2.0 to 7.0. About 51% to 63% of its original activity remains after incubation at 75 ℃ to 95 ℃ for 10 min. Due to heavy glycosylation, the expressed fusion phytase shows a broad and diffuse band in SDS-PAGE （sodium dodecyl sulfate-polyacrylamide gel electrophoresis）. After deglycosylation by endoglycosidase H （EndoHf）, the enzyme has an apparent molecular size of 95 kDa. The characterization of the fusion phytase was compared with those ofphyCs andphyA^m.     

Adiponectin levels are associated with the number and activity of circulating endothelial progenitor cells in patients with coronary artery disease

Objective: To study the relationship between plasma adiponectin concentration and the functional activities of circulating endothelial progenitor cells (EPCs) in patients with coronary artery disease (CAD). Methods: Circulating EPCs were enumerated as AC133⁺/KDR⁺ cells via flow cytometry and identified by co-staining with DiI-acLDL and fluorescein isothiocyanate (FITC)-conjugated lectin under a fluorescent microscope. The migratory capacity of EPCs was measured by modified Boyden chamber assay. Adhesion capacity was performed to count adherent cells after replating EPCs on six-well culture dishes coated with fibronectin. Results: The number of circulating EPCs (AC133⁺/KDR⁺ cells) decreased significantly in CAD patients, compared with control subjects [(74.2±12.3) vs (83.5±12.9) cells/ml blood, P<0.01]. In addition, the number of EPCs also decreased in CAD patients after ex vivo cultivation [(54.4±8.6) vs (71.9±11.6) EPCs/field, P<0.01]. Both circulating EPCs and differentiated EPCs were positively correlated with plasma adiponectin concentration. The functional activities of EPCs from CAD patients, such as migratory and adherent capacities, were also impaired, compared with control subjects, and positively correlated with plasma adiponectin concentration. Conclusion: The study demonstrates that the impairment of the number and functional activities of EPCs in CAD patients is correlated with their lower plasma adiponectin concentrations.     

Use of suppression subtractive hybridization strategy for cloning and identifying specifically expressed genes of renal cell carcinoma

Using suppression subtractive hybridization, a renal cell carcinoma (RCC) cDNA subtractive library which only contains differently expressed cDNAs between human RCC and normal kidney has been constructed. 200 clones were picked out randomly to perform enzyme digest analysis, a part of them underwent sequence analysis and Northern blot to identify RCC specially expressed genes. Results showed that 190 clones contain 50—400 bp inserts respectively. Sequence analysis was performed in 10 clones. All the 10 sequences were unknown before and derived from 6 unique novel genes among which the cDNA insert RCC18 has five copies. Northern blot analysis showed that RCC18 cDNA expressed highly in RCC, but there was no signal detected in normal kidney, and the full length of RCC18 was about 2.5 kb. The constructed cDNA subtractive library of human RCC is a highly efficient one and lays the solid foundation for large-scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specially expressed genes provided an important clue for researching the mechanism of the occurrence and development of RCC.     

用差减cDNA文库筛选由热激导致的小鼠睾丸中差异表达的基因

采用抑制性差减杂交（Suppression Subtractive Hybridization, SSH）方法,构建正常和热激小鼠睾丸组织的差减 cDNA 文库,以期 筛选出小鼠生精过程中对热敏感的基因。分别从正常和热激小鼠睾丸组织提取总RNA,反转录成cDNA,以正常小鼠睾丸组织cDNA作为待检组织(tester),以热激小鼠睾丸组织cDNA作为驱动组织(driver), 经过2轮杂交和抑制性PCR扩增,产物与T载体连接,经蓝白斑筛选,提取质粒,经EcoRI酶切鉴定插入片段并测序,由此构建了2种组织间差异表达基因的差减cDNA文库。用半定量RT-PCR方法,进一步验证 了该文库中差减出的基因基本上为差异表达的基因。对随机挑选其中的932个克隆测序,对有效测序的565个基因序列与GenBank数据库中发布的序列进行同源性比较,大部分片段都可以检索到同源序列。 结果显示,cPGES/p23等13个基因热激后表达量上调;septin2等120个基因热激后表达量下调。其中cPGES/p23为本研究中首次发现的小鼠生精过程中对热敏感的基因。     

