miR-106b-5p靶向KLF4调控山羊肌内前体脂肪细胞分化

旨在明确miR-106b-5p对山羊肌内前体脂肪细胞分化的影响，并确定这种作用是通过靶向KLF4来实现的。本研究利用实时荧光定量PCR （quantitative real-time PCR，qRT-PCR）技术检测miR-106b-5p在山羊肌内前体脂肪细胞分化过程中的表达模式，通过脂质体转染技术将miR-106b-5p mimic和miR-106b-5p inhibitor转入体外培养的山羊肌内前体脂肪细胞，油红O染色法从形态学验证miR-106b-5p对脂肪细胞中脂滴积聚的影响，qRT-PCR检测预测的靶标基因KLF4和脂肪分化标志基因的表达情况，利用双荧光素酶报告系统鉴定miR-106b-5p与KLF4的靶标关系。qRT-PCR结果显示，miR-106b-5p在山羊肌内前体脂肪细胞诱导分化第3天时表达量最高。在山羊肌内脂肪细胞中干扰miR-106b-5p后油红O染色显示脂滴聚积减少，过表达miR-106b-5p后脂滴聚积增加。在山羊肌内前体脂肪细胞中转染miR-106b-5p inhibitor后PPARγ表达量显著降低（P<0.05），而KLF4表达量极显著升高（P<0.01）；转染miR-106b-5p mimic后LPL和PPARγ表达量极显著升高（P<0.01）。荧光素酶活性试验结果显示，过表达miR-106b-5p可显著抑制KLF4荧光活性。miR-106b-5p通过靶向并负调节KLF4的表达促进山羊肌内脂肪细胞分化。     

Effects of the severity and timing of basal leaf removal on the amino acids profiles of Sauvignon Blanc grapes and wines

The effects of the severity and timing of leaf removal(LR) on the amino acids of Sauvignon Blanc grapes and wines were studied during the 2017 growing season. High-performance liquid chromatography(HPLC) was used to analyze the amino acids profiles of grape berries and wines. The basal leaves were removed at three time points(40, 56 and 72 days after flowering, named LR40, LR56 and LR72, respectively) at two severity levels(one at which the first, third, and fifth basal leaves of each shoot were removed(50% level); and another at which the first six basal leaves were removed(100% level)). The results showed that leaf removal had little impact on total soluble solids(°Brix), titratable acidity, pH or berry weight. The LR72-50% treated grapes had higher berry weight, titratable acidity and °Brix than those of the other treatments. The highest concentrations of total amino acids and of total amino acids except proline were detected in LR72-50% treated grapes(2 952.58 and 2 764.36 mg L~(-1), respectively); the lowest were detected in LR72-100% treated grapes(2 172.82 and 2 038.71 mg L~(-1), respectively). LR72-50% treatment significantly promoted the synthesis of aspartic acid, serine, arginine, alanine, aminobutyric acid and proline at both severity levels for grapes, the concentrations of all of these amino acids were increased relative to the control concentrations. The LR72-50%, LR40-100% and LR72-100% treated wines had higher total amino acids concentrations and higher concentrations of some individual amino acids, such as arginine, alanine and serine, than did the control wines. Of all the amino acids studied, glycine, tyrosine, cysteine, methionine and lysine were not significantly influenced by the timing or severity basal defoliation in grapes and wines. The present study reveals the effects of the timing and severity of leaf removal on the amino acids profiles of grapes and wines.     

Reduction in cadmium accumulation in japonica rice grains by CRISPR/Cas9-mediated editing of OsNRAMP5

Cadmium (Cd) intake is harmful to human health and Cd contamination in rice grains represents a severe threat to those consuming rice as a staple food. Knockout of Cd transporters is a promising strategy to reduce Cd accumulation in rice grains. OsNRAMP5 is the major transporter for Cd and manganese (Mn) uptake in rice. Nevertheless, it is uncertain whether knockout of OsNRAMP5 is applicable to produce low Cd rice without affecting plant growth and grain yield. In this study, we adopted CRISPR/Cas9-based gene editing technology to knock out OsNRAMP5 in two japonica varieties. We generated three independent transgene-free osnramp5 mutants and investigated the effect of osnramp5 mutations on Cd accumulation and plant growth. Hydroponic experiments showed that plant growth and chlorophyll content were significantly reduced in osnramp5 mutants at low Mn conditions, and this defective growth in the mutants could be fully rescued by supply of high levels of Mn. Cd and Mn accumulation in both roots and shoots was markedly reduced in the mutants compared to that in wild-type plants. In paddy field experiments, although Cd in flag leaves and grains was greatly reduced in osnramp5 mutants, some agronomic traits including plant height, seed setting rate, and grain number per panicle were affected in the mutants, which ultimately caused a mild reduction in grain yield. The reduced plant growth in the mutants can be attributed to a marked decrease in Mn accumulation. Our results reveal that the manipulation of OsNRAMP5 should be treated with caution: When assessing the applicability of osnramp5 mutants, soil pH and soil water content in paddy fields need to be taken into consideration, since they might affect the levels of available Mn in the soil and consequently determine the effect of the mutation on grain yield.     

