艾纳香脱氢酶基因家族的生物信息学分析

采用RT-PCR和RACE（Rapid amplification of cDNA ends）技术,从艾纳香（Blumea balsamifera (L.) DC.）叶片中克隆到4条编码艾纳香脱氢酶（BbADH1、BbADH2、BbADH3、BbADH4）基因的cDNA序列,并对4条核苷酸及其编码的氨基酸序列进行生物信息学分析。结果显示:4条艾纳香脱氢酶序列开放阅读框均在900 bp左右,蛋白质等电点（pI）值在5.0~9.0之间,含量最多的氨基酸为亮氨酸（Leu）,最少的为色氨酸（Try）,具有明显的疏水区和亲水区,N端未发现信号肽,且无跨膜区;同源性比对结果显示,艾纳香BbADH蛋白与其他植物中ADH蛋白具有高度的相似性,且具有脱氢酶的特征功能域;系统发育分析表明,艾纳香（B. balsamifera (L.) DC.）BbADH1和BbADH3同处于一个分支,且与胡杨（Populus euphratica Oliv.）PeADH物种亲缘关系较近,而艾纳香BbADH4和BbADH2处于不同分支,且分别与葡萄（Vitis vinifera）VvADH和烟草（Nicotiana tabacum）NtADH物种亲缘关系较近。     

豫南茶树种质资源籽实脂肪含量及脂肪酸组成分析

以豫南地区42份茶树种质资源的茶籽为研究对象,分析了茶籽含仁率及茶籽仁的脂肪含量,范围分别在33.53%~71.60%、17.77%~38.39%。利用气-质联用技术（GC-MS）检测了茶籽仁中的脂肪酸组成和含量,共检测到21种脂肪酸,其中主要成分棕榈酸、硬脂酸、顺-油酸、亚油酸和α-亚麻酸的含量分别在2.64%~5.70%、0.21%~1.11%、7.33%~17.29%、0.09%~8.44%和0.01%~0.15%;相关性分析显示,顺-油酸与十三碳酸、十四碳酸和顺-二十碳二烯酸的含量呈极显著负相关,亚油酸与反-油酸、顺-二十二碳六烯酸含量呈极显著负相关;除了水仙7202茶籽仁中的饱和脂肪酸、单不饱和脂肪酸和多不饱和脂肪酸含量比值为3.08∶12.16∶1,其余茶树种质资源茶籽仁中,这三类脂肪酸的比值均约为1∶2.69∶1.37。中黄1号、农抗早、迎霜、鄂茶11号、龙井43、中选8号、崂山3号和黄金叶8个茶树品种,不仅含仁率和脂肪含量较高,而且茶籽仁脂肪酸的组分和含量丰富、均衡,可初步筛选出来作为豫南地区叶籽两用茶园的候选品种。     

微波真空干燥对铁皮石斛品质特性影响的研究

本研究以新鲜铁皮石斛为原料,采用微波真空干燥方法干燥,通过测定铁皮石斛的多糖含量、色泽、多酚氧化酶（PPO）、水分分布状态、组成氨基酸以及多酚的DPPH?和?OH清除率,研究微波真空干燥对铁皮石斛品质特性的影响。结果表明：相比于新鲜样品,当微波强度为9 W/g,干燥后样品的多糖含量最高可达52.21%;氨基酸分析仪分析结果表明,微波真空干燥后铁皮石斛的总游离氨基酸和风味氨基酸含量显著提高。在微波强度9 、12 、15 W/g下,微波真空干燥的铁皮石斛的PPO活性分别在干燥16、12、6 min后不再存在,表明微波真空干燥能够在较短时间内钝化铁皮石斛的PPO活性,且微波强度越大时间越短。干燥过程的非酶促褐变主要由Maillard反应引起。低场核磁共振结果表明,自由水驰豫时间T₂₃和A₂₃均显著降低。自由基清除能力研究结果表明,铁皮石斛多酚仍具有较强的羟基自由基清除能力。通过观察干燥后样品的微观结构发现,随着微波功率密度的增大,样品孔状结构受到更严重破坏,微波强度越大,铁皮石斛的复水特性越好。     

菠萝酸性转化酶基因家族初步生物信息学及表达分析

酸性转化酶（acid invertase, AIN）在菠萝采后蔗糖降解过程中起着重要作用,基于菠萝全基因组数据库,预测菠萝AIN家族基因并进行生物信息学分析,解析其在采后菠萝不同贮藏温度下的表达变化情况,为阐明AIN基因在采后菠萝果实贮藏特性中的作用奠定基础。以水稻AIN家族基因为探针,在菠萝全基因组中鉴定到2个菠萝细胞壁酸性转化酶基因（cell wall acid invertase, CWIN）和2个液泡酸性转化酶基因（vacuolar acid invertase, VIN）,分别命名为AcCWIN1、AcCWIN2、AcVIN1、AcVIN2,设计编码区引物进行测序验证,并进行生物信息学分析。进化分析结果表明,AcCWIN1、AcCWIN2和AcVIN1、AcVIN2蛋白分别归于细胞壁酸性转化酶和液泡酸性转化酶2个进化支上,且均属于糖基水解酶家族GH32,基因结构、保守域和保守基序均一致。荧光定量分析结果表明,菠萝果肉中AcVIN1和AcVIN2在果实采后贮藏过程中表达量升高,且AcVIN1在发生黑心病的部位大量表达,而AcCWIN1和AcCWIN2在采后贮藏过程中表达量逐渐降低,且随着贮藏温度的升高其表达量降低,预示AcVIN1、AcVIN2较AcCWIN1、AcCWIN2在菠萝采后蔗糖降解和黑心病的发生方面发挥着更为重要的作用。     

