哺乳动物附植前胚胎的基因表达调控

哺乳动物胚胎附植前期包括:合子的形成,胚胎基因组的激活和细胞分化的开始。在这个时期,发育由母源物质控制转为合子基因控制,在此过程,同时形成染色质介导的转录抑制时期,要解除抑制必须经过胚胎基因组的激活。通过对体内、外附植前胚胎的mRNA的表达特点以及它们与成功发育联系的研究,可以筛选出最佳的体外培养条件,设计最佳的核移植方案。     

苜蓿银纹夜蛾核多角体病毒在家蚕蛹体内的复制及重组病毒外源基因的表达

家蚕蛹在复眼着色期,经4℃冷藏24小时后,可被Ac NPV(苜蓿银纹夜蛾核多角体病毒)感染。在蛹体内复制出的多角体大小差异较大,且比在昆虫Sf—21细胞中繁殖的Ac NPV和在蚕蛹体内形成的Bm NPV多角体小。在蛹体内繁殖的Ac NPV的游离病毒可以回返感染Sf—21细胞。由蚕蛹内分离的Ac NPV基因组DNA的限制性内切酶图谱与野生型的Ac NPV相同。以上结果证明Ac NPV可以在蚕蛹体内复制。含HBsAg基因(人乙肝表面抗原)的重组Ac NPV亦可感染家蚕蛹,并能正确表达外源基因。此外,还发现化蛹后第2天的家蚕蛹被注射约5×10~5PFU的Ac NPV,可诱导“人工滞育蛹”现象的发生,在25℃经过30天后蛹仍存活,发育停滞在复眼着色前阶段。     

香蕉ACC氧化酶基因(MAO3)的克隆及其表达特性分析

 根据同源扩增得到香蕉(Musa acuminata) ACC氧化酶基因(MAO3) 的核心部分, 再通过3'和5'RACE扩增上、下游序列以及Genome-Walker的方法得到启动子部分, 共获得3 718 bp长度的序列。将所得结果进行聚类分析, 发现香蕉中ACC氧化酶的氨基酸序列非常保守, 各序列间的同一性高达99% ,与单子叶植物和双子叶植物中ACC氧化酶的氨基酸序列的同源性在66.7%～71.8%之间。组织原位杂交试验表明MAO3基因的表达具有组织特异性, 初步认为是在韧皮部筛管组织中特异表达。运用实时荧光定量PCR技术, 实时监测到了MAO3基因和香蕉乙烯受体基因ERS2受机械伤诱导的定量变化。     

H5N1亚型禽流感病毒NS1基因的原核表达及其ELISA检测方法的建立

采用RT-PCR技术扩增了禽流感病毒（AIV）A／Goose／HLJ／p46／2003（H5N1）的NSl基因，将其克隆于融合蛋白表达载体pMAL-c2X上，转化DH5α大肠埃希氏菌感受态细胞，经BamHⅠ和HindⅢ双酶切鉴定及序列分析，表明筛选到了重组质粒pc2X-NS1。SDS-PAGE电泳结果显示，重组质粒转化TB1大肠埃希氏菌后，经0．3mmol／L的IPTG诱导，融合蛋白MBP-NS1得到大量表达，融合蛋白以可溶形式存在，分子质量约为67ku。Western-blotting检测结果表明，融合蛋白MBP-NS1能够与H5N1亚型AIV活病毒感染康复鸭血清发生特异性反应，而不能够与H5N1亚型AIV灭活疫苗免疫鸭血清发生反应。试验初步建立了以纯化的融合蛋白MBP-NS1为包被抗原的间接ELISA检测方法，为AIV灭活疫苗免疫家禽与AIV感染家禽的鉴别诊断奠定了基础。     

鸡传染性腔上囊病病毒VP2基因在昆虫细胞中的表达

为了深入研究鸡传染性腔上囊病病毒（IBDV）VP2基因的结构和功能,利用Bac-to-Bac系统研制出含有VP2基因的重组杆状病毒rBac-VP2,将rBac-VP2感染Sf9细胞,并用抗IBDV VP2特异性单克隆抗体经间接免疫荧光试验检测,证实感染重组病毒Sf9细胞能高效表达IBDV VP2基因产物;Western-blotting分析结果表明,VP2基因表达产物的分子质量约为40 ku.     

Analysis of gene expression profile of multidrug resistant MCF/DOX cell line after benflumetol derivative LY980503 treatment

AIM:To investigate the effect of LY980503(a benflumetol derivative)on multidrug resistance of tumor cell line using DNA microarray.METHODS:Total RNA was extracted from multidrug resistant MCF/DOX cell line. cDNA microarray containing 320 cDNAs was used to detect the gene expression profile.RESULTS:9 down-regulated genes and 1 up-regulated gene were identified after multidrug resistant MCF/DOX cells were treated with LY980503.CONCLUSION:LY980503 can effectively reverse the resistance of MCF/DOX to DOX in vitro by adjusting the expression of multi-genes.     

