Roles of protein phosphatase 4 in down-regulation of eNOS Ser633 phosphorylation induced by palmitic acid in human umbilical vein endotheli?al cells

AIMTo investigate the roles of protein phosphatase 4 (PP4) in down-regulation of endothelial nitric oxide synthase (eNOS) Ser633 phosphorylation induced by palmitic acid (PA). METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with PA at 25 μmol/L, 50 μmol/L, 100 μmol/L and 200μmol/L for 36 h, or treated with PA at 100 μmol/L for 12 h, 24 h, 36 h and 48 h. Protein phosphatase 2A (PP2A) family inhibitor fostriecin (FST, 20 nmol/L) or okadaic acid (OA, 5 nmol/L) was selected to pretreat the HUVECs for 30 min. Protein phosphatase 4 catalytic subunit (PP4c) siRNA or protein phosphatase 2A catalytic subunit (PP2Ac) siRNA was transfected into the HUVECs. The protein expression levels of of eNOS, PP4c and PP2Ac, as well as the level of eNOS Ser633 phosphorylation, were detected by Western blot. The intracellular nitric oxide (NO) content was measured by DAF-FM DA. RESULTS(1) Compared with control group, the levels of eNOS Ser633 phosphorylation were decreased in PA groups in which the HUVECs were treated with 25 μmol/L, 50 μmol/L, 100 μmol/L and 200 μmol/L PA for 36 h (P<0.05) and 100 μmol/L PA for 24 h, 36 h and 48 h (P<0.05). No significant difference in the level of total eNOS protein expression among all the groups was observed. (2) Compared with control group, both FST and OA pretreatment reversed the reduction of eNOS Ser633 phosphorylation (P<0.05) and the decrease in intracellular NO content (P<0.05) induced by PA. No significant difference in the level of total eNOS protein expression among all the groups was observed. (3) Compared with si-Control group, the PP4c protein expression was significantly reduced (P<0.05), while the level of eNOS Ser633 phosphorylation was significantly increased in si-PP4c group (P<0.05). Although the levels of PP2Ac protein expression declined significantly (P<0.05), the level of eNOS Ser633 phosphorylation remained unchanged in si-PP2Ac group. No significant differencein the level of total eNOS protein expression among all the groups was found. CONCLUSION PA significantly reduces the level of eNOS Ser633 phosphorylation and the content of NO in the HUVECs, which may be due to PA inducing the activation of the PP2A family member PP4 rather than PP2A.     

利用CRISPR/Cas9系统敲除葡萄中VviEDR2提高对白粉病的抗性

利用CRISPR/Cas9系统定点编辑葡萄白粉病感病基因VviEDR2（Enhanced disease resistance 2），在VviEDR2的DUF1336结构域设计靶位点VviEDR2-T1，构建CRISPR/Cas9敲除载体，通过农杆菌介导法转化‘无核白’葡萄胚性愈伤组织。对PCR阳性植株进行靶位点扩增测序，结果表明，共有8个转基因植株在靶位点处发生不同类型的双等位基因突变，编辑效率为32%；突变体植株生长势较弱，叶片较小，茎秆丛生、细弱。进一步对突变体植株进行抗病检测，结果表明，接种葡萄白粉菌（Erysiphe necator Schw.）5 d后，突变体植株叶片上白粉菌孢子仅能萌发出少量较短初级菌丝，表皮细胞产生大量明显的H2O2，而野生型叶片中白粉菌萌发出大量初级菌丝、次级菌丝和吸器，无明显H2O2产生。这些结果表明，可以利用CRISPR/Cas9技术编辑葡萄感病基因VviEDR2，提高葡萄白粉菌抗性。     

依据显微结构及光合特性探讨蝴蝶兰花芽分化的时期

以蝴蝶兰‘大辣椒’为试验材料，对花芽分化进程及期间光合特性和碳水化合物、可溶性蛋白及激素含量的变化进行研究。结果表明：花芽长度为0、2、4、8、16和24 cm时，分别处于花芽分化初始期、花序原基分化期、花原基分化期、萼片原基分化期和花瓣原基分化期（16和24 cm）。蝴蝶兰叶片的净CO2吸收速率在花芽发育前期（0 ~ 4 cm）没有显著变化，花芽8 cm时显著降低。花芽中的碳水化合物和可溶性蛋白的含量显著高于叶片，碳水化合物在花芽长度为4 cm时达到稳定水平，可溶性蛋白含量在花芽8 cm时达到叶片与花芽的平衡；赤霉素（GA）的含量在花芽2 cm时达到最大值，生长素（IAA）含量在花芽4 cm时显著升高，玉米素（ZT）含量在花芽8 cm时显著降低，而ABA含量在花芽发育的过程中并没有显著变化。由此可知，当蝴蝶兰花芽开始分化萼片原基（8 cm）时，光合生理及生化物质基本达到一个相对稳定的水平，此阶段的蝴蝶兰花芽已彻底完成成花分化。     

