Efficient transformation and expression of gfp gene in the edible mushroom Pleurotus nebrodensis

An efficient transformation method mediated by PEG-protoplasts was developed for the newly commercial edible mushroom Pleu-rotus nebrodensis. Two plasmids were used to co-transform protoplasts of P. nebrodensis. One plasmid is pAN7-1 containing a positive selectable marker gene hph conferring hygromycin B resistance. Another plasmid is pBIue-GFP containing a reporter gene gfp conferring green fluorescent protein. PCR and Southern blot analysis showed that hph gene or/and gfp gene were integrated into the genome of P.nebrodensis transformants. The transformation efficiency of the positive selectable marker gene hph was 3 transformants per microgram of plasmid pAN7-1 DNA, which was about 30 times higher than that previously reported in thoroughly studied Pleurotus species such as Pleurotus ostreatus. The transformation efficiency of the reporter gene gfp was 9 transformants per microgram of plasmid pBlu-GFP DNA. The co-transformation efficiency was 23.68%. This is the first report that a "reporter" gene, green fluorescent protein gene can be successfully stably expressed in this Pleurotus species.     

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum

Agrobacterium tumefaciens-mediated transformation （ATMT） system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction （PCR） amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.     

真菌纤维二糖水解酶基因的克隆与表达研究

采用能够转化细菌和真菌的双功能质粒ｐＤＬ１，与部分酶切的糙皮侧耳（Ｐｌｅｕｒｏｔｕｓｏｓｔｒｅａｔｕｓ）基因组ＤＮＡ片段进行体外重组。再用重组质粒ＤＮＡ转化玉米黑粉菌（Ｕｓｔｉｌａｇｏ，ｍａｙｄｉｓ）的原生质体，在平皿上筛选抗新霉素的转化子，转化率达８００转化子／μｇＤＮＡ，由此在玉米黑粉菌中建立了糙皮侧耳的基因文库。随机选出１５个转化子提取其ＤＮＡ，以３２Ｐ标记的ｎｅｕｒ基因酶切片段作为探针进行打点杂交，全部显示阳性，证明抗性菌落不是来源于敏感细胞的回复突变。以李氏木霉（Ｔｒｉｃｈｏｄｅｒｍａｒｅｅｓｅｉ）的纤维二糖水解酶（Ｃｅｌｌｏｂｉｏｈｙｄｒａｓｅｓ，简称ＣＢＨ）ＣＢＨⅡ基因末端片段为探针，从阳性克隆菌株中提取ＤＮＡ进行Ｓｏｕｔｈｅｒｎ转移并杂交，结果证明本研究已成功地克隆了糙皮侧耳纤维二糖水解酶ＣＢＨⅡ基因。基因的表达检测证明，克隆菌株具有纤维二糖水解酶活力。     

Green fluorescent protein （GFP） as a vital marker for studying the interaction of Phytophthora sojae and soybean

Transgenic Phytophthora sojae strains that produce green fluorescent protein （GFP） were obtained after stable DNA integration using the Hsp70 promoter and the Ham34 terminator of Bremia lactucae. The expression of GFP during different developmental stages of P. sojae was observed using fluorescent microscopy. Based on this reporter system, the histopathologic events caused by the pathogen in soybean leaves, hypocotyls and roots were monitored. Meanwhile, the difference in resistance between different soybean cultivars against P. sojae was analyzed microscopically in roots. The results indicate that GFP can be stably expressed in zoosporangia, zoospores, cysts, hyphae and oospores of P. sojae. Using the GFP marker, the infecting pathogens in leaves, hypocotyls and roots of host could be distinctly visualized. The germ tube length of cysts germinating on the roots of resistant cultivar Nannong 8848 was longer than that on the roots of susceptible cultivar Hefeng 35. These results show for the first time that this eukaryotic reporter can be used in P. sojae as a stable and vital marker, allowing the study of genetics of this hemibiotrophic pathogen.     

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic loach (Misgurnus anguillicaudatus Cantor) have been studied. TheGFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of theGFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.     

