循环包装回收技术在筛选肝癌细胞相关耐药基因中的应用

肝癌先天性多表达多药耐药基因,严重影响肝癌的化疗效果,筛选肝癌细胞中的耐药基因,研究其耐药机制有助于提高肝癌化疗效果,提高肝癌的治愈率。首先构建肝癌细胞逆转录病毒的cDNA文库,感染成纤维细胞,使得逆转录病毒基因整合进细胞,加药筛选,存活细胞中的基因再次包装成病毒,用于下一轮筛选。采用循环包装回收(Cyclical packaging rescue,CPR)技术进行肝癌细胞耐药基因的筛选即是通过病毒包装将基因从细胞中钓取出来,相比于常规筛选方法,仅通过一轮筛选可能会出现很多假阳性基因,采用CPR技术则是通过多轮筛选,很大程度减少了假阳性细胞的出现。通过该方法经过四轮筛选获得核糖体蛋白S11(RPS11)、核糖体蛋白L6(RPL6)、核糖体蛋白L11(RPL11)、核糖体蛋白L24(RPL24)等几种基因,经初步检测,增加了细胞的耐药性。     

利用自裂解多肽T2A在牛耳皮肤成纤维细胞中实现多基因共同表达

目的:探讨利用自裂解多肽2A构建的多顺反子载体能否在牛耳皮肤成纤维细胞中实现多基因的有效表达。方法:利用来自一点褐翅蛾病毒(TaV)的2A元件(T2A)将GFP和Neo基因连接到同一载体中,构建pCMV-GFP-T2A-Neo质粒,将其转染牛耳皮肤成纤维细胞,以FACS检测GFP基因的表达,RT-qPCR检测GFP、T2A和Neo的表达。结果:由T2A连接的GFP和Neo基因在mRNA水平上都有显著表达,且表达水平相当。结论:以T2A连接的基因在转入细胞后能正常翻译和表达,显示T2A在牛耳皮肤成纤维细胞中具有自裂解功能,可作为一种构建多顺反子载体的有效工具用于牛耳皮肤成纤维细胞的基因转移,为其将来在转基因牛研制中的应用奠定了基础。     

水稻幼芽细胞生物膜上的赤霉素结合蛋白的结合特性

在水稻 (Oryza sativa)幼芽中存在膜结合的赤霉素结合蛋白 ,其与 GA3 结合的平衡解离常数(Kd)为 6.5× 1 0 -8mol/ L,总浓度为 0 .3 pmol· mg-1 蛋白质。结合蛋白与 GA3 结合活力在 0℃时比 2 5℃时高 1 4 0 %。它与 GA3 结合的最适 p H为 5。 GA3 与此结合蛋白的结合量随反应时间延长而增加 ,1 h达最大值 ,以后又逐渐下降。 IAA、ABA可与 GA3 竞争赤霉素结合蛋白。     

Chromosome‐level genome assembly for the horned‐gall aphid provides insights into interactions between gall‐making insect and its host plant

The aphid Schlechtendalia chinensis is an economically important insect that can induce horned galls, which are valuable for the medicinal and chemical industries. Up to now, more than twenty aphid genomes have been reported. Most of the sequenced genomes are derived from free‐living aphids. Here, we generated a high‐quality genome assembly from a galling aphid. The final genome assembly is 271.52 Mb, representing one of the smallest sequenced genomes of aphids. The genome assembly is based on contig and scaffold N50 values of the genome sequence are 3.77 Mb and 20.41 Mb, respectively. Nine‐seven percent of the assembled sequences was anchored onto 13 chromosomes. Based on BUSCO analysis, the assembly involved 96.9% of conserved arthropod and 98.5% of the conserved Hemiptera single‐copy orthologous genes. A total of 14,089 protein‐coding genes were predicted. Phylogenetic analysis revealed that S. chinensis diverged from the common ancestor of Eriosoma lanigerum approximately 57 million years ago (MYA). In addition, 35 genes encoding salivary gland proteins showed differentially when S. chinensis forms a gall, suggesting they have potential roles in gall formation and plant defense suppression. Taken together, this high‐quality S. chinensis genome assembly and annotation provide a solid genetic foundation for future research to reveal the mechanism of gall formation and to explore the interaction between aphids and their host plants.     

一步法扩增克隆IBDV上海超强毒VP2-4-3基因

分离、纯化了鸡传染性法氏囊病病毒超强毒 (vvIBDV) 上海株SH95的病毒核酸dsRNA,应用随机引物将RNA反转录成cDNA,以此为模板一步扩增出A片段前体融合蛋白基因即VP2-4-3基因,将其克隆入pGEM-T载体,并进行序列分析,其与超强毒株HK46的核苷酸序列的同源性达98%,整个基因有5个氨基酸差异,同源性达99.51%(1007/1012).     

An eukaryotic translation initiation factor,AteIF5A‐2, affects cadmium accumulation and sensitivity in Arabidopsis

Cadmium (Cd) is one of the most toxic elements and can be accumulated in plants easily; meanwhile, eIF5A is a highly conserved protein in all eukaryotic organisms. The present work tried to investigate whether eIF5A is involved in Cd accumulation and sensitivity in Arabidopsis (Arabidopsis thaliana L.) by comparing the wild‐type Columbia‐0 (Col‐0) with a knockdown mutant of AteIF5A‐2, fbr12‐3 under Cd stress conditions. The results showed that the mutant fbr12‐3 accumulated more Cd in roots and shoots and had significantly lower chlorophyll content, shorter root length, and smaller biomass, suggesting that downregulation of AteIF5A‐2 makes the mutant more Cd sensitive. Real‐time polymerase chain reaction revealed that the expressions of metal transporters involved in Cd uptake and translocation including IRT1, ZIP1, AtNramp3, and AtHMA4 were significantly increased but the expressions of PCS1 and PCS2 related to Cd detoxification were decreased notably in fbr12‐3 compared with Col‐0. As a result, an increase in MDA and H₂O₂ content but decrease in root trolox, glutathione and proline content under Cd stress was observed, indicating that a severer oxidative stress occurs in the mutant. All these results demonstrated for the first time that AteIF5A influences Cd sensitivity by affecting Cd uptake, accumulation, and detoxification in Arabidopsis.