枣AP2/ERF转录因子鉴定及其响应枣疯病植原体表达分析

利用生物信息学方法，从植物转录因子数据库和NCBI数据库中分别得到175和164个候选的枣AP2/ERF转录因子序列，使用DNAMAN软件进行序列比对、去除重复序列，采用SMART软件预测蛋白结构域发现，枣基因组中包含有145个AP2/ERF基因，其中ERF、AP2、RAV亚家族分别含有116个、23个、5个，另有1个独立基因。预测枣AP2/ERF转录因子氨基酸数量在111 ~ 692之间，分子量在12 446.87 ~ 76 154.10之间，pI在4.31 ~ 10.11之间。鉴定出的145个AP2/ERF转录因子，105个分别定位到12条染色体上，40个未能定位。在此基础上，利用嫁接病芽方法将枣疯病植原体转至健康枣植株，通过转录组测序和qRT-PCR手段，分析了AP2/ERF转录因子对枣植原体侵染的响应，在植原体侵染枣的6个不同时期，枣AP2/ERF表达数量和表达量均不相同，共有48个差异表达基因，其中ZjAP2*9、ZjERF49和ZjERF91是响应枣疯病植原体最为重要的AP2/ERF转录因子。     

大花君子兰叶绿体基因组及其特征

采用Illumina MiSeq测序平台对大花君子兰（Clivia miniata）叶片总DNA进行测序，通过组装获得了其叶绿体基因组（cpDNA）全长序列（158 114 bp）。对其cpDNA注释得到135个基因，包含87个蛋白编码基因、40个tRNA基因和8个rRNA基因。采用生物信息学方法对获得的cpDNA进行简单序列重复（SSR）分析和密码子偏好性分析。结果显示：①大花君子兰cpDNA中共有61个SSR位点，其中单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸和六核苷酸重复数分别为38、9、2、8、3和1个，多数SSR分布在基因间隔区；②大花君子兰cpDNA密码子偏爱以A或U（T）结尾，亮氨酸使用频率最高，半胱氨酸使用频率最低。基于24种植物的cpDNA全长和23种植物的叶绿体ycf2基因序列进行系统发育分析，结果显示大花君子兰与石蒜科植物在同一分支，显示最近的亲缘关系，支持大花君子兰属于石蒜科。基于叶绿体ycf2的系统发育分析结果与基于cpDNA全长的系统发育分析研究结果大部分相同，支持ycf2基因可以代替cpDNA全长用于植物系统发育分析。     

脱二氧化碳装置对循环水养殖系统pH的影响

为了调节循环水系统中养殖水体的pH,根据气体交换原理,设计一种脱二氧化碳(CO_2)装置。采用该装置去除养殖水体中的CO_2,并对由于CO_2含量累积造成的pH下降进行调节,使养殖鱼类处在一个适宜的pH环境中。试验时水温控制在(25±0.5)℃,每1 h取水样测1次pH,每4 h测1次碱度。水样取自养鱼桶内的水,检测前先对水样用40μm孔径针头过滤器进行过滤处理,实验周期24 h。结果显示,循环水系统加装脱二氧化碳装置能有效去除CO_2,使水体稳定在一个适宜的pH范围(7.39~7.42);CO_2质量浓度呈降低趋势,24 h后由开始的13.16 mg/L降低到7~8 mg/L,降低近50%,而不加装脱二氧化碳装置的循环水系统CO_2质量浓度持续上升,24 h后增加到37 mg/L左右,pH持续降低,最终降低到6.72~6.81。研究表明,脱二氧化碳装置能够有效去除水体中的CO_2,使水体pH维持在一个适宜鱼类生长的范围。     

大豆PAL2-3基因的克隆及转化研究

大豆异黄酮是苯丙氨酸代谢途径中合成的一类次级代谢产物,在苯丙氨酸代谢途径中,苯丙氨酸解氨酶(PAL)是关键酶和限速酶。本课题组前期研究发现PAL基因的相对表达量与大豆异黄酮含量具有明显的协同增减趋势,并且在PAL基因家族成员时空表达模式分析中发现,PAL2-3(XM_003542493)是PAL基因家族中相对表达量较高的主要表达成员之一。本试验首先通过克隆大豆中PAL2-3基因;然后构建pCAMBIA3301-GmPAL2-3植物过表达载体,将构建好的pCAMBIA3301-GmPAL2-3表达载体重组质粒转化到根癌农杆菌EHA105中;采用农杆菌介导的大豆子叶节转化体系获得转化植株,并对T1代转化植株进行PPT检测,外源标记基因Bar检测以及荧光定量PCR检测,对转基因阳性植株进行大豆籽粒异黄酮含量的测定。结果表明T_1代转基因植株中PAL2-3基因的表达量是对照的5.11~11.24倍,总异黄酮含量最高的(2 587.63μg·g~(-1))是对照(1 616.90μg·g~(-1))的1.6倍。因此在大豆中过量表达PAL2-3基因可以提高大豆籽粒中异黄酮含量。     