人MiTERF3基因启动子的克隆及其荧光素酶报告基因载体的构建

人MiTERF3基因编码线粒体基因转录和能量代谢的负调控因子。采用PCR技术对人MiTERF3基因5''侧翼上游1251 bp启动子序列进行扩增,并将其克隆至荧光素酶表达载体pGL6-TA,构建人MiTERF3基因启动子荧光素酶报告基因质粒。经酶切、测序鉴定后,将其用脂质体转染体外培养的HEK293细胞株,利用双荧光素酶测定系统检测其表达活性。研究结果表明,克隆获得的1251 bp DNA序列与GenBank报道的一致,且插入方向正确。含人MiTERF3基因启动子的报告基因荧光素酶的表达活性显著提高（P<0.05）,约为对照组（空载体pGL6-TA）的9.8倍。本研究通过对人MiTERF3基因启动子的克隆及其荧光素酶表达载体构建与表达活性的测定,为进一步阐明人MiTERF3基因表达的调控机制奠定实验基础。     

Introduction of exogenous DNA into cotton via the pollen-tube pathway with GFP as a reporter

The green fluorescent protein (GFP) gene from the jellyfishAequorea victoria as a vital reporter for gene expression in plants is considered to have several advantages over other reporter genes. The pBIN35S-mGFP4 plasmid DNA has been introduced into cotton embryos by the pollen-tube pathway method. A transformed seedling has been verified according to its GFP-related fluorescence and Southern blotting analysis. The results provided direct and convincing facts in cytology and molecular biology for the pollen-tube pathway method, an efficient transformation technique used in plants.     

产丁醇大肠杆菌工程菌的构建

【目的】为了在大肠杆菌(Escherichia coli)中导入改良的丁醇合成途径,使非生产菌株大肠杆菌具备产丁醇的能力。【方法】克隆大肠杆菌乙酰转移酶基因atoB和丙酮丁醇梭菌(Clostridium acetobutylicum)丁醇合成途径关键酶基因(crt、hbd、adhE),构建多顺反子表达质粒pSE380-atoB-adhE-crt-hbd;克隆齿垢密螺旋体(Treponema denticola)反式烯酰辅酶A还原酶基因ter,构建表达质粒pSTV29-ter,并将双质粒导入到大肠杆菌。【结果】构建的工程菌能半厌氧发酵产微量丁醇,产量为0.08g/L。【结论】大肠杆菌中的丁醇合成途径导入成功,构建了产丁醇的大肠杆菌工程菌。     

The expression of vasa gene during zebrafish ( Danio rerio ) oogenesis

vasa gene expression pattern during oogenesis of zebrafish was examined usingin situ hybridization and fluorescent quantitative RT-PCR. During zebrafish oogensis,vasa mRNA is expressed strongly and uniformly distributed in the cytoplasm in stage II oocytes, followed by a distribution among vacuome in stage III. Later in stage IV and V,vasa mRNA is enriched at the cortex and finally localized at the cortex. The fluorescent quantitative RT-PCR shows that the quantity ofvasa mRNA decreases from stage II to stage III, but remains relatively invariable from stage III to stage V. The observed differences invasa mRNA expression in the different stages of zebrafish oogenesis suggest thatvasa gene plays an important role during oogenesis. Foundation item: Supported by the National Natural Science Foundation of China (30370744, 30150005) Biography: XIANG Fang (1979-), male, Master candidate, research direction: molecular development of animals.     

