粪肠球菌Ace重组蛋白的表达、纯化及黏附活性研究

构建含粪肠球菌表面蛋白Ace保守序列A的重组载体,在大肠埃希菌M15中进行诱导表达、纯化表达产物,研究其黏附活性。以粪肠球菌标准株JH2-2为模板进行PCR扩增ace基因保守序列A,构建重组质粒pQE30/Ace,转化至表达宿主菌M15中进行诱导表达,利用SDS-PAGE和Western blot进行分析和鉴定表达结果,Ni-NTA亲和层析柱纯化重组蛋白,同时用纯化的Ace重组蛋白免疫新西兰兔,用所得相应的抗血清分别进行黏附和黏附抑制实验。结果可见,构建的重组质粒经酶切鉴定和测序鉴定证明其中插入片段为ace基因保守序列A,测序结果与Genbank上登录序列完全一致;SDS-PAGE分析显示,重组工程菌表达了一相对分子质量(Mr)约为37 ku的目的蛋白条带,Western blot检测其能与6×His单克隆抗体发生特异性反应。粪肠球菌JH2-2能够黏附于胶原蛋白Ⅰ表面;抗Ace多克隆抗体可抑制46℃培养的JH2-2对胶原蛋白Ⅰ的黏附,这种抑制作用与其稀释度呈负相关。构建的原核表达载体PQE30/Ace在E.coli M15中成功地表达,纯化的重组Ace蛋白具有胶原黏附活性。     

In vitro differentiation of rat embryonic stem cells into functional cardiomyocytes

The recent breakthrough in the generation of rat embryonic stem cells (rESCs) opens the door to application of gene targeting to create models for the study of human diseases. In addition, the in vitro differentiation system from rESCs into derivatives of three germ layers will serve as a powerful tool and resource for the investigation of mammalian development, cell function, tissue repair, and drug discovery. However, these uses have been limited by the difficulty of in vitro differentiation. The aims of this study were to establish an in vitro differentiation system from rESCs and to investigate whether rESCs are capable of forming terminal-differentiated cardiomyocytes. Using newly established rESCs, we found that embryoid body (EB)-based method used in mouse ESC (mESC) differentiation failed to work for the serum-free cultivated rESCs. We then developed a protocol by combination of three chemical inhibitors and feeder-conditioned medium. Under this condition, rESCs formed EBs, propagated and differentiated into three embryonic germ layers. Moreover, rESC-formed EBs could differentiate into spontaneously beating cardiomyocytes after plating. Analyses of molecular, structural, and functional properties revealed that rESC-derived cardiomyocytes were similar to those derived from fetal rat hearts and mESCs. In conclusion, we successfully developed an in vitro differentiation system for rESCs through which functional myocytes were generated and displayed phenotypes of rat fetal cardiomyocytes. This unique cellular system will provide a new approach to study the early development and cardiac function, and serve as an important tool in pharmacological testing and cell therapy.     

神经细胞粘附分子与记忆

记忆的形成阶段包含着神经元突触的可塑性变化过程.近年来的研究表明,神经细胞粘附分子可同时增进突触的可塑性和维持突触结构的稳定性.许多研究证实神经细胞粘附分子对与学习和记忆相关的过程起着一定的调节作用.     

越南篦齿苏铁传粉媒介的研究

利用排除法和引入昆虫的方法相结合,研究越南篦齿苏铁(Cycas elongata)的传粉媒介.结果表明,越南篦齿苏铁靠风媒传粉和虫媒传粉的雌株结实率分别55.3%和57.2%,自然传粉的结实率为63.5%,说明风和象鼻虫都是越南篦齿苏铁的有效传粉媒介.越南篦齿苏铁散粉高峰在白天,夜间散粉很少,在3.0 m以内风传花粉的密度较高,3.0 m之外的密度急剧下降.可见,风媒和虫媒都是越南篦齿苏铁的有效传粉途径,与泽米铁类植物为专一寄主性昆虫传粉的结论不一致.     

大孔树脂对三孢布拉霉菌产番茄红素的吸附性能研究

为利用大孔吸附树脂分离纯化三孢布拉霉菌发酵液番茄红素,考察了4种大孔树脂(HP20, HP2MG, SP825, SP207)对番茄红素的吸附 解吸特性,筛选出最佳树脂。结果表明,HP20大孔树脂对番茄红素具有较好的吸附 解吸性能,且吸附迅速,能在1h内达到吸附平衡。用含55%和60%异丙醇的丙酮不连续梯度洗脱,回收率可达89.55%,重结晶后可获得纯度95%以上的番茄红素,经红外、质谱、核磁等鉴定为天然型番茄红素。该方法简单,可操作性强,极具工业应用价值。     

Biodetoxification of toxins generated from lignocellulose pretreatment using a newly isolated fungus, Amorphotheca resinae ZN1, and the consequent ethanol fermentation

Background

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.

Results

When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.

Conclusion

The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.     

Genome‐wide distribution and functions of the AAE complex in epigenetic regulation in Arabidopsis

Heterochromatin is widespread in eukaryotic genomes and has diverse impacts depending on its genomic context. Previous studies have shown that a protein complex, the ASI1‐AIPP1‐EDM2 (AAE) complex, participates in polyadenylation regulation of several intronic heterochromatin‐containing genes. However, the genome‐wide functions of AAE are still unknown. Here, we show that the ASI1 and EDM2 mostly target the common genomic regions on a genome‐wide level and preferentially interacts with genetic heterochromatin. Polyadenylation (poly(A) sequencing reveals that AAE complex has a substantial influence on poly(A) site usage of heterochromatin‐containing genes, including not only intronic heterochromatin‐containing genes but also the genes showing overlap with heterochromatin. Intriguingly, AAE is also involved in the alternative splicing regulation of a number of heterochromatin‐overlapping genes, such as the disease resistance gene RPP4. We provided evidence that genic heterochromatin is indispensable for the recruitment of AAE in polyadenylation and splicing regulation. In addition to conferring RNA processing regulation at genic heterochromatin‐containing genes, AAE also targets some transposable elements (TEs) outside of genes (including TEs sandwiched by genes and island TEs) for epigenetic silencing. Our results reveal new functions of AAE in RNA processing and epigenetic silencing, and thus represent important advances in epigenetic regulation.     

黄麻吸附重金属Cr(Ⅵ)专用种质筛选

首次以国内外23个代表性黄麻种质为研究对象,用主茎嫩梢和一、二级分枝嫩梢制成天然重金属吸附剂,测定其对溶液中Cr(Ⅵ)离子的去除率,并对与黄麻吸附能力及产量密切相关的功能性状:生育期动态、株高、分枝习性、各级分枝嫩梢的产量进行调查、方差分析和相关性分析。结果表明,不同黄麻种质制成的生物吸附剂对Cr(Ⅵ)的吸附能力不同,去除率在85.25%~96.88%之间;不同级次分枝嫩梢的产量及对Cr(Ⅵ)的去除率不同;种质间除出苗速度外所有调查性状均存在较大差异;去除率与株高呈极显著负相关(P<0.01)。从产量和去除率方面综合考虑,J001和J011 2个种质表现优良,适宜作为吸附重金属专用品种推广种植。