Influences of chronic hypoxia on the gene expression of Kv1.3,Kv2.1 and Kv3.1 induced by acute hypoxia in PASMC of rats

AIM and METHODS: Total RNA was extracted from 6th rat subcultured pulmonary artery smooth muscle cells(PASMC) exposed to continual chronic hypoxia or normoxia and the effects of chronic hypoxia on the changes of Kv1.3,Kv2.1,Kv3.1 mRNA in cultured PASMC induced by acute hypoxia were studied by semiquantitative RT-PCR in vitro. RESULTS:①Kv1.3,Kv2.1,Kv3.1 genes were found to be expressed in PASMC of rats exposed either to hypoxia or normxia.②The expression of Kv2.1 and Kv3.1 in 6th subcultured of PASMC in normaxia group could be upregulated by exposure to acute hypoxia,the levels of Kv2.1 and Kv3.1 mRNA were significantly increased from 0.646±0.092, 0.782±0.104 to 1.059±0.134, 0.985±0.116,respectively (P<0.01,n=5). ③PASMC cultured continuously in chronic hypoxia for 6 subcultures and then exposed to normoxia for 12 h,thereafter the expression of Kv2.1 and Kv3.1 were downregulated by acute hypoxia for 6 hours.The level of Kv2.1 mRNA was significantly decreased from 1.008±0.117 to 0.649±0.097 (P<0.01,n=5). CONCLUSION:Kv2.1,Kv3.1 genes might be oxygen sensitive genes.Chronic hypoxia might change the response of these Kv genes of PASMC to acute hypoxia and down-regulate its expression,which might probably decrease the role of Kv in HPV.     

IL-12 induces T lymphocytes apoptosis and influences the expression and signal transduction of Fas/FasL

AIM: IL-12 acts upon Tlymphocytes and activates its receptor complexes of β1/β2,and so IL-12 can regulate TH1/TH2 balance. Our study is aimed at IL-12-inducing apoptosis of Tcells and expression and signal transduction of Fas/FasLduring Tcell apoptosis. METHODS: The apoptosis of Tcells was detected by Annexin Vstaining cytometry and the expression of Fas/FasLunder different inhibitors were detected by semi-quantitative PCR. RESULTS: IL-12 induced the human leukemic Tcell line(TIB-152) and the human lymphoma Tcell line(HTB-176) and the normal human Tcells to undergo apoptosis. The FasLexpression at 6 hours after treatment with IL-12 increased apparently, and reached the max at 24 hours, and FasLexpression induced by IL-12 was inhibited by PKCinhibitor. But IL-12 did not influence Fas expression. CONCLUSIONS: IL-12 can induce Tcells to undergo apoptosis which is characterized by early membrane changes, the inducing effect is correlated with the concentration of IL-12 and the maturation of Tcells. FasLparticipates in the progression of Tcell apoptosis as a apoptosis mediator, and the effect of IL-12 on FasLexpression may be related with PKCpathway.     

Functional Analysis of ABC Transporter Genes From Botrytis cinerea Identifies BcatrB as a Transporter of Eugenol

The role of multiple ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes from the plant pathogenic fungus Botrytis cinerea in protection against natural fungitoxic compounds was studied by expression analysis and phenotyping of gene-replacement mutants. The expression of 11 ABC (BcatrA–BcatrK) and three MFS genes (Bcmfs1, Bcmfs2 and Bcmfs4) was studied. All genes showed a low basal level of expression, but were differentially induced by treatment with cycloheximide and the plant defence compounds camptothecin, eugenol, psoralen, resveratrol and rishitin. The latter compounds induced expression of BcatrB at a high level. Eugenol was more toxic to BcatrB gene-replacement mutants than to the control isolates. Eugenol also caused an instantaneous increase in mycelial accumulation of the fungicide fludioxonil, a known substrate of BcatrB. However, there was no difference in virulence between the wild-type and BcatrB gene-replacement mutants on Ocimum basilicum, a plant known to contain eugenol. The results indicate that BcatrB is a transporter of lipophilic compounds, such as eugenol, but its role in virulence remains uncertain.     

植物苯丙氨酸解氨酶基因的表达调控与研究展望

苯丙氨酸解氨酶(phenylalanineammonia-lyase,PAL,EC4.3.1.5)是催化苯丙烷代谢途径第一步反应的酶,也是这个途径的关键酶,对植物有非常重要的生理意义。根据有关文献综述了植物PAL的分布与定位、酶学性质,总结了生长发育、钝化因子与调节因子、末端产物等内部因素及光、温、机械损伤与生长调节剂等外部因素对PAL的调控作用,得出外部因子是在转录水平上对酶活性实施调控的结论,并运用酶学和分子生物学方面的知识阐述了其调控机理。还着重阐述了PAL酶在果树上的研究现状与进展,并对其今后的研究及在果树上的应用进行了展望。