基于数据库分析FOXG1在非小细胞肺癌中的表达和临床意义及稳定过表达FOXG1肺癌细胞株A549的构建

通过TCGA数据库分析FOXG1在非小细胞肺癌中的表达及预后相关性，建立稳定过表达人源FOXG1基因的肺癌细胞株A549。利用TCGA数据库中下载基因表达数据和临床信息，分析FOXG1在非小细胞肺癌和正常组织的表达差异、FOXG1表达水平与临床病理特征及生存预后的相关性并进行基因集富集分析。通过HEK-293T包装慢病毒表达载体，收集病毒上清液侵染A549细胞,嘌呤霉素筛选稳定过表达FOXG1的A549细胞株。细胞核染色鉴定外源FOXG1表达定位，Western blot检测外源FOXG1的表达情况。结果发现FOXG1在非小细胞肺癌组织中高表达，且FOXG1高表达能够降低患者总体生存率。FOXG1的表达水平与患者年龄相关，与性别，分级以及TMN分期无关；细胞周期、P53、Notch等信号通路在高表达FOXG1的非小细胞肺癌组织中被激活；慢病毒表达载体共转染HEK-293T细胞成功；病毒上清液侵染A549细胞，24 h后可见绿色荧光表达，72 h后对照组空载体病毒颗粒侵染效率高达80%左右，实验组过表达FOXG1病毒颗粒侵染效率为50%~60%；经嘌呤霉素筛选培养后，对照组和实验组荧光效率均达到90%以上；细胞核染色外源FOXG1基因定位在细胞核中，Western blot结果显示外源FOXG1在细胞中正确表达。因此可以认为FOXG1基因在非小细胞肺癌患者中高表达，其表达水平与患者总体预后有关且稳定表达FOXG1的A549肺癌细胞株构建成功。     

菠菜叶绿体基因组定点整合表达载体构建及原核表达

分析菠菜叶绿体基因组全序列,选用了rbcL基因和accD基因的间隔区作为外源基因的定点整合位点,并从菠菜叶绿体基因组中克隆了rbcL基因全长和accD基因的5′端部分,长度分别为1956 bp和1320 bp。以这2个DNA片段作为同源重组片段,以烟草叶绿体基因的启动子Prrn和终止子psbA3′控制外源基因的转录,构建了包含筛选标记基因aadA基因(编码氨基糖苷-3′-腺苷酸转移酶,具有壮观霉素和链霉素抗性)和报告基因GFP(编码绿色荧光蛋白)的菠菜叶绿体基因组定点整合表达载体pRAGA。酶切结果显示构建正确。将该载体转化大肠杆菌,在激光扫描共聚焦显微镜下用488 nm蓝光激发,发现大肠杆菌发出强烈的绿色荧光,而对照菌体没有荧光,表明GFP基因在原核大肠杆菌中已经成功表达。实验结果说明构建的菠菜叶绿体定点整合表达载体pRAGA可以用于菠菜叶绿体转化。     

海绵Pseudoceratina sp.共栖细菌多样性及其抑制甘蔗鞭黑粉菌活性研究

【目的】探讨广西斜阳岛附近海域海绵共附生细菌的多样性,并筛选对甘蔗鞭黑粉菌有抑制作用的活性物质,为发现潜在的细菌新物种及开发农用生防菌肥奠定基础。【方法】采用稀释涂布法,通过6种复合营养分离培养基,从海绵Pseudoceratina sp.中分离可培养的共附生细菌。采取细菌形态学观察去除冗余,通过PCR扩增菌株的16SrRNA基因序列,并通过构建系统发育树分析物种多样性。采用牛津杯法进行抑制甘蔗鞭黑粉菌活性筛选实验。【结果】从海绵Pseudoceratina sp.中共分离到可培养细菌54株,经16SrRNA基因序列对比后,获得24株细菌,隶属于13科14属。其中,菌株BGMRC 2118与已发表有效菌株的16SrRNA基因序列的最高相似率为96.46%,可能为潜在新种。抑菌筛选实验结果显示,有8株细菌的发酵粗提物对甘蔗鞭黑粉菌具有很强的抑菌活性。【结论】广西斜阳岛附近海域中海绵共附生细菌具有较高的物种多样性,蕴藏着丰富的新物种资源,富含生物活性菌株。     

