碱性成纤维细胞生长因子在苜蓿中的表达

为了降低bFGF(basic fibroblast growth factor)的生产成本,结合植物生物反应器的优点,就bFGF在转基因苜蓿中的表达进行了探索.将bFGF插入植物表达载体pBⅡ21中,获得了含有bFGF基因的植物表达pBIcbFGF,再将pBIcbFGF利用冻融法转到农杆菌中.利用农杆菌介导法将基因转化保定苜蓿,转基因苜蓿在TM-1培养基+20 mg/L卡那霉素(Kan)+200 mg/L特美汀(Tim)中诱导分化,在生根培养基中生根,获得再生植株.再生植株通过PCR检测、RT-PCR及Western blot证实外源基因已经在苜蓿中成功表达.获得含有目的蛋白的阳性植株.为苜蓿作为植物生物反应器生产bFGF奠定了理论及技术基础.     

种子老化诱导小麦染色体畸变及大麦醇溶蛋白带型频率变化的研究

通过时20份小麦种质发芽率和根尖细胞染色体畸变的测定,结果表明同一品种贮藏于中期库的低发芽率种质,其染色体畸变率明显高于贮藏于长期库的高发芽率种质.从总体上看,种子根尖细胞染色体畸变率与发芽率呈负显著相关,而与贮藏年限无显著相关.但对长期库和中期库的种质分别进行统计分析,种子根尖染色体畸变率与发芽率和贮藏年限均无显著相关.对大麦地方品种"普乃干木"的醇溶蛋白分析表明,该品种有4种biotype,随着发芽率的下降,其群体中的4种bio-type的频率发生了变化,当发芽率从95%降到34%时,其中一种biotype出现了消失,而有一种biotype频率从83%上升至95%,此结果表明,种子生活力下降和不同biotype种子存活能力存在差异,可能导致了异质种质材料遗传选择和漂变的发生.     

miR-22 regulates C2C12 myoblast proliferation and differentiation by targeting TGFBR1

Recently, miR-22 was found to be differentially expressed in different skeletal muscle growth period, indicated that it might have function in skeletal muscle myogenesis. In this study, we found that the expression of miR-22 was the most in skeletal muscle and was gradually up-regulated during mouse myoblast cell (C2C12 myoblast cell line) differentiation. Overexpression of miR-22 repressed C2C12 myoblast proliferation and promoted myoblast differentiation into myotubes, whereas inhibition of miR-22 showed the opposite results. During myogenesis, we predicted and verified transforming growth factor beta receptor 1 (TGFBR1), a key receptor of the TGF-β/Smad signaling pathway, was a target gene of miR-22. Then, we found miR-22 could regulate the expression of TGFBR1 and down-regulate the Smad3 signaling pathway. Knockdown of TGFBR1 by siRNA suppressed the proliferation of C2C12 cells but induced its differentiation. Conversely, overexpression of TGFBR1 significantly promoted proliferation but inhibited differentiation of the myoblast. Additionally, when C2C12 cells were treated with different concentrations of transforming growth factor beta 1 (TGF-β1), the level of miR-22 in C2C12 cells was reduced. The TGFBR1 protein level was significantly elevated in C2C12 cells treated with TGF-β1. Moreover, miR-22 was able to inhibit TGF-β1-induced TGFBR1 expression in C2C12 cells. Altogether, we demonstrated that TGF-β1 inhibited miR-22 expression in C2C12 cells and miR-22 regulated C2C12 cell myogenesis by targeting TGFBR1.     

Improving the Secretory Expression of an α-Galactosidase from Aspergillus niger in Pichia pastoris

α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1’ residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Mut^s) strain of AGA-I with the specific P1’ site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.     

A novel RGD-toxin protein,Lj-RGD3, from the buccal gland secretion of Lampetra japonica impacts diverse biological activities

RGD (Arg-Gly-Asp) motif toxin proteins from snake venoms, saliva glands secretion of leech or tick have typical characteristics of inhibiting platelet aggregation, angiogenesis, and tumor growth. Here we report cloning and characterization of a novel RGD-toxin protein from the buccal gland of Lampetra japonica. In an attempt to study the activities of anticoagulant in the buccal gland secretion of L. japonica, we established buccal gland cDNA library and identified a gene encoding a predicted protein of 118 amino acids with 3 RGD motifs. The predicted protein was named Lj-RGD3. We generated the cDNA of Lj-RGD3 and obtained the recombinant protein rLj-RGD3. The polyclonal antibodies against rLj-RGD3 recognized the native Lj-RGD3 protein in buccal gland secretion in Western blot analyses. The biological function studies reveal that rLj-RGD3 inhibited human platelet aggregation in a dose-dependent manner with IC₅₀ value at 5.277 μM. In addition, rLj-RGD3 repressed bFGF-induced angiogenesis in the chick chorioallantoic membrane model. rLj-RGD3 also inhibited the adhesion of ECV304 cells to vitronectin. Furthermore, rLj-RGD3 induced apoptosis and significantly inhibited proliferation, migration, and invasion evoked by bFGF in ECV304 cells. Taken together, these results suggested that rLj-RGD3 is a novel RGD-toxin protein possessing typical functions of the RGD-toxin protein.     

Behavioral stress causes mitochondrial dysfunction via ABAD up-regulation and aggravates plaque pathology in the brain of a mouse model of Alzheimer disease

Basic and clinical studies have reported that behavioral stress worsens the pathology of Alzheimer disease (AD), but the underlying mechanism has not been clearly understood. In this study, we determined the mechanism by which behavioral stress affects the pathogenesis of AD using Tg-APPswe/PS1dE9 mice, a murine model of AD. Tg-APPswe/PS1dE9 mice that were restrained for 2h daily for 16 consecutive days (2-h/16-day stress) from 6.5months of age had significantly increased Aβ(1-42) levels and plaque deposition in the brain. The 2-h/16-day stress increased oxidative stress and induced mitochondrial dysfunction in the brain. Treatment with glucocorticoid (corticosterone) and Aβ in SH-SY5Y cells increased the expression of 17β-hydroxysteroid dehydrogenase (ABAD), mitochondrial dysfunction, and levels of ROS, whereas blockade of ABAD expression by siRNA-ABAD in SH-SY5Y cells suppressed glucocorticoid-enhanced mitochondrial dysfunction and ROS accumulation. The 2-h/16-day stress up-regulated ABAD expression in mitochondria in the brain of Tg-APPswe/PS1dE9 mice. Moreover, all visible Aβ plaques were costained with anti-ABAD in the brains of Tg-APPswe/PS1dE9 mice. Together, these results suggest that behavioral stress aggravates plaque pathology and mitochondrial dysfunction via up-regulation of ABAD in the brain of a mouse model of AD.     

辣椒素及其受体

可以感受痛觉刺激的初级感觉神经元的周围末梢被称为伤害性感受器。这些小直径神经元的末梢可将化学、机械和热刺激信号转化为动作电位,并将这些信息上传到中枢,最后使机体产生痛觉或不舒服的感受。但到目前为止,人们对这些可探测到伤害性刺激的分子所知甚少。1997年成功克隆的辣椒素受体亚型1（vanilloid receptor subtype1,VR1)是近年来科学家们研究的“热点分子”,它是表达于伤害性感受器上的非选择性阳离子通道,已有诸多证据表明其可探测和整合诱发痛觉的化学和热刺激信号,基因敲除小鼠的研究分析也有力证明了该离子通道参与了疼痛及组织损伤后痛觉过敏的产生,而且是热诱发疼痛发生过程的关键分子。