两种复合肥在叶菜类蔬菜上的应用效果比较

在山东省莱阳和寿光两地露地及保护地生长的芹菜和大白菜上进行新型复合肥 2 1 8 11S肥效研究 ,同时与市场上较为流行的 15 15 15S复合肥进行比较。结果表明 ,2 1 8 11S复合肥比 15 15 15S复合肥在增加产量、提高品质方面效果更好。 2 1 8 11S复合肥最佳用量大白菜为 14 14～ 170 0kg·hm-2 ,芹菜为 2 0 6 2～ 2 4 78kg·hm-2 。     

Relationship of p21WAF1 gene polymorphisms with protein expression in breast carcinomas

AIM: To investigate the relationship between p21^WAF1gene polymorphisms and protein expression in breast carcinoma. METHODS: Polymerase chain reaction single-strand conformation polymorphisms technique (PCR-SSCP) and immunohistochemical assay of S-P immunostaining technique were used to study polymorphisms of p21^WAF1 and protein expression respectively on the specimen of paraffin-embedded tissues in 100 cases of breast carcinomas and 40 benign breast diseases as control. RESULTS: Two p21^WAF1 gene polymorphisms were found in 18% (18/100) of breast carcinomas and 5% (2/40) of control samples. The difference between the two groups was statistically significant (χ²=3.94, P＜0.05). The positive immunohistochemical reaction of p21^WAF1 protein were found in 50% (50/100) of breast carcinomas and 12.5% (5/40) of control samples. The difference between the two groups was statistically significant (χ²=16.84, P＜0.01). The positive immunohistochemical reaction of p21^WAF1 protein were found in 100% (18/18) of breast carcinomas with p21^WAF1 gene polymorphisms and 39% (32/82) of no p21^WAF1 gene polymorphisms. The difference between two groups was statistically significant (χ²=21.95, P＜0.01). The p21^WAF1 gene polymorphisms were correlated with the protein expression in breast carcinomas (r=0.576, P＜0.01). CONCLUSION: p21^WAF1 gene polymorphisms may create the different copies of mRNA and may make relevant protein molecules.     

miR-18a-5p在鸡不同生长时期的表达及其生物信息学分析与靶基因预测

为探究miR-18a-5p在鸡不同生长时期组织表达变化规律及其生物信息学特点,本研究以苏禽3号鸡为试验动物,利用实时荧光定量PCR技术检测不同生长时期鸡miR-18a-5p的组织表达变化。通过文献和miRBase检索脊椎动物的miR-18a-5p序列,利用Ensembl数据库确定miR-18a-5p在基因组中的位置,根据成熟序列构建系统进化树;使用miRmap、microT、miRanda和TargetScan网站预测miR-18a-5p靶基因,并进行GO和KEGG分析。组织表达分析结果显示,miR-18a-5p序列在鸡心脏、脾脏、肾脏和下丘脑中的表达量显著高于除大脑以外的其他组织(P<0.05)。与3日龄雏鸡相比,90日龄鸡心脏、脾脏、肾脏、腿肌和下丘脑中miR-18a-5p的表达量均显著上升(P<0.05)。在50种脊椎动物中共发现52条miR-18a-5p序列,几乎所有物种都只有1条成熟序列;基因定位分析发现,鸡miR-18a-5p位于1号染色体上的基因间隔区。多序列比对分析表明,不同物种miR-18a-5p的成熟序列同源性较高,物种间较为保守。系统进化树分析发现,鸡miR-18a-5p与原鸽、斑胸草雀等其他鸟类聚为一类,这表明miR-18a-5p进化过程中是保守的。靶基因预测和功能分析发现,miR-18a-5p共有121个靶基因。GO分析结果显示,靶基因主要富集到蛋白质泛素化、细胞周期阻滞、白细胞介素-6产生的负调控等功能。KEGG 通路分析表明,靶基因主要富集到细胞周期及Wnt和FoxO信号通路等。多个与肌肉生长及细胞增殖等相关的基因富集到相关通路之上。综上,鸡miR-18a-5p是组织广泛表达的miRNA,其可能通过Wnt及FoxO信号通路调控肌肉生长及细胞增殖分化。本研究为miR-18a-5p功能及调控机制的深入研究提供参考依据。     

miR-122靶向调控鸡BZW2基因的表达

miR-122(MicroRNA-122)是在肝脏中高表达的miRNA,在肝脏的生长发育和脂类代谢等过程发挥重要作用,BZW2 (basic leucine zipper and W2 domains 2)主要参与蛋白质合成代谢,本研究目的是确定BZW2是否为miR-122调控的靶基因.通过生物信息学预测miR-122的靶基因,并分析BZW2的3-UTR区域与miR-122种子区的配对情况,再用双荧光素酶报告系统和突变实验证明miR-122作用于BZW2的3'UTR.用荧光定量PCR检测转染miRNA-122mimic的鸡(Gallus gallus)肝癌细胞系(leghorn hepatocellar,LMH)细胞和转染LNA-antimiR-122的鸡原代肝细胞中BZW2的表达.鸡BZW2的3'-UTR区域与miR-122种子区互补配对,荧光素酶报告基因和突变实验分析表明,miR-122能够通过与BZW23'-UTR区结合抑制基因的表达;将miR-122在LMH细胞中过表达后,发现BZW2 mRNA表达水平显著下降;利用LNA-antimiR-122抑制鸡肝脏原代细胞中的miR-122后,BZW2 mRNA表达水平呈显著性上升.结果证明BZW2是miR-122的靶基因,其在mRNA水平上受到miR-122的负性调控,本研究为揭示miR-122在鸡肝脏中的广泛的功能提供了理论依据.     