金针菇杏鲍菇素肠的制作工艺研究

以金针菇和杏鲍菇为原料，选择菌菇质量比、大豆蛋白添加量、玉米淀粉添加量、卡拉胶添加量等四个因素设计单因素及正交实验，通过感官评定确定金针菇杏鲍菇素肠的最佳工艺条件，同时分析其硬度、弹性和咀嚼性等指标。金针菇杏鲍菇素肠的最佳制作配方：金针菇与杏鲍菇质量比3：3、大豆蛋白6 g、玉米淀粉18 g、卡拉胶1.6 g。该条件下制得的金针菇杏鲍菇素肠组织紧密，有弹性，具有适宜的金针菇和杏鲍菇香气，咸香可口。     

Overexpression of rice OsLOL2 gene confers disease resistance in tobacco to Pseudomonas syringae pv. Tabaci

LSD1-related proteins of Arabidopsis with LSDl-like zinc finger domains regulate disease resistance and programmed cell death （PCD）. We cloned a rice OsLOL2 gene, orthologous to LSD1 of Arabidopsis and expressed it in a tobacco plant. Transgenic tobacco lines displayed enhanced disease resistance to a virulent bacterium Pseudomonas syringae pv. tabaci （Pst）. RT-PCR analysis showed that overexpression of OsLOL2 in transgenic tobacco lines resulted in upregulation of two pathogenesis-related （PR） protein genes, PR2 and PR5. Our results suggest that overexpression of OsLOL2 in transgenic tobacco enhances the resistance through the induction of PR proteins and hypersensitive response-like reaction.     

小单孢菌40027菌株内源质粒pJTU101的特性及其抗性标记

从稀有放线菌小单孢菌40027菌株中分离到2个内源质粒p JTU101和p JTU112,用Bam H I单酶切质粒p JTU101确定其大小,通过同源片段之间发生单交换实现内源质粒p JTU101的抗性标记,并对其稳定性进行了初步研究.结果表明:小单孢菌40027菌株内源质粒p JTU101拷贝数为1~2,大小约为36.6 kb,被阿泊拉霉素抗性基因标记且在小单孢菌40027菌株中比较稳定.     

酸处理对栽培条斑紫菜及其附生缘管浒苔的影响

酸处理是清除条斑紫菜(Neopyropia yezoensis)栽培过程中附生绿藻的主要方法之一，酸处理时间与pH值是影响条斑紫菜与绿藻活性的重要因素。本研究以半浮式紫菜筏架上的条斑紫菜及其附生缘管浒苔(Ulva linza)为研究对象，研究40 s浸泡时间下不同pH值的盐酸海水溶液对条斑紫菜和缘管浒苔的影响。结果显示，pH值对缘管浒苔的影响大于条斑紫菜，盐酸海水溶液浸泡40 s时间下杀灭紫菜附生缘管浒苔的最佳pH值为1.9。酸处理7 d后，条斑紫菜pH 1.9处理组的超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性、丙二醛(MDA)含量、最大光化学量子效率(Fv/Fm)值以及叶绿素a(Chl a)与类胡萝卜素(Car)含量与酸处理前相比均无显著差别，而缘管浒苔的SOD、POD活性和MDA含量均显著降低，Fv/Fm值下降到0.2以下，Chl a与Car含量下降为0;在pH 1.7及以下处理组中，条斑紫菜与缘管浒苔以上生理参数与物质含量均显著低于初始值。据此并总结前人研究，推导出在pH值为0.3-1.9时，杀灭条斑紫菜附生缘管浒苔并保持紫菜活性的线性回归方程y(时间，s)=21.6...     

Tup1基因缺失对酿酒酵母耐高糖性状的影响

研究Tup1基因对酿酒酵母（Saccharomyces cerevisiae）细胞耐高糖性状的影响。利用Cre/loxP系统对单倍体细胞中的转录因子Tup1基因进行敲除,单倍体细胞复倍后研究基因缺失菌株的性状变化。结果表明,Tup1基因缺失虽然减弱了菌株的呼吸能力,但却增强了菌株在高浓度葡萄糖培养基中的生长和发酵能力。Tup1基因可能通过调节酿酒酵母细胞呼吸强度来调控细胞的高糖应激反应。