微生物流式分选的单细胞样品制备及存活率提高的方法优化

【背景】以流式细胞技术为代表的高通量筛选技术能够高效筛选具有目标性状的微生物工程菌株。在流式分选中微生物的粘连会造成分析数据不准确，分选纯度降低，因此快速简便的单细胞样品制备是流式检测的关键。优势菌大多是通过筛选偶联荧光蛋白的随机突变库获得，阳性率低，杂质和死细胞的自发荧光较强，容易混入分选门内造成存活率降低，亟须提高分选存活率的方法。【目的】建立一种简便的微生物流式分选的单细胞样品制备方法，并通过碘化丙啶(propidium iodide, PI)染色提高分选样品存活率。【方法】分别在大肠杆菌、枯草芽孢杆菌、谷氨酸棒状杆菌和酵母菌4种底盘细胞中探索超声波、消化酶、表面活性剂及超声-表面活性剂联合作用4种方式对单细胞制备效率的影响。提高微生物流式分选存活率，用常压室温等离子诱变(atmospheric and room temperature plasma, ARTP)技术处理含有绿色荧光蛋白(green fluorescent protein, GFP)的酿酒酵母HZ848 (简称HZ848-GFP)，形成不同强度GFP文库后，按照GFP强度分选全细胞和PI染色阴性细胞的前0.5%，统计单细胞存活率。【结果】酵母细胞分散条件为：0.01% Tween-80联合超声1 min，单细胞率达到88%以上，PI染色细胞破损率＜1.4%。谷氨酸棒状杆菌单细胞分散条件为：0.01% Tween-80联合超声5 min，单细胞率达到97%以上，PI染色细胞破损率＜1%。分选存活率结果表明，未用PI染色的酿酒酵母分选后单细胞存活率是4.3%，用PI染色去除死细胞后再分选单细胞存活率是18.3%，后者是前者的4.3倍，且具有显著性差异。【结论】本研究为微生物流式分选建立了一套简单快捷的单细胞样品制备方法，证实了PI染色法能够显著提高分选样品存活率。     

Genetic relationship between parasitized and non-parasitized Haloxyion ammodendron in the Alxa Desert

Haloxylon ammodendron (C. A. Mey.) Bunge is a host for the holoparasitic plant Cistanche deserticola Y. C. Ma, the original source of medicinal material known as Herba Cistanchis. The inter-simple sequence repeat marker was used to assess the genetic variations and relationships among six accessions of H. ammodendron with a total of 120 individuals collected from three localities in the Alxa Desert, Inner Mongolia, China. At each locality, individuals both parasitized (PP) by C. deserticola and non-parasitized (NP) were sampled. The results showed that Nei's gene diversity and Shannon's index of PP accessions were higher, but were not significantly different, from those of NP accessions. An unweighted pair-group method arithmetic average dendrogram showed two clusters, one that included all PP accessions, and the other the NP accessions. Genetic differentiation therefore existed between PP and NP accessions, which might be attributed to low gene flow between the NP and PP groups (Nm< 1). However, the relationship between genetic distance and geographic distance within each group, although not statistically significant in this study, might be associated with high gene flow in both the NP and PP groups.     

Up-Regulation of Epithelial Membrane Protein-1 in the Temporal Neocortex of Patients with Intractable Epilepsy

Epithelial membrane protein-1 (EMP-1), called Tumor-associated membrane protein, is the marker of a drug-resistant tumor and take part in the drug-resistant mechanism of tumor, with the relationship of epidermal growth factor receptor (EGFR). Because there are some similarities between the pathogenesis and the drug resistance mechanism of tumor and the drug resistance mechanisms in epilepsy. EMP1 expression may be connected with the drug-resistance mechanism of epilepsy. We detected EMP-1 by gene scanning and immunohistochemistry staining, comparing the IE group and the control group, and we investigated the relationship between EMP-1 and EGFR by double-label immunofluorescence staining in the IE group. We found expression of EMP-1 mRNA was higher in IE per the gene scanning, EMP-1 immunoreactivity was apparent in neurons of IE patients but not in the control group, and the expression of EMP-1 and EGFR occurred in the same neuron. We confirm EMP-1 is abnormally expressed in IE and suggest the interaction of EGFR and EMP-1 plays a role in the mechanism of drug resistance in epilepsy and may be a new gene for drug resistance.