Sublytic C5b-9 induces protective autophagy in cultured podocytes

AIM: In podocytes, autophagy occurs at a high basal level and dysregulated autophagy is associa-ted with a variety of podocytopathies. This paper is to investigate the role of autophagy in sublytic C5b-9-induced podocyte injury. METHODS: Sublytic complement C5b-9 stimulation was used as an in vitro model. Autophagosomes were confirmed using monodansylcadaverine (MDC) staining. Immunoblotting was used to measure the change of autophagy-related markers. Cellular morphological changes were observed by Wright-Giemsa staining. Immunofluorescence staining and confocal microscopy were used to detect the expression and distribution of nephrin. The cell viability was assessed by methylthiazol tetrazolium (MTT) assay. The cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/PI staining. RESULTS: For ensuring sublytic complement injury, the maximal amounts of anti-podocyte antiserum and 160×-diluted normal human serum were used without inducing cell lysis (defined as >5% LDH release). Sublytic C5b-9 promoted autophagy of podocytes in vitro. The proautophagic effect of sublytic C5b-9 manifested in the form of accumulated MDC-labeled vesicles and enhanced the expression of LC3-Ⅱ. Autophagy inhibitor 3-methyladenosine (3-MA) promoted sublytic C5b-9-induced podocyte morphological abnormalities. Compared with the sublytic C5b-9-injured podocytes, 3-MA exposure further decreased the expression of nephrin. 3-MA enhanced sublytic C5b-9-induced podocyte apoptosis. CONCLUSION: Sublytic C5b-9 attack induces autophagy, which may play a protective role against complement-mediated podocyte injury.     

水产调味品鲫鱼汁的研制

随着人们生活水平的提高,对食品质量的要求也在不断的提高,调味品在食品质量中扮演着非常重要的角色。本实验以普通养殖鲫鱼为主要原料,研究水产调味品鲫鱼汁的最佳工艺。通过正交试验和感官评价,最终得到水产调味鲫鱼汁的最佳配方:黄原胶0.04%,鲫鱼汁30%,盐15%,糖5%,谷氨酸钠5%,5′-肌苷酸0.25%,叶黄素0.03%,鱼味香精0.03%,山梨酸钾0.05%,均质压力35MPa。水产调味品鲫鱼汁质地均匀,口感细腻,鱼鲜风味突出,色泽淡黄,气味芬芳,符合人们对于风味和营养的需求,具有广阔的市场前景。     

谷子杂交种与常规种光合和叶绿素荧光特性的比较

为了明确杂交谷的光合优势是否是其产量高于常规谷的一大主因,本研究于2013年对常规谷晋谷21号和杂交谷张杂谷5号的光合和叶绿素荧光特性进行比较。采用完全随机设计,每个品种种植5个小区,在拔节期和灌浆期分别测定各项光合指标。结果表明,在拔节期,张杂谷5号的净光合速率、PSⅡ实际光化学量子产量显著高于晋谷21号;但在灌浆期,张杂谷5号和晋谷21号之间的总叶绿素含量、净光合速率、蒸腾速率、气孔导度、胞间CO2浓度、PSⅡ实际光化学量子产量、光化学淬灭参数和非光化学淬灭参数都存在显著差异。总体杂交谷在拔节期和灌浆期的光合性能高于常规谷。     

荔枝FKBP16-2基因启动子的克隆与瞬时表达分析

前期研究中发现了一个果肉低表达而叶片中高表达的荔枝基因FKBP16-2。本文克隆了该基因1 578 bp的启动子片段并对其功能进行了初步分析,结果表明:荔枝FKBP16-2基因启动子序列中含有大量的TATA-box和CAAT-box保守元件,以及TCA-element,ARE,HSE,GCN4_motif,O2-site等各种转录调控相关的顺式作用元件。该启动子能驱动GUS基因在荔枝的花、叶、根、果皮以及种子中表达而在果肉中不表达,表达具有组织特异性。     

