一种拟南芥基因高效编辑体系研究

CRISPR/Cas9基因编辑系统操作简单易行,无需引入外源基因,生物安全性高。但怎样快速筛选获得不含外源转化元件的基因编辑后代是一个关键技术问题。本研究创造性的将拟南芥种皮特异性启动子At2S3与荧光筛选标记基因mCherry组装进植物基因组定点编辑CRISPR载体pHDE中,以拟南芥as1为靶基因,构建一种通过荧光标记筛选、实现转化后代中Cas9 Free的基因高效编辑体系。结果表明,通过同源重组方法构建的带有筛选标记的CRISPR载体与设计相符,外源插入片段正确。挑选转化后种皮上带有红色荧光标记的阳性种子培育得到T₁代植株,经PCR验证,成功获得as1定点敲除的纯合突变植株,纯合子比率达到40%;挑选T₁代纯合突变上不带荧光的种子,培育得到的T₂代植株中,PCR检测不到Cas9片段,实现了编辑后代的Cas9 Free。本研究构建的一种带有可视化筛选标记的基因高效编辑体系,成功实现编辑后代中无外源插入的Cas9等转化元件,生物安全性高,为基因组定点编辑技术在植物遗传资源改良中的高效利用提供了借鉴与参考。     

毛蕊异黄酮通过抑制calpain-1的表达发挥抗脑缺血再灌注损伤的研究

目的:探讨毛蕊异黄酮抗脑缺血再灌注损伤的作用是否与抑制calpain-1的表达有关。方法:将SD大鼠随机分为假手术组、模型组以及药物组,采用线栓法建立大鼠大脑中动脉阻断(MCAO)模型,于缺血再灌注前30 min腹腔注射给予20 mg/kg毛蕊异黄酮或等体积的溶剂。再灌注24 h后,行神经功能学评分、脑梗死面积以及神经元凋亡检测;再灌注12 h、24 h时,采用免疫组化和蛋白印迹技术检测大鼠脑皮层calpain-1的表达。结果:与假手术组大鼠比较,MCAO模型组大鼠再灌注24 h后神经功能学评分、梗死面积、神经元凋亡率及calpain-1的表达均明显升高(P0.05),而毛蕊异黄酮能够降低模型组大鼠再灌注24 h后神经功能学评分、梗死面积、神经元凋亡率以及calpain-1的表达(P0.05)。结论:毛蕊异黄酮可能通过抑制calpain-1的表达发挥抗脑缺血再灌注损伤作用。     

SOD2在姜黄素减轻氧糖剥夺所致神经元损伤中的作用

目的:探讨二型超氧化物歧化酶(Mn-SOD,SOD2)是否介导了姜黄素(Curcumin,Cur)对氧糖剥夺模型(Oxygen-Glucose Deprivation,OGD)损伤神经元的保护作用。方法:本研究采用HT22神经元细胞暴露于OGD环境中3 h模拟神经元缺血缺氧损伤,SOD2-si RNA抑制神经元SOD2蛋白表达后,通过噻唑蓝法(Methylthiazolyldiphenyl-tetrazolium bromide,MTT)检测细胞活力,比色法测量培养基乳酸脱氢酶(Lactic Dehydrogenase LDH)水平,流式细胞仪计算细胞凋亡率,Western blot测定凋亡蛋白Cleaved Caspase-3表达,并观察细胞形态和线粒体功能。结果:与正常培养的Control组相比,OGD组细胞活力显著降低,LDH释放明显增加,细胞凋亡率和Cleaved Caspase-3表达显著上升,细胞形态破坏并降低线粒体膜电位(MMP)和线粒体复合物1(Mitochondrial Complex 1 Activity)的活力(P0.05),100 ng/ml的Cur可显著减轻OGD诱导的神经元细胞的上述损伤性改变(P0.05)。而SOD2-si RNA显著逆转Cur对OGD诱导的神经元细胞损伤的保护作用(P0.05),SC-si RNA则未对Cur产生的神经保护作用造成显著干扰(P0.05)。结论:Cur可能通过上调SOD2的表达,减轻OGD对神经元细胞的损伤。     