甘蓝型油菜MYB28转录因子对根肿菌和水杨酸的响应特征

MYB转录因子参与植物的生长发育和逆境胁迫过程,在植物防御相关信号反应中起着重要的调控作用。 为了明确MYB28在油菜与根肿菌互作中的作用,对感病甘蓝型油菜中双11中MYB28家族基因在根肿菌接种以及 水杨酸处理后的表达模式进行了分析。不同组织表达模式分析显示,相比于根和叶,BnaMYB28 在茎和种子中的表 达水平更高;BnaMYB28 基因在根肿菌和外源水杨酸处理下均有不同程度的表达响应,外源水杨酸处理下Bna⁃ MYB28-1 和BnaMYB28-3 上调表达,BnaMYB28-2 表达则受抑制。根肿菌侵染后,BnaMYB28-1 在整个侵染期表达 量均显著上调,但在加入水杨酸后表达被显著抑制;BnaMYB28-3 表达量在接菌后7 d和28 d显著上调,在关键皮层 侵染期（接菌后14 d）表达量与对照组相比没有变化,但在施加水杨酸处理后表达量显著上调。综上,外源水杨酸能 正向调控BnaMYB28-1 和BnaMYB28-3 表达,BnaMYB28-3 可能通过水杨酸信号路径参与油菜与根肿菌的互作。     

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.     

Effects of apelin-13 on oxidative stress induced by high uric acid in adipocytes

AIM: To study the effects of apelin-13 on oxidative stress induced by high uric acid in 3T3-L1 adipocytes and its underlying mechanisms. METHODS: 3T3-L1 adipocytes were stimulated with uric acid at 10 mg/dL for 48 h. Some of the adipocytes were administered with 1 μmol/L apelin-13 in the presence of uric acid at 10 mg/dL. The adipocytes stimulated with 100 μmol/L H₂O₂ were served as positive controls. The intracellular reactive oxygen species (ROS) concentrations were detected by flow cytometry. The biochemical kits were used to measure the activities of superotide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and NADPH oxidase (NOX) activity, and the content of malondialdehyde (MDA) in the cell lysate and the supernatant. The mRNA levels of renin-angiotensin system (RAS) components, including angiotensinogen (AGT), angiotensin-converting enzyrne1 (ACE1), angiotensin II type 1 receptor (AT1R) and AT2R, as well as angiotensin II receptor -like 1 (APJ) were measured by real-time PCR. The concentrations of angiotensin II (AngⅡ) in the cell lysate and the supernatant were measured by ELISA. RESULTS: Adipocytes stimulated with uric acid at 10 mg/dL had lower activities of antioxidant enzymes (SOD, GSH-PX and CAT) and higher levels of NOX activity and MDA content (P < 0.05). Accordingly, the intracellular ROS levels were found to be dramatically increased. However, apelin-13 administration attenuated uric acid-induced oxidative stress in the 3T3-L1 adipocytes. Uric acid at 10 mg/dL upregulated the mRNA expression of local RAS, enhanced AngⅡ concentrations both in the cell lysate and the supernatant, and down-regulated the mRNA level of APJ in the adipocytes (P < 0.05). Conversely, apelin-13 partially reversed these parameters. CONCLUSION: Apelin-13 attenuates oxidative stress induced by uric acid, may be via down-regulation of local RAS expression in the 3T3-L1 adipocytes.     

马铃薯晚疫病菌效应子PITG_16427.2的基因序列分析及功能验证

 马铃薯晚疫病的病原是致病疫霉菌(Phytophthora infestans),该病原菌在侵染马铃薯过程中分泌大量RxLR型效应子,但目前绝大多数RxLR效应子的功能和作用机制尚不明确。本研究成功克隆了致病疫霉菌的一个RxLR效应子PITG_16427.2,在本氏烟中瞬时表达PITG_16427.2,发现该效应子能够抑制6种激发子(INF1、PsojNIP、BAX、SIF2、Avh238、Avh241)激发的植物免疫反应。进一步研究发现,效应子PITG_16427.2在晚疫病菌侵染马铃薯早期上调表达。在15个致病疫霉菌株和3个同属菌株中克隆该基因,克隆到氨基酸序列一致性超过93%的同源基因,这些同源基因均能在本氏烟中抑制INF1和Avh241引起的HR,揭示了该效应子在病原卵菌中序列和功能的高度保守性。在本氏烟和马铃薯感病品种Désirée中瞬时表达PITG_16427.2,发现该效应子能够显著促进晚疫病菌的侵染。通过qRT-PCR方法检测发现,乙烯信号的相关基因ERF1显著上调,而水杨酸信号相关基因PR1b显著下调,表明PITG_16427.2在晚疫病菌侵染过程中可抑制寄主的SA信号途径,促进晚疫病菌侵染。因此,RxLR型效应子PITG_16427.2是致病疫霉菌中一个重要的侵染致病因子。