牛羊胎盘免疫调节因子的提取及对PANC-Ⅰ增殖和迁移影响的比较

胎盘免疫调节因子(placenta immunoregulating factor,PF)是从胎盘提取的小分子多肽,对病毒性疾病、免疫缺陷疾病及恶性肿瘤等具有潜在的临床应用价值.为检测牛(Bos taurus)和绵羊(Ovis aries)胎盘免疫调节因对人(Homo sapiens)胰腺癌细胞(pancreatic cancer cells-Ⅰ,PANC-Ⅰ)增殖与迁移的影响,本研究对从屠宰场获得的牛和绵羊胎盘进行分离,利用双酶水解法制备PF,将质量浓度为100～2 000 ng/mL的牛、羊PF作用于体外培养的PANC-Ⅰ细胞,以3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲酯基)-2-(4-磺苯基)-2H-四唑(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,MTS)法观察细胞增殖抑制率,应用流式细胞技术检测其对PANC-Ⅰ细胞周期的影响.并利用Transwell迁移实验检测添加PF对PANC-Ⅰ细胞迁移能力的影响,同时通过实时荧光定量PCR法检测肿瘤抑制基因(tumor suppressor gene,PS3)和细胞周期依赖性蛋白激酶抑制因子1A基因(cyclin-dependent kinaseinhibitor 1A,P21)的表达情况.结果显示,17.11 g绵羊胎盘制得0.588 mg PF,6.61 g牛胎盘制得0.312 mgPF,胎盘质量与所得PF的质量比约为20 000∶1.在体外培养的PANC-Ⅰ细胞培养液中添加PF,从形态上观察,牛羊PF对PANC-Ⅰ细胞没有明显的影响.绵羊PF处理后PANC-Ⅰ细胞增殖抑制率为9.75％～22.89％,牛PF处理后PANC-Ⅰ细胞增殖抑制率为-3.81％～23.56％,增殖抑制率最显著的绵羊PF浓度为500 ng/mL,牛PF浓度为1 000 ng/mL.绵羊PF在浓度为500 ng/mL时,PANC-Ⅰ细胞的PI和SPF最低,而牛PF各浓度下的胰腺癌细胞增殖指数(proliferation index,PI)和增殖活性(S-phase fraction,SPF)没有明显的变化.同时,牛羊PF对PANC-Ⅰ细胞的迁移能力无显著影响.牛羊PF使PANC-Ⅰ细胞P53表达上调而P21表达下调.上述结果表明,在一定浓度下,PF对体外培养的PANC-Ⅰ细胞增殖有负调控作用,且牛羊胎盘免疫调节因子对胰腺癌细胞的影响不同,可能因为其存在的活性成分有区别.本研究为胰腺癌的治疗提供了新的思路.     

pGH和IGF-Ⅰ双基因共表达载体的构建及转双基因猪的获得与检测

旨在构建可高效表达pGH基因和IGF-Ⅰ基因的双基因共表达载体,制备转双基因(pGH+IGF-Ⅰ)猪,以期探索pGH基因和IGF-Ⅰ基因对猪生长发育的影响,为节粮型高瘦肉率新品种猪的培育奠定理论基础。从长白猪耳样中提取总RNA,经反转录RT-PCR获得pGH基因不含终止密码子的编码序列和IGF-Ⅰ基因完整的编码序列,经酶切连接克隆至pc DNA3.1(+)真核表达载体上,构建pc DNA3.1(+)-pGH-IGF-Ⅰ双基因共表达载体。将其转染PK15细胞,Q-PCR检测2个目的基因在PK15细胞中的表达情况。将构建的双基因共表达载体用纳米材料包裹后转染长白猪精子,采用精子载体法制备转双基因猪。PCR及测序鉴定转双基因阳性个体,Q-PCR检测2个目的基因在转双基因猪体内的表达情况。PCR及测序鉴定追踪检测转双基因猪体内pGH基因和IGF-Ⅰ基因的稳定情况。RT-PCR及测序结果表明,成功克隆了长白猪的pGH基因和IGF-Ⅰ基因的编码序列。酶切和测序分析表明成功构建了双基因真核共表达载体,转染PK15细胞后,Q-PCR检测表明,pGH基因和IGF-Ⅰ基因均在mRNA水平成功表达。母猪妊娠获得13头仔猪,经PCR及测序检测,其中4头仔猪为转双基因阳性,转双基因阳性率为30.76%。Q-PCR检测外源pGH基因与IGF-Ⅰ基因在转双基因猪体内成功表达。1~7月龄均可检测到外源pGH基因与IGF-Ⅰ基因,证明2个外源基因在转双基因猪体内稳定存在,并未随着生长而丢失。在转双基因公猪的精液中均能检测到2个外源基因,证明外源基因存在稳定传代的可能。     

Mapping soil salinity and pH across an estuarine and alluvial plain using electromagnetic and digital elevation model data

Increasing pressures from agriculture and urbanization have resulted in drainage of many floodplains along the eastern Australian coastline, which are underlain by sulphidic sediments, to lower water tables and reduce soil salinity. This leads to oxidation of the sediments with a rapid decline in pH and an increase in salinity. Accurately mapping soil salinity and pH in coastal acid sulphate soil (CASS) landscapes is therefore important. One required map is the extent of highly acidic (i.e. pH < 4.5) areas, so that the application of alkaline amendments (e.g. lime) to neutralize the acid produced can be specifically targeted to the variation in pH. One approach is to use digital soil mapping (DSM) using ancillary information, such as an EM38, digital elevation models (DEM – elevation) and trend surface parameters (east and north). We used an EM38 in the horizontal (EM38h) and vertical (EM38v) modes together with elevation data to develop multiple linear regressions (MLR) for predicting EC_1:5 and pH. For pH, best results were achieved when the EM38 EC_a data were log‐transformed. By comparing MLR models using REML analysis, we found that using all ancillary data was optimal for mapping EC_1:5, whereas the best predictors for pH were north, log‐EM38v and elevation. Using residual maximum likelihood (REML), the final EC_1:5 and pH maps produced were consistent with previously defined soil landscape units, particularly CASS. The DSM approach used is amenable for mapping saline soils and identifying areas requiring the application of lime to manage acidic soil conditions in CASS landscape.