Expression and mechanisms of hsa_circ_087631 in patients with primary biliary cholangitis

AIM To explore the expression and mechanisms of circular RNA hsa_circ_087631 in the patients with primary biliary cholangitis （PBC）. METHODS RT-qPCR was used to detect the expression of hsa_circ_087631 in PBC patients and healthy controls. Hsa-miR-346-overexpressing lentiviral vector pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS was constructed and transfected into human acute T cell leukemia Jurkat cells， and then the expression levels of hsa_circ_087631， Bcl-6 mRNA and interleukin-21 （IL-21） mRNA were detected by RT-qPCR. Dual-luciferase reporter assay was performed to identify the interactions between hsa_circ_087631 and hsa-miR-346. RESULTS The expression of hsa_circ_087631 in the PBC patients was significantly increased compared with the healthy subjects. Hsa-miR-346-overexpressing lentiviral vector pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS was successfully constructed. The expression of hsa-miR-346 was significantly increased after the hsa-miR-346-overexpressing lentiviral vector was transfected into the Jurkat cells， while the expression levels of hsa_circ_0087631， Bcl-6 mRNA and IL-21 mRNA were significantly decreased. After wild-type or mutant hsa_circ_087631 vector and hsa-miR-346 mimics were transfected into 293T cells， the results of dual-luciferase reporter assay showed that hsa-miR-346 significantly decreased the luciferase activity of wild-type hsa_circ_087631 （P<0.01）， but the regulation did not change significantly after mutation of the predicted binding site. CONCLUSION Peripheral blood hsa_circ_087631 level is elevated in the PBC patients. The hsa_circ_087631/hsa-miR-346/Bcl-6 signaling may take effect in human T cells. Hsa-miR-346 significantly reduces the expression of hsa_circ_087631， but it may not be regulated by predicted binding sites.     

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。     

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.     

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。     

鸡脂肪组织TCF21基因启动子区DNA甲基化与其表达的关系

旨在研究鸡脂肪组织中TCF21基因启动子区DNA甲基化水平与其表达的关系。以东北农业大学高、低腹脂双向选择品系（简称高、低脂系）第24世代7周龄肉鸡为试验材料，利用RT-qPCR检测高、低脂系肉鸡腹部脂肪组织中TCF21基因的mRNA表达水平；利用生物信息学和双荧光素酶报告系统分析TCF21基因启动子的结构与功能；利用Sequenom MassARRAY飞行质谱检测高、低脂系肉鸡腹部脂肪组织中TCF21基因启动子区CpG位点的甲基化水平；利用CpG甲基转移酶处理TCF21启动子报告基因质粒，分析DNA甲基化对TCF21基因启动子活性的影响。结果显示，高脂系肉鸡腹部脂肪组织中TCF21基因的mRNA表达水平极显著高于低脂系（P<0.001）；TCF21基因的启动子区存在40个CpG位点，且在启动子的近端和远端均有分布，但不存在CpG岛；将TCF21基因的启动子划分为5个功能区域，分别为R1区域（-2 000~-1 500 bp）、R2区域（-1 500~-1 000 bp）、R3区域（-1 000~-500 bp）、R4区域（-500~-200 bp）和Core区域（-200~-100 bp）；高脂系R2、R3和R2+R3区域的DNA甲基化水平显著或极显著高于低脂系（P<0.05或P<0.001）；R2、R3、R2+R3区域的DNA甲基化水平与TCF21基因mRNA表达水平呈显著正相关（R2区域：r=0.438，P<0.05；R3区域：r=0.371，P<0.05；R2+R3区域：r=0.489，P<0.05）；R2区域的DNA甲基化显著抑制其转录活性（P<0.05）。综上所述，TCF21基因在高、低脂系肉鸡腹部脂肪组织中的表达水平主要与其启动子R2区域的DNA甲基化水平有关。     

microRNA-146a通过靶向MAPK4和Myosin Va基因调控羊驼黑色素细胞增殖、迁移

旨在研究microRNA-146a(miR-146a)对羊驼黑色素细胞增殖和迁移的调控及其分子机制。本研究使用双荧光素酶试验验证MAPK4和Myosin Va是miR-146a的靶基因;利用荧光定量PCR和蛋白质免疫印迹试验检测在羊驼黑色素细胞中过表达miR-146a对相关下游基因表达的影响;利用CCK8和Transwell检测miR-146a过表达对羊驼黑色素细胞增殖和迁移的影响。结果显示,与对照组相比,将miR-146a和MAPK4或Myosin Va共转染293T细胞,双荧光素酶活性分别极显著下降36%和30%(P<0.001);MAPK4和Myosin Va基因转录水平分别极显著下调67%和47%(P<0.001,P<0.01);蛋白质水平的表达量分别显著或极显著下调38%和69%(P<0.05,P<0.01);增殖和迁移相关的基因CREB、MITF、MLPH和Rab27a在转录水平和蛋白水平的表达均极显著下调(P<0.01,P<0.001);CCK8和Transwell结果显示,过表达miR-146a使羊驼黑色素细胞的增殖和迁移能力极显著下调(P<0.01)。综上所述,miR-146a通过靶向调控MAPK4和Myosin Va,使增殖和迁移相关的基因MEK1、CREB、MITF、MLPH和Rab27a的表达下调,从而对羊驼黑色素细胞的增殖和迁移起抑制作用。