Assessment of volumetric bone mineral density of the femoral neck in postmenopausal women with and without vertebral fractures using quantitative multi-slice CT

Objective: To demonstrate the validity and reliability of volumetric quantitative computed tomography (vQCT) with multi-slice computed tomography (MSCT) and dual energy X-ray absorptiometry (DXA) for hip bone mineral density (BMD) measurements, and to compare the differences between the two techniques in discriminating postmenopausal women with osteoporosis-related vertebral fractures from those without. Methods: Ninety subjects were enrolled and divided into three groups based on the BMD values of the lumbar spine and/or the femoral neck by DXA. Groups 1 and 2 consisted of postmenopausal women with BMD changes <−2SD, with and without radiographically confirmed vertebral fracture (n=11 and 33, respectively). Group 3 comprised normal controls with BMD changes ≥−1SD (n=46). Post-MSCT (GE, LightSpeed16) scan reconstructed images of the abdominal-pelvic region, 1.25 mm thick per slice, were processed by OsteoCAD software to calculate the following parameters: volumetric BMD values of trabecular bone (TRAB), cortical bone (CORT), and integral bone (INTGL) of the left femoral neck, femoral neck axis length (NAL), and minimum cross-section area (mCSA). DXA BMD measurements of the lumbar spine (AP-SPINE) and the left femoral neck (NECK) also were performed for each subject. Results: The values of all seven parameters were significantly lower in subjects of Groups 1 and 2 than in normal postmenopausal women (P<0.05, respectively). Comparing Groups 1 and 2, 3D-TRAB and 3D-INTGL were significantly lower in postmenopausal women with vertebral fracture(s) [(109.8±9.61) and (243.3±33.0) mg/cm³, respectively] than in those without [(148.9±7.47) and (285.4±17.8) mg/cm³, respectively] (P<0.05, respectively), but no significant differences were evident in AP-SPINE or NECK BMD. Conclusion: the femoral neck-derived volumetric BMD parameters using vQCT appeared better than the DXA-derived ones in discriminating osteoporotic postmenopausal women with vertebral fractures from those without. vQCT might be useful to evaluate the effect of osteoporotic vertebral fracture status on changes in bone mass in the femoral neck.     

杉木炭疽菌遗传转化及附着胞发育过程的细胞核行为观察

【目的】建立高效的杉木炭疽菌遗传转化体系,并观察附着胞发育过程中的细胞核行为。【方法】通过菌丝体酶解的方法制备原生质体,PEG介导的原生质体转化法将含有伯莱霉素抗性的细胞核表达质粒NL1::GFP转入受体材料杉木炭疽菌SMCG1#C菌株,通过荧光显微镜跟踪观察附着胞发育过程中的孢子、芽管和附着胞等结构中的细胞核行为。【结果】获得了稳定表达NL1::GFP质粒的杉木炭疽菌阳性转化子; 杉木炭疽菌附着胞发育过程伴随着细胞的有丝分裂与细胞核的转移; 附着孢发育成熟后,孢子和芽管菌丝中的细胞核仍然保持完整。【结论】建立了高效的杉木炭疽菌遗传转化体系; 杉木炭疽菌与稻瘟病菌、希金斯炭疽菌的附着胞发育过程的细胞生物学特性显著不同。     

Identification of antiviral-relevant genes in the cultured fish cells induced by UV-inactivated virus

UV-inactivated grass carp hemorrhage virus (GCHV) can induce high titer of interferon in cultured CAB (Crucian carp (Carassius auratus L.) blastulae )cells,and thus defend host cells against the virus invasion ,The mechanism is proposed that an antiviral state should be established in the host cells by activating expression of a set of antiviral-relevant genes,In this study ,suppressive subtractive hybridization is applied to constructing a subtracted ,cDNA library with mRNAs isolated from UV-inactivated GCHV infected and mock-infected CAB cells,272 differential cDNA fragments are identified by both PCR and dot blot from the subtractive cDNA library .Sequencing analysis reveals 69 genes,including 46 known gene homlologues,and 23 unknown putative genes,The known genes include the gemes involved in interferon signaling pathways,such as Stat1 and Jak1,the antiviral gences,such as Mx and Vipering,and a set of interferon-stimulated genes observed in mammalian cells. Most of the unknow putative genes contain AU-rich element in their sequences,Differential expressions of these genes are further confirmed by virtual Northern blot and RT-PCR,The data imply that UV-inactivated GCHV is not only able to induce production of interferon in the infected CAB cells,but also leads to the expression of a series of antiviral-relevant genes or immune-releveant genes,and therefore reveals that the signaling pathway of interferon system and antiviral mechanism in fish are similar to those in mammals.     

