Circular RNA profiling reveals chi_circ_0008219 function as microRNA sponges in pre-ovulatory ovarian follicles of goats (Capra hircus)

Circular RNAs (circRNAs) are a new class of non-coding RNAs in animals and are a novel target of non-coding RNA (ncRNA) regulation. The mechanism and function of circRNAs have been reported in some species and tissues. However, there is little available information on the functions of circRNAs in the goat reproductive system. In the present study, we deeply sequenced and analyzed circRNAs through bioinformatics to reveal the expression profiles, and predicted 13,950 circRNAs in the pre-ovulatory ovarian follicles of goats for the first time. Thirty-seven circRNAs were differentially expressed in the Boer goat compared with the Macheng black goat. The chi_circ_0008219 was involved in a vast circRNA-miRNA-mRNA co-expression network. Via a luciferase activity assay, chi_circ_0008219 is observed to sponge to 3 ovarian follicle-related miRNAs. These findings demonstrate that circRNAs have potential effects in the ovarian follicles of ewes and may represent a promising new research field in ovarian follicular development.     

文心兰乙烯不敏感基因EIN2的克隆及表达分析

该研究采用PCR、RACE方法,对文心兰‘南茜’的乙烯不敏感蛋白基因（ethylene insensitive 2,EIN2）进行克隆及生物信息学分析,并采用qRT PCR技术,对该基因在文心兰不同组织器官和不同花期中的表达模式进行分析。结果表明：（1）成功克隆得到文心兰_EIN2基因序列,命名为OnEIN2（MH497388）;该基因cDNA序列全长为4 177 bp,其中开放阅读框3 879 bp,编码1 292个氨基酸,3′非编码区长208 bp,5′非编码区长90 bp。（2）生物信息学分析显示OnEIN2是一个不稳定的疏水蛋白,含有跨膜结构,分子式为C₆₄₀₆H₉₉₈₈N₁₆₇₀O₁₈₉₇S₄₇;蛋白质分子量142.22 kD,理论等电点5.80。多序列比对和系统进化分析表明,文心兰EIN2与铁皮石斛EIN2的相似度最高（81.98%）,二者亲缘关系也最为接近。（3）实时荧光定量PCR分析发现,OnEIN2基因在根、茎、叶花中均有表达,在花中表达量最高,茎中表达量最低;而在不同花期中,盛开期表达量最高,其次是衰老期。研究表明,文心兰OnEIN2基因在开花和衰老过程中可能有重要的作用。     

Exploitation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> as a robust workhorse for production of heterologous proteins and beyond

Bacillus subtilis, belonging to the type species of Bacillus, is a type of soil-derived, low %G+C, endospore-forming Gram-positive bacterium. After the discovery of B. subtilis 168 that displayed natural competence, this bacterium has been intensively considered to be an ideal model organism and a robust host to study several basic mechanisms, such as metabolism, gene regulation, bacterial differentiation, and application for industrial purposes, such as heterologous protein expression and the overproduction of an array of bioactive molecules. Since the first report of heterologous overproduction of recombinant proteins in this strain, the bulk production of a multitude of valuable enzymes, especially industrial enzymes, has been performed on a relatively large scale. Since B. subtilis can non-specifically secrete recombinant proteins using various signal peptides, it has tremendous advantages over Gram-negative bacterial hosts. Along with the report of the complete genome sequence of B. subtilis, a number of genetic tools, including diverse types of plasmids, bacterial promoters, regulatory elements, and signal peptides, have been developed and characterized. These novel genetic elements tremendously accelerated genetic engineering in B. subtilis recombinant systems. In addition, with the development of several complex gene expression systems, B. subtilis has performed a number of more complex functions. This ability enables it to be a substantial chassis in synthetic biology rather than just a workhorse for the overproduction of recombinant proteins. In this review, we review the progress in the development of B. subtilis as a universal platform to overproduce heterologous diverse high-value enzymes. This progress has occurred from the development of biological parts, including the characterization and utilization of native promoters, the fabrication of synthetic promoters and regulatory elements, and the assembly and optimization of genetic systems. Some important industrial enzymes that have been produced in large quantities in this host are also summarized in this review. Furthermore, the ability of B. subtilis to serve as a cellular tool was also briefly recapitulated and reviewed.     

Synthesis and evaluation of a novel ‘off-on’ chemical sensor based on rhodamine B and the 2,5-pyrrolidinedione moiety for selective discrimination of glutathione and its bioimaging in living cells

A new “turn-on” fluorescent probe, RDMBM, based on the rhodamine B dye and the 2,5-pyrrolidinedione moiety was synthesized and characterized. Its sensing behavior toward various amino acids was evaluated via UV–vis and fluorescence spectroscopic techniques. The observed spectral changes showed that RDMBM displays high selectivity and sensitivity toward GSH in MeOH/H₂O (1:2, v/v, pH 7.40, Tris-HCl buffer, 1?mM) solution and that it undergoes 1:1 covalent binding with GSH. More importantly, the hydrogenation and ring-opening of the nitrogen atom in the spirane structure of rhodamine B derivatives were tightly bound to the induction effects of different groups. Furthermore, fluorescence imaging applications demonstrated that RDMBM can be successfully used for the detection of GSH in human breast cancer cells MCF-7.     