Effect of l‐ascorbyl‐2‐polyphosphate supplementation on growth performance,body composition,antioxidative capacity and salinity stress tolerance of juvenile Pacific white shrimp,Litopenaeus vannamei

An 8‐week feeding trial was conducted to evaluate the effects of ascorbic acid (AsA), in the form of l ‐ascorbyl‐2‐polyphosphate (LAPP) on growth performance, body composition, antioxidative capacity and salinity stress tolerance of juvenile Pacific white shrimp, Litopenaeus vannamei. Five practical diets (46% crude protein and 7.6% lipid) supplemented with graded levels of AsA (14.64, 48.55, 84.98, 308.36 and 639.27 mg kg^?1 diet) were fed to five replicate groups of L. vannamei (mean initial wet weight 0.57 g). No significant differences were found on growth performance among all treatments. However, whole body lipid content significantly decreased with dietary AsA levels increasing. Activities of total antioxidant capacity, glutathione reductase and glutathione peroxidase were significantly affected by dietary AsA levels. Shrimp fed LAPP‐free diet had higher malondialdehyde content than those fed the diets supplemented with LAPP. Dietary AsA levels higher than 308.36 mg kg^?1 diet increased the survival of shrimps after 1, 2 and 3 h of acute salinity change. Broken‐line regression analysis on survival after 3 h of salinity stress and second‐degree polynomial regression analysis on glutathione reductase data indicated that the optimal dietary AsA requirement of L. vannamei was estimated to be 306.39, 319.75 mg kg^?1 diet respectively.     

Transgenic sorghum with suppressed synthesis of kafirin subclasses: Effects on flour and dough rheological characteristics

Arising from work showing that conventionally bred high protein digestibility sorghum types have improved flour and dough functionality, the flour and dough properties of transgenic biofortified sorghum lines with increased protein digestibility and high lysine content (TG-HD) resulting from suppressed synthesis of several kafirin subclasses, especially the cysteine-rich γ-kafirin, were studied. TG-HD sorghums had higher flour water solubility at 30 °C (p < 0.05) and much higher paste viscosity (41% higher) than their null controls (NC). TG-HD doughs were twice as strong as their NC and dynamic rheological analysis indicated that the TG doughs were somewhat more elastic up to 90 °C. CLSM of doughs and pastes indicated that TG-HD had a less compact endosperm protein matrix surround the starch compared to their NC. The improved flour and dough functional properties of the TG-HD sorghums seem to be caused by reduced endosperm compactness resulting from suppression of synthesis of several kafirin subclasses which modifies protein body and protein matrix structure, and to improved protein-starch interaction through hydrogen bonding specifically caused by reduction in the level of the hydrophobic γ-kafirin. The improved flour functionality of these transgenic biofortified sorghums can increase their commercial utility by complementing their improved nutritional quality.     

华南6水系与澜沧江-湄公河攀鲈线粒体ND2基因的遗传多样性分析

测定了中国华南6水系及澜沧江(云南勐腊)-湄公河流域(柬埔寨洞里萨湖)的125尾攀鲈(Anabas testudineus)线粒体部分ND2基因1 010 bp序列,分析发现39个变异位点和12个单倍型,总遗传多样性较低(h=0.369,π=0.003 8),推测可能经历过严重的瓶颈效应;中国攀鲈群体遗传多样性更低(h=0.282,π=0.000 4),处于边缘区的中国攀鲈群体是造成低遗传多样性的主要原因。在单倍型网络图中柬埔寨和中国攀鲈各自聚类,具有明显地理结构和谱系结构,推测地质运动和气候变化导致基因交流受阻所致。核苷酸错配图和中性检验表明中国群体经历过种群扩张,时间约为(5.94~4.13)万年前。华南水系群体间基因交流通畅,不存在明显分化;但与云南澜沧江群体间分化大而显著(FST=0.775,P0.01),AMOVA分析显示变异主要来自组群间(77.41%),推测二者分化时间约为(4.0~2.8)万年前,云南群体受末次冰期的影响,基因交流受阻而出现分化。中国群体和柬埔寨群体可作为2个管理单位进行保护;就中国群体而言,韩江水系群体遗传多样性最高,建议优先保护;澜沧江与华南水系间群体分化显著且遗传多样性极低,建议对澜沧江水系群体进行保护,以避免种质资源灭绝。