基于离散时序基因表达数据的双聚类算法

目前应用于基因表达数据上的双聚类算法大多是基于真实数据提出的, 因此易受噪声干扰, 且这些算法很少考虑样本间的时序性。提出了一种有效的时间点连续的双聚类挖掘算法DTCB, 从离散的时序基因表达数据中挖掘出时间点连续的最大共表达双聚类。该算法使用了一种新的数据离散化方法, 同时提出了三种在离散数据集下基因间的共表达关系; 为了提高挖掘效率, DTCB使用了有效的剪枝和输出策略, 可以在不产生候选集的情况下一次性挖掘出所有的最大共表达双聚类。通过实验分析, 证明DTCB具有高效的性能和良好的鲁棒性, 且结果具有较好的统计和生物意义。     

Expression and Iron Storage Properties of Recombinant Human Ferritins in Escherichia Coli: Relevance for Functional Ferritins in Food Systems

Ferritin is an iron storage protein found in most living organisms. Human heavy chain (H-) and light chain (L-) ferritin were amplified from human heart cDNA library. Each ferritin gene was inserted down stream of the T7 promoter of bacterial expression, and finally four types of H-, L-, and co-expression vectors were constructed. Recombinant human ferritins were overexpressed and identified with SDS-PAGE and western blotting. The expression levels of recombinant ferritin proteins ranged about 29–36% of whole cell total protein. From atomic absorption spectrometry (AAS) analysis, the rate of iron uptake for H-ferritin was significantly faster than that for the HL-, LH-, and L‐ferritin, respectively. From AAS analysis, the levels of iron content in cells progressively increased in transformants with 0–30 mM ferric citrate in the media. Among these ferritin transformants, the highest amount of cellular iron was observed with H‐ferritin transformant. The total amounts of iron content in E. coli could be sequentially ranked as H-ferritin > HL-ferritin > LH-ferritin > L-ferritin > negative transformants. Expressed recombinant human ferritins indicated that their assembled subunits were able to store iron in the cells. The results of this study further enhances our understanding of iron uptake properties in vivo and suggest similar strategies for using food-grade ferritins for functional foods or iron-fortified food ingredients.     

共表达谷氨酰-tRNA还原酶增强染料脱色过氧化物酶在大肠杆菌中的表达活性

染料脱色过氧化物酶(dye-decoloorizing peroxidase,DyP)属于以血红素为辅基的新型过氧化物酶类,常因缺乏辅因子而导致催化活性低。将来源于褐色嗜热裂孢菌(Thermobifida fusca)的染料脱色过氧化物酶基因(TfuDyP)与大肠杆菌谷氨酰-tRNA还原酶基因(hemA),构建重组质粒phemA-DyP,转化至E.coli BL21中进行共表达。分别以2,2-联氮-二(3-乙基苯并噻唑-6-磺酸)二铵盐(ABTS)和顽固性染料活性蓝19(RB19)、溴酚蓝、溴甲酚绿为底物检测TfuDyP的催化活力以及染料脱色效率。结果表明,诱导后的共表达菌株pAD胞内血红素含量为9. 8μmol/L,而单独过表达基因TfuDyP的菌株pD仅为3. 4μmol/L。TfuDyP纯酶的全波长扫描分析表明,在菌株pAD中DyP酶与血红素的结合度相比pD有较大幅度的提升。pAD菌株表达的DyP酶活力较pD菌株提高了110%,酶活力的提高使其在染料脱色应用方面也得到增强。在pAD菌株培养基中分别添加谷氨酸(Glu)、FeCl2使得胞内血红素含量、DyP酶活力和染料脱色效率比未添加时进一步提高。以上结果为TfuDyP的功能开发奠定了基础,同时也为其他血红素依赖性过氧化物酶的研发提供借鉴。     

Identification of the Potential Function of circRNA in Hypertrophic Cardiomyopathy Based on Mutual RNA-RNA and RNA-RBP Relationships Shown by Microarray Data

The pathogenesis of hypertrophic cardiomyopathy (HCM) is very complicated, particularly regarding the role of circular RNA (circRNA). This research pays special attention to the relationships of the circRNA-mediated network, including RNA-RNA relationships and RNA-RNA binding protein (RNA-RBP) relationships. We use the parameter framework technology proposed in this paper to screen differentially expressed circRNA, messenger RNA (mRNA), and microRNA (miRNA) from the expression profile of samples related to HCM. And 31 pairs of circRNA and mRNA relationship pairs were extracted, combined with the miRNA targeting database; 145 miRNA-mRNA relationship pairs were extracted; 268 circRNA-mRNA-miRNA triads were established through the common mRNA in the 2 types of relationship pairs. Thus, 268 circRNA-miRNA regulatory relationships were deduced and 30 circRNA-RBP relationship pairs were analyzed at the protein level. On this basis, a circRNA-mediated regulatory network corresponding to the two levels of RNA-RNA and RNA-RBP was established. And then the roles of circRNA in HCM were analyzed through circRNA-mRNA, circRNA-miRNA, and circRNA-RBP, and the possible role in disease development mas inferred.     