Optimization of fermentation conditions for an <Emphasis Type="Italic">Escherichia coli</Emphasis> strain engineered using the response surface method to produce a novel therapeutic DNA vaccine for rheumatoid arthritis

Background

Fermentation condition optimization and nutrients screening are of equal importance for efficient production of plasmid DNA vaccines. This directly affects the downstream purification and final quality and yield of plasmid DNA vaccines. The present study aimed to optimize the fermentation conditions for high-throughput production of therapeutic DNA vaccine pcDNA-CCOL2A1 by engineered Escherichia coli DH5α, using the response surface method (RSM).

Results

We hypothesized that optimized fermentation conditions significantly increase the yield of pcDNA-CCOL2A1 therapeutic DNA vaccine, a novel DNA vaccine for treating rheumatoid arthritis (RA). Single-factor analysis was performed to evaluate the optimal basal culture medium from LB, 2?×?YT, TB, M9 (Glycerol) and M9 (Glucose), respectively. Thereafter, the Plackett-Burman design (PBD) was used to ascertain the three most significant factors affecting the vaccine yields, followed by the paths of steepest ascent to move to the nearest region of maximum response. Initial screening through the PBD revealed that the most key factors were peptone, mannitol, and inoculum concentration. Subsequent use of RSM was further optimized for the production of therapeutic DNA vaccine pcDNA-CCOL2A1 through Box-Behnken design (BBD). The final optimized fermentation conditions were as follows: peptone, 25.86 g/L; mannitol, 8.08 g/L; inoculum concentration, OD?=?0.36. Using this statistical experimental design, the yield of therapeutic DNA vaccine pcDNA-CCOL2A1 markedly increased from 223.37 mg/L to339.32 mg/L under optimal conditions, and a 51.9% increase was observed compared with the original medium.

Conclusions

The present results provide a basis for further production of high-quality and high-yield therapeutic DNA vaccine pcDNA-CCOL2A1 in pilot-scale and even industrial-scale.

     

细枝木麻黄气生瘤的人工形成

细枝木麻黄气生瘤的人工形成邹铧,张忠泽,张成刚,丁鉴,王育英,付莉（中国科学院沈阳应用生态研究所,沈阳１１００１５）ＡｒｔｉｆｉｃｉａｌｆｏｒｍａｔｉｏｎｏｆａｅｒｉａｌｎｏｄｕｌｅｓｏｎＣａｓｕａｒｉｎａｃｕｎｎｉｎｇｈａｍｉａｎａ￥ＺｏｕＨｕａ;...     

结香化学成分的研究

从结香(Edgeworthia chrysantha)的花蕾中分离得出3个香豆索,2个黄酮。用波谱等方法确定其结构为伞形花内酯(umbelliferone,1),6-甲氧基-7-羟基双香豆素-3,7’-醚(daphnoretin,2),7,7’-二羟基双香豆素-8,8’-醚-7-α-L-鼠李糖甙(edgeworoside c,5),4’,5,7-三羟基黄酮醇-3-O-β-D-(6′′-对羟基桂皮酰基)-葡萄糖甙(tiliroside,3),山萘酚-3-O-β-D-葡萄糖甙(kaempferol-3-O-β-D-glucoside,4),以上化合物均为首次从结香花蕾中得到。     

抗HPV16 E7-ribozyme在CV-1细胞中的稳定表达

构建了由RSV—LTR启动子带动并能在细胞内稳定复制的Ribozyme的自身修剪表达质粒pRSV—Rz523、Ribozyme反义对照质粒pRSV—AE7及人增殖细胞核抗原基因(PCNA)启动子带动的HPVl6 E7片段(+554～+686)的真核表达质粒pPCNA—E7。经G418抗性筛选获得了稳定表达Ribozyme的CV-1细胞克隆,其表达水平约为9．Opmol／lO6个细胞,其中活性Ribozyme的量大于50fmol／lO6个细胞,分离得到的Ribozyme可在体外特异切割E7靶RNA片段。通过共转染Ribozyme(或反义对照)和底物表达质粒并筛选出细胞克隆．研究了Ribozyme在细胞中对底物表达水平的影响。初步结果显示．Rihozyme的导人可使细胞内底物E7的RNA表达水平降低了90％(反义对照使E7 RNA表达降低20％)。上述结果提示：在CV-1细胞中表达的Ribozyme不仅在体外,同时在细胞内具有一定的生物学活性,有可能应用于逆转官颈癌细胞的恶性表型。