黄纹长腹扇蟌胸部色斑变异研究

不同种群的雄性黄纹长腹扇蟌胸部色斑存在较大的变异,主要表现在前胸背板斑纹的有无、合胸背面及侧面斑纹的面积大小、形状及数量等方面。本文通过几何形态学方法,以及对前胸背板斑纹的观察比较,对34个地里样点共319头雄性黄纹长腹扇蟌的胸部斑纹变异情况进行分析。几何形态学分析显示,依据合胸斑纹轮廓形状可将黄纹长腹扇蟌划分为东南(Southeast)与西北(Northwest)两群组,且在分布重叠区的种群中同时存在两种特征的个体;对前胸背板斑纹的观察统计显示,几乎所有分布于西北群组的样本前胸背板具一对蓝色斑纹,而东南群组的样本几乎都缺失这对斑纹。本研究使用的两方面证据所揭示的变异趋势相吻合,均证明雄性黄纹长腹扇蟌胸部斑纹随地理分布存在明显的变异规律。研究结果显示雄性黄纹长腹扇蟌胸部斑纹存在明显变异多样性,几何形态学可用于蜻蜓目昆虫色斑变异规律的研究,也为该物种的演化与地理格局的形成等后续研究提供证据支持。     

Analysis of the contribution of the globin and reductase domains to the ligand-binding properties of bacterial haemoglobins

Bacterial Hbs (haemoglobins), like VHb (Vitreoscilla sp. Hb), and flavoHbs (flavohaemoglobins), such as FHP (Ralstonia eutropha flavoHb), have different autoxidation and ligand-binding rates. To determine the influence of each domain of flavoHbs on ligand binding, we have studied the kinetic ligand-binding properties of oxygen, carbon monoxide and nitric oxide to the chimaeric proteins, FHPg (truncated form of FHP comprising the globin domain alone) and VHb-Red (fusion protein between VHb and the C-terminal reductase domain of FHP) and compared them with those of their natural counterparts, FHP and VHb. Moreover, we also analysed polarity and solvent accessibility to the haem pocket of these proteins. The rate constants for the engineered proteins, VHb-Red and FHPg, do not differ significantly from those of their natural counterparts, VHb and FHP respectively. Our results suggest that the globin domain structure controls the reactivity towards oxygen, carbon monoxide and nitric oxide. The presence or absence of a reductase domain does not affect the affinity to these ligands.     

多指标记录的1962—2008年长白山园池泥炭地地表湿度变化

使用AMS ¹⁴C和¹³⁷Cs方法建立年代序列,通过植物残体、有壳变形虫和腐殖化度3个指标的综合分析,重建了长白山园池泥炭地地表的湿度变化,并探讨其对气候的响应。结果表明: 园池泥炭地地表50 cm泥炭植物残体以藓类为主,利用去趋势对应分析(DCA)发现,第一轴得分代表沼泽表面湿度变化,将植物残体揭示的1962—2008 A.D.间泥炭地地表湿度与有壳变形虫-水位间转换函数得到的水位埋深以及腐殖化度进行对比,发现植物残体、有壳变形虫和腐殖化度揭示的沼泽表面湿度变化总体趋势一致,即40～50 cm层(1962—1975 A.D.)湿度较大;27～40 cm层(1975—1987 A.D.)湿度变小,处于干湿交替期;0～27 cm层(1987—2008 A.D.)湿度较小。将3种指标重建的地表湿度与二道气象站数据对比发现,气候代用指标揭示的沼泽地表湿度变化与温度的变化趋势基本吻合,沼泽地地表湿度降低发生在夏季温度和年均温偏高的年份。由此推测,近46年来沼泽地地表湿度变化主要是对温度升高引起的有效降水减弱的响应。     

Taurine supplementation in conjunction with exercise modulated cytokines and improved subcutaneous white adipose tissue plasticity in obese women

Amino Acids - Interventions that can modulate subcutaneous white adipose tissue (scWAT) function, such as exercise training and nutritional components, like taurine, modulate the inflammatory...     