重组大肠杆菌全细胞催化D,L-扁桃酸对映选择性制备L-苯甘氨酸

借助pACYCDuet-1和pET28a双质粒共表达系统,构建携带Agrobacterium radiobacter来源扁桃酸消旋酶（Ar mandelate racemase,ArMR）、Lactobacillus harbinensi来源D-扁桃酸脱氢酶（Lh D-mandelate dehydrogenase,LhDMDH）和Exiguobacterium sibiricum DSM 17290来源L-亮氨酸脱氢酶（Es L-leucine dehydrogenase,EsLeuDH）编码基因的重组大肠杆菌,将其命名为E. coli BL21（DE3）/pACYCDuet-1-EsLeuDH-LhDMDH:pET28a-ArMR。在低温、低浓度诱导剂的诱导下,该重组菌成功表达了具有各自催化活性的3 种重组酶,其发酵液中LhDMDH、EsLeuDH和ArMR的活性分别为195.8、56.2 U/mL和174.5 U/mL。以诱导后的全细胞为催化剂、D,L-扁桃酸为底物,在D,L-扁桃酸初始浓度50 mmol/L、pH 9.5的500 mmol/L NH4Cl-NH3·H2O缓冲液体系下,180 r/min、30 ℃反应48 h后,L-苯甘氨酸得率可达77.48%,其对映体过量值大于99%。本研究具有较大的产业化潜力,为实现L-苯甘氨酸规模化的生物合成奠定了坚实的基础。     

串联表达NAD激酶和谷氨酸脱氢酶基因对L-谷氨酸产量的影响

由ppnk和gdh编码的NAD激酶和谷氨酸脱氢酶是L-谷氨酸合成途径中的两个重要酶。以谷氨酸生产菌CN1021基因组为模板PCR扩增ppnk和gdh基因并转化至该菌中,检测上述两个基因单独表达和串联表达对L-谷氨酸产量的影响。结果表明,当两个基因单独过表达时,其酶活性分别提高2.4和2.1倍,L-谷氨酸产量分别提高7.9%和1.4%。当将两个基因串联表达时,其酶活性分别提高2.0和1.5倍,L-谷氨酸的产量却提高了13.2%。说明过表达ppnk和gdh能够有效提高L-谷氨酸产量,且具有协同作用。     

从基因表达数据中有效挖掘差异共表达双聚类：DiCluster算法

双聚类是一种可以同时在基因和条件两个维度上分析基因表达数据的方法,它可以找出在部分条件下具有相似表达趋势的基因。已有的方法都是从一个数据集中挖掘双聚类。从生物意义上分析,从不同基因表达数据集中挖掘差异表达双聚类可以发现具有生物意义的转录因子等信息。因此,提出一种挖掘不同数据集上差异共表达双聚类的算法——DiCluster。该算法采用深度优先基因扩展方法,并引入了剪枝策略,有效挖掘最大差异表达双聚类。实验结果表明,DiCluster不仅比已有算法具有更高的效率,而且挖掘出的结果具有更好的统计学和生物学意义。     

内切葡聚糖酶与木聚糖酶在毕赤酵母中的共分泌表达

为实现内切葡聚糖酶和木聚糖酶在毕赤酵母中的共分泌表达,进而降低酶制剂的生产成本,构建含木聚糖酶基因的重组表达载体pPICZαA-Aoxyn11A,经SacI线性化后,电转化至含内切葡聚糖酶基因Aucel12A的重组毕赤酵母GSC7中,获得双重重组毕赤酵母GSCX8。经甲醇诱导表达后,GSCX8发酵上清液中内切葡聚糖酶和木聚糖酶的活性分别为47.77IU/mL和192.71IU/mL,为单独表达菌株GSC7和GSX5的85%和80%。酶学性质分析显示,内切葡聚糖酶的最适pH为4.0,在pH3.0~8.5稳定;最适温度为50℃,在60℃以下稳定。木聚糖酶的最适pH为5.5,在pH3.0~10.0稳定;最适温度为55℃,在50℃以下稳定。GSCX8遗传稳定性测试结果表明,内切葡聚糖酶和木聚糖酶在毕赤酵母中实现了稳定的共表达。