miR-142-3p在MCF-7细胞中靶向调节AKT pathway活性机理

为探究miR-142-3p在MCF-7细胞中作用机理,采用RNA免疫共沉淀技术和双荧光素酶报告基因技术筛选及验证miR-142-3p作用靶基因;蛋白质免疫印迹技术验证靶基因及其介导的PI3K-AKT-mTOR信号通路蛋白表达量及通路活性;应用实时荧光定量PCR验证靶基因PTEN对miR-142-3p调节关系。结果表明,miR-142-3p靶向调节AKT和PTEN表达;miR-142-3p过表达组中PI3K-AKT-mTOR通路活性显著降低;miR-142-3p抑制组中PI3K-AKT-mTOR通路活性显著上升;抑制PTEN表达显著提高miR-142-3p表达量。因此miR-142-3p靶向调节AKT和PTEN表达继而抑制PI3K-AKT-mTOR信号通路活性,PTEN与miR-142-3p存在调节关系。     

马铃薯低温响应的ScmiR390-5p及其靶基因表达与结构分析

【目的】研究马铃薯富含亮氨酸重复序列(leucine-rich repeat receptor-like protein kinase LRR-RLK)类受体蛋白激酶基因SCLRRK1受miR390（ScmiR390-5p）调控响应非生物胁迫的机理。【方法】通过低温响应马铃薯miRNA测序分析与靶基因预测,发现ScmiR390-5p通过调控1个可能的LRR类受体蛋白激酶基因对低温做出响应;利用实时荧光定量PCR技术（Quantitative Real-time PCR,RT-qPCR）验证ScmiR390-5p和SCLRRK1响应低温胁迫的表达情况;利用RLM-5′RACE确定ScmiR390-5p作用于SCLRRK1的切割位点;利用PCR技术克隆得到SCLRRK1的DNA序列和CDS序列,生物信息学分析SCLRRK1的结构和功能;利用RT-qPCR分析ScmiR390-5p/SCLRRK1在马铃薯各组织中的表达情况及其响应各种非生物胁迫的表达情况。【结果】RT-qPCR结果表明,低温诱导ScmiR390-5p表达,SCLRRK1的表达则受到低温的抑制,ScmiR390-5p和SCLRRK1的表达在低温胁迫下呈负相关性;RLM-5′RACE分析表明,ScmiR390-5p作用于SCLRRK1的切割位点是ATTCCT//CCTGAGTT,马铃薯ScmiR390-5p/SCLRRK1调控模块在低温响应中起作用。克隆结果表明SCLRRK1的CDS序列长度为2 685 bp,编码894个氨基酸,DNA序列长度为3 549 bp,含有1个内含子、2个外显子、3′非编码区和5′非编码区,ScmiR390-5p的靶位点位于SCLRRK1 CDS序列内部（960—981 bp,GGAACTATTCCTCCTGAGTTT）。生物信息学显示SCLRRK1是1个富含亮氨酸重复序列(leucine-richrepeat,LRR)的类受体蛋白激酶基因,编码的蛋白属于跨膜分泌蛋白。马铃薯组织表达RT-qPCR分析显示,ScmiR390-5p在叶中的表达量最高,根次之,茎中（地上茎、块茎和匍匐茎）表达量较低;而SCLRRK1的表达情况与ScmiR390-5p相反,其在叶中的表达最低,在茎中表达最高（匍匐茎>块茎>地上茎）。多种非生物胁迫结果显示,ScmiR390-5p和SCLRRK1表达在NaCl胁迫下呈负相关,ScmiR390-5p受到NaCl胁迫诱导表达。与对照相比,ABA和6-BA处理下ScmiR390-5p表达先下降之后呈波浪小幅回调;6-BA处理下SCLRRK1表达持续升高,ABA处理下SCLRRK1表达先上升后下降。GA₃、PEG和IAA处理8 h时,ScmiR390-5p的表达达到峰值,GA₃、PEG和IAA处理都能诱导SCLRRK1表达,但其变化趋势与ScmiR390-5p相关性不强。【结论】SCLRRK1具备编码LRR类受体蛋白激酶的核酸、氨基酸序列和结构基础,是ScmiR390-5p的靶基因;ScmiR390-5p/SCLRRK1调控模块在马铃薯组织器官中具备明显作用;ScmiR390-5p在转录后水平通过抑制马铃薯SCLRRK1的表达对低温胁迫做出响应,同时,马铃薯ScmiR390-5p/SCLRRK1调控模块在NaCl和6-BA胁迫响应中起作用,但不对ABA、GA₃、IAA和PEG胁迫做出响应,ScmiR390-5p和SCLRRK1分别在转录水平受到ABA、GA₃、IAA和PEG胁迫信号调控。     