Using Xenopus to analyze neurocristopathies like Kabuki syndrome

Neurocristopathies are human congenital syndromes that arise from defects in neural crest (NC) development and are typically associated with malformations of the craniofacial skeleton. Genetic analyses have been very successful in identifying pathogenic mutations, however, model organisms are required to characterize how these mutations affect embryonic development thereby leading to complex clinical conditions. The African clawed frog Xenopus laevis provides a broad range of in vivo and in vitro tools allowing for a detailed characterization of NC development. Due to the conserved nature of craniofacial morphogenesis in vertebrates, Xenopus is an efficient and versatile system to dissect the morphological and cellular phenotypes as well as the signaling events leading to NC defects. Here, we review a set of techniques and resources how Xenopus can be used as a disease model to investigate the pathogenesis of Kabuki syndrome and neurocristopathies in a wider sense.     

黑龙江流域积雪覆盖时空变化遥感监测

利用MODIS双星数据对黑龙江流域2003—2012年的积雪覆盖面积进行提取和验证,然后基于合成的数据分析研究区积雪覆盖面积的季节和年际变化.结果表明: 双星合成降低了云的影响,总体精度>91%,可以满足分析和研究需求.研究期间,黑龙江流域积雪覆盖面积存在显著的季节变化,7、8月的积雪最少,几乎为零,1月积雪覆盖面积最大,占流域的80%以上.2003—2004、2009—2010年冬季积雪覆盖面积较高(>180×10⁴ km²),2011年冬季最大积雪覆盖面积较低(150×10⁴ km²).积雪覆盖的年际变化与年平均气温和平均降水量的波动存在一定的对应关系:积雪覆盖面积较低年份对应的年降水量较少、平均气温较高,反之亦然.2003—2012年,研究区5、6月的积雪覆盖面积呈减少趋势,降水量增加和气温的升高与积雪覆盖面积减少紧密相连.     

CHD7, the gene mutated in CHARGE syndrome,regulates genes involved in neural crest cell guidance

Heterozygous loss of function mutations in CHD7 (chromodomain helicase DNA-binding protein 7) lead to CHARGE syndrome, a complex developmental disorder affecting craniofacial structures, cranial nerves and several organ systems. Recently, it was demonstrated that CHD7 is essential for the formation of multipotent migratory neural crest cells, which migrate from the neural tube to many regions of the embryo, where they differentiate into various tissues including craniofacial and heart structures. So far, only few CHD7 target genes involved in neural crest cell development have been identified and the role of CHD7 in neural crest cell guidance and the regulation of mesenchymal-epithelial transition are unknown. Therefore, we undertook a genome-wide microarray expression analysis on wild-type and CHD7 deficient (Chd7 ^Whi/+ and Chd7 ^Whi/Whi ) mouse embryos at day 9.5, a time point of neural crest cell migration. We identified 98 differentially expressed genes between wild-type and Chd7 ^Whi/Whi embryos. Interestingly, many misregulated genes are involved in neural crest cell and axon guidance such as semaphorins and ephrin receptors. By performing knockdown experiments for Chd7 in Xenopus laevis embryos, we found abnormalities in the expression pattern of Sema3a, a protein involved in the pathogenesis of Kallmann syndrome, in vivo. In addition, we detected non-synonymous SEMA3A variations in 3 out of 45 CHD7-negative CHARGE patients. In summary, we discovered for the first time that Chd7 regulates genes involved in neural crest cell guidance, demonstrating a new aspect in the pathogenesis of CHARGE syndrome. Furthermore, we showed for Sema3a a conserved regulatory mechanism across different species, highlighting its significance during development. Although we postulated that the non-synonymous SEMA3A variants which we found in CHD7-negative CHARGE patients alone are not sufficient to produce the phenotype, we suggest an important modifier role for SEMA3A in the pathogenesis of this multiple malformation syndrome.