Magnaporthe oryzae MTP1 gene encodes a type III transmembrane protein involved in conidiation and conidial germination

In this study the MTP1 gene, encoding a type III integral transmembrane protein, was isolated from the rice blast fungus Magnaporthe oryzae. The Mtp 1 protein is 520 amino acids long and is comparable to the Ytp 1 protein of Saccharomyces cerevisiae with 46% sequence similarity. Prediction programs and MTP1-GFP （green fluorescent protein） fusion expression results indicate that Mtp 1 is a protein located at several membranes in the cytoplasm. The functions of the MTP1 gene in the growth and development of the fungus were studied using an MTP1 gene knockout mutant. The MTP1 gene was primarily expressed at the hyphal and conidial stages and is necessary for conidiation and conidial germination, but is not required for pathogenicity. The Amtpl mutant grew more efficiently than the wild type strain on non-fermentable carbon sources, implying that the MTP1 gene has a unique role in respiratory growth and carbon source use.     

好好芭叶片RNA提取方法和cDNA干旱消减文库的构建

介绍了从好好芭成熟叶片中提取总RNA的方法;用含有SDS的裂解液裂解细胞,用DNase除去可能出现的DNA干扰,再经植物提取试剂盒纯化后,获得了高质量的完整RNA,可用于基因编码序列的克隆和基因表达的mRNA分析等精细研究,并进一步构建了好好芭干旱消减文库.     

Construction of cDNA library from iron-deficiency induced maize roots and screening and identification of iron-stress geneFdr3

To isolate Fe-deficient related (Fdr) genes, an expression cDNA library of 4.5×10⁵ pfu/μg has been constructed from maize roots in iron-stress. 6 clones have been screened from the cDNA library by differential hybridization screening. It is proved that anFdr3 cDNA clone expressed stronger under iron-deficient condition than under iron-sufficient one by Northern blot and Western blot.     

通过差减文库筛选山羊羔肠道淋巴集结特异表达基因

羊羔肠道淋巴集合淋巴结（Peyer's patch，以下简称PP），是B淋巴细胞的主要来源地和发育、成熟场所，兼具中枢淋巴器官和周围淋巴器官的功能.采用抑制性差减杂交（suppression subtractive hybridization，SSH）方法，构建山羊羔肠道淋巴集结与其非PP肠壁组织的差减 cDNA 文库，以期找到在山羊羔肠道淋巴集结中特异表达的与免疫相关的基因. 分别从山羊羔PP与非PP肠壁组织提取总RNA，反转录成cDNA，以PP作为待检组织(tester)，非PP肠壁作为驱动组织(driver)，经过两轮杂交和抑制性PCR扩增，产物与T载体连接，经蓝白斑筛选，提取质粒,经EcoRI酶切鉴定插入片段并测序,由此构建了两种组织间差异表达基因的差减cDNA文库. 对其中160个克隆测序，得到几个可能与免疫相关的基因，为进一步从中挑选和表达活性蛋白基因用于生产免疫增强剂奠定了基础.     

Resistance to rice blast (Pyricularia oryzae) caused by the expression of trichosanthin gene in transgenic rice plants transferred through agrobacterium method

The gene of trichosanthin has been transferred into rice plants through agrobacterium method. The single copy insertion and the expression of foreign gene have been proved in regenerated plants. In antifungal assay the degrees of rice blast (Pyricularia oryzae) infection of the transgenic plants expressing trichosanthin and expressingGUS gene as control have been evaluated. The differences such as the time of disease symptom observed, the number of infected plants and damaged leaves, the growth of infected plants of the two transgenic plants after being inoculated by rice blast (Pyricularia oryzae) are significant. The transgenic plants with trichosanthin gene grew faster than the plants withGUS gene, even when humidity environment was removed. The results show that the transgenic plants that expressed trichosanthin are able to delay the infection of rice blast compared with the plants as control. In addition, no damage caused by the expression of trichosanthin gene in transgenic plants has been observed.