利用绿色荧光蛋白GFP研究稻瘟病菌与水稻的互作

稻瘟病菌Magnaporthe oryzae严重威胁水稻的产量与质量,明确稻瘟病菌与水稻互作过程及机理,对防治稻瘟病具有重要意义。本研究利用稻瘟病菌常用致病菌株GUY11和ZB25,构建了绿色荧光蛋白GFP的过量表达菌株,并通过荧光显微观察菌株侵染寄主水稻过程中侵染结构的形成与发育,包括孢子萌发、附着胞形成、侵染钉形成、侵染菌丝增殖、坏死斑形成及产孢。另外,通过比较过量表达菌株对稻瘟病高抗水稻和易感水稻的侵染过程,发现侵染过程的差异主要集中于侵染钉的穿透和侵染菌丝的定殖。本研究为分析稻瘟病菌对寄主水稻的定殖规律提供了一种有效工具。     

苏云金芽孢杆菌营养期杀虫蛋白Vips研究进展

苏云金芽孢杆菌Bt营养期杀虫蛋白Vips主要分为Vip1、Vip2和Vip33种。Vip1和Vip2构成二元毒素,对叶甲科昆虫具有特异杀虫活性。Vip3对鳞翅目昆虫具有广谱杀虫活性,该蛋白广泛存在于Bt中,与已知杀虫晶体蛋白ICPs没有序列相似性。Vip3通过诱发细胞凋亡,最终导致昆虫死亡,这与Btδ-内毒素的作用机理完全不同,Vips的发现对于Bt的进一步开发利用具有重要意义。本文主要对营养期杀虫蛋白Vips的分布、特性、作用机制及应用等进行报道。     

SIRT1 promotes autophagy of pancreatic cancer cells induced by hypoxia via regulating FOXO1/RAB7 signaling pathway

AIM: To investigate the effect of SIRT1 on the autophagy of pancreatic cancer cells under hypoxia condition, and to analyze the underlying mechanism of regulating FOXO1/RAB7 signaling pathway. METHODS: Western blot and immunofluorescence methods were used to determine the expression of SIRT1 in the pancreatic cancer cells. The small interfering RNA targeting SIRT1 and SIRT1 over-expression plasmid were transfected into the pancreatic cancer Panc-1 cells. Confocal microscopy was used to detect the LC3 expression. Western blot was used to analyze the protein levels of LC3, p62 and FOXO1/RAB7 signaling pathway-related molecules. Co-immunoprecipitation was used to detected the protein interaction between SIRT1 and FOXO1. RESULTS: The expression level of SIRT1 in the nucleus of Panc-1 cells was increased under hypoxia condition. Compared with negative control under hypoxia condition, knock-down of SIRT1 expression attenuated the autophagy flux in the pancreatic cancer Panc-1 cells (P<0.05). Over-expression of SIRT1 increased the protein levels of FOXO1 and RAB7. On the contrary, knock-down of SIRT1 expression inhibited the protein levels of FOXO1 and RAB7. The protein interaction between SIRT1 and FOXO1 in the pancreatic cancer cells was observed. CONCLUSION: SIRT1 in pancreatic cancer Panc-1 cells under hypoxia condition is over-expressed in the nucleus. Down-regulation of SIRT1 inhibits autophagy and its mechanism may be related to FOXO1/RAB7 signaling pathway.     

Hemin induces and sustains the phosphorylation of Erk1/2 in human umbilical vascular endothelial cells

AIM: To investigate the relationship between hemin and Erk1/2 activation in human umbilical vascular endothelial cells (HUVECs). METHODS: Cultured HUVECs were separately incubated with hemin or H₂O₂ for different times. Subsequently Erk1/2 phosphorylation and total Erk1/2 were determined by Western blotting assay. Flow cytometry was employed to determine the cell cycle distribution. RESULTS: Hemin at the concentration of 1-10 μmol/L induced the phosphorylation of Erk1/2 in HUVECs, and sustained the phosphorylation of Erk1/2 for three hours. The duration of phospho-Erk1/2 induced by hemin was much longer than that in H₂O₂ control (3 h vs 30 min). CONCLUSION: Hemin induces and sustains the phosphorylation of Erk1/2 in HUVECs, which indicates that the effect of hemin on the Erk1/2 activation may be one of pharmacological target of hemin.     

Expression of natural killer cell stimulatory receptor NKG2D and its ligand MICA in Tibetan patients with gastric cancer at Qinghai plateau

AIM: To investigate the expression of natural killer cell stimulation receptor D(NKG2D) in peripheral blood and the expression of major histocompatibility complex class I chain-related protein A(MICA) on the gastric carcinoma tissue and the neighboring non-cancerous tissue in Tibetan patients with gastric cancer at Qinghai plateau. METHODS: Samples of 33 patients and 20 healthy persons were collected to detect NKG2D in peripheral blood by Flow cytometry analysis. The expressions of MICA in part of the correspondent tissues were examined by RT-PCR and immunohistochemistry.RESULTS: The expression of NKG2D in the plateau of Tibetan patients with gastric cancer group (13.47 ±5.26)% was significantly lower than that in healthy groups (32.62±10.08)%. The mRNA level of MICA in gastric cancer was significantly higher than that in neighboring non-cancerous tissue (P<0.05). The expressions of MICA proteins showed a significant difference between the neighboring non-cancerous tissue (21.21%, 7/33) and the gastric carcinoma tissue (78.79%, 26/33), between the high differentiation (60.00%, 3/5) and the moderate (72.73%, 8/11), low differentiation (88.24%, 15/17). The expressions of MICA were not correlated with tumor size, sex, age, lymph node metastasis of the patients (P>0.05). Spearman rank correlation displayed that the expressions of MICA mRNA were positively correlated with the MICA protein level (r=0.903, P<0.01).CONCLUSION: The immune escape in Tibetan patients with gastric cancer at Qinghai plateau probably relates to the down-regulation of NKG2D and the up-regulation of its ligand MICA. The activity of NK cells and the anti-cancer cellular immunity level are descending in the plateau of Tibetan patients with gastric cancer. The decrease in the receptor NKG2D is a reason for the activity of NK cell descending.     

GFP标记生防细菌B579及其定殖能力检测

 利用绿色荧光蛋白（Green fluorescent protein, GFP）标记靶标微生物是目前研究微生物和宿主互作的重要手段。在本研究中,作者用电击转化的方法将携带质粒gfpmut3a基因的穿梭载体pGFP4412导入生防枯草芽孢杆菌（Bacillus subtilis）B579中,并得到成功表达GFP的枯草芽孢杆菌B579-gfp。用抗生素平板回收结合激光扫描共聚焦显微镜（LSCM）观察对枯草芽孢杆菌B579-gfp在盆栽的黄瓜根表定殖情况进行了研究,结果表明：标记菌株在激发光波长为488 nm的蓝光下可观察到亮绿色的荧光;B579-gfp菌体能够定殖在黄瓜根部,在根基部和中部都有菌体聚集而形成膜状结构,在根的分叉和根冠处可观察到大量B579-gfp定殖;浸种、蘸根以及灌根接种均可回收到大量的B579-gfp,分别为4.0×10³、1.0×10⁴和2.0×10² cfu/g。     

Molecular characterization of A2 and D strains of Soybean mosaic virus, which caused a recent virus outbreak in soybean cultivar Sachiyutaka in Chugoku and Shikoku regions of Japan

Coat protein sequences of two isolates in strain A₂ and five isolates in strain D of Soybean mosaic virus (SMV), which caused a recent mosaic outbreak in soybeans (cv. Sachiyutaka) in Chugoku and Shikoku in Japan, were compared to published data on 15 other Asian-origin isolates. Sequence comparison and cluster analysis showed that SMV isolates of strain A₂ from these districts were closely related, as were those of strain D, but strains A₂ and D were not. Thus, the two strains may have different origins and be carried through seed transmission.     

芽胞杆菌CQBS03抑菌蛋白TasA基因的克隆及原核表达

为了探讨柑桔溃疡病生防菌芽胞杆菌Bacillus CQBS03菌株TasA基因的功能,采用PCR方法从CQBS03基因组DNA中扩增出编码TasA基因的全长DNA序列,并构建pEASY-E1/TasA原核表达载体,经大肠杆菌Escherichia coli表达获得TasA基因的融合表达蛋白,纸碟法检验融合蛋白对柑桔溃疡病菌Xanthomonas citri citri的抑制作用。结果显示,CQBS03菌株的TasA基因包含1个786 bp的完整开放阅读框（GenBank登录号为JQ309841）,编码261个氨基酸残基;该序列与来源于解淀粉芽胞杆菌B.amyloliquefaciens的1个已知同源TasA基因序列FJ713580的相似性达99.75%。原核表达产物经SDS-PAGE分析,检测到约31 kD的融合蛋白;纯化后的融合蛋白对柑桔溃疡病菌有明显的抑制作用,72 h后抑菌圈直径达11.5 mm。研究表明TasA基因是生防菌芽胞杆菌CQBS03抑制柑桔溃疡病菌的功能基因之一,并且该基因对原核表达宿主没有抑制作用,具有较好的开发利用前景。     

砂梨扩展蛋白基因cDNA克隆及全序列分析

设计植物EXP扩展蛋白简并引物,以砂梨果实cDNA为模板,克隆得到EXP基因cDNA片段,该片段长465 bp。根据该片段序列,分别设计2条5’和3’末端扩增的特异引物,利用RACE技术,获得了该片段的5’端和3’端序列。用DNAMAN5.22软件对3个序列进行拼接和分析,获得了该基因的cDNA全长,命名为Pyp-EXP。该cDNA全长为1 395 bp,5’端起始密码子ATG始于72 bp,3’端终止子TAG止于830 bp,Ploy+(A)从1 363～1 395 bp。该基因已在GenBank基因数据库注册,注册号为EF602031。Pyp-EXP核苷酸序列有一个759 bp完整的开放阅读框,编码区与西洋梨、苹果、湖北海棠、桃的同源性分别为96%,96%,94%和86%;该cDNA推导编码252个氨基酸,含有1个组氨酸(His_Phe_Asp,HFD)功能域,与西洋梨、苹果、湖北海棠、桃中相应序列同源性分别为98%,97%,95%和93%。该基因的克隆为研究扩展蛋白的时空表达及其在果实发育和成熟过程中的作用奠定了基础。