麋鹿非繁殖期生境适宜度评价指标探讨

根据麋鹿生境分布特点,将影响麋鹿生境适宜度的因素分为非生物因子、生物因子和干扰因子,运用麋鹿的生物学、生态学研究成果,对我国麋鹿生境适宜度评价指标进行了探讨。在众多影响麋鹿生存的因子中,植被、食物、水源、外界干扰、生境类型、生态安全等被确定为影响麋鹿生境适宜的主要评价因子,制定了麋鹿生境适宜度评估模型,并赋予了相应分值,可为我国麋鹿栖息地评价提供参考。     

不同处理褐煤腐殖酸对磷吸附特性的研究

采用吸附等温线的试验方法,研究了平衡溶液中磷浓度、反应时间及介质pH对不同处理的2种褐煤腐殖酸吸附磷的影响及其规律性。结果表明:在试验pH范围内,pH的升高减缓了吸附反应进行的速度,对于未经硝酸处理的样品S1,在pH为4.7时,吸附量和分配系数有一最大值,而样品S2,随着pH的升高,吸附量和分配系数一直减小;经硝酸处理后,样品对磷的吸附能力降低,一定条件下其单位吸附量仅为处理前的5.33%。适宜的固液比例可提高2个不同处理供试样品对磷的单位吸附量,其等温吸附规律可用Langmuir方程来加以描述;其吸附动力学用Elovich方程描述最佳。     

绵羊甲状腺miR-370-3p靶向COL4A3基因调控繁殖力性状的初步研究

为探讨小尾寒羊甲状腺组织中与繁殖功能相关的miR-370-3p与COL4A3的靶向关系,本研究利用miRDB与Targetscan网站预测了miR-370-3p的靶基因,并取其交集进行功能富集分析,通过RNAhybrid与Targetscan网站预测绵羊miR-370-3p与候选靶基因的结合位点;随后检测了miR-370-3p与COL4A3基因在小尾寒羊甲状腺组织中的相对表达量。构建psiCHECK2-COL4A3-3′UTR-野生型/突变型双荧光素酶载体,并将其与miR-370-3p mimics/mimics NC共转染至HEK293T细胞并检测荧光活性。结果显示：1)miR-370-3p的靶基因可富集到孕酮介导的卵母细胞成熟、卵母细胞减数分裂、HIF-1信号通路、MAPK信号通路、mTOR信号通路、Ras信号通路和FoxO信号通路等与动物繁殖有关的信号通路中。2)miR-370-3p可与COL4A3基因3′UTR区域结合,且两者在小尾寒羊甲状腺组织中表达呈相反趋势。3)共转染psiCHECK2-COL4A3-3′UTR-野生型和miR-370-3p mimics组的荧光素酶活性显...     

Effect of DNA methylation of miR-30a-5p promoter region on hepatic injury induced by homocysteine

AIM: To explore the role of DNA methylation of microRNA-30a-5p(miR-30a-5p) promoter region in hepatic injury. METHODS: Four-week-old normal mice and cystathionine β-synthase (CBS) single gene knockout mice were used and divided into normal (CBS^+/+, n=12) group and single gene knockout (CBS^+/-, n=12) group, and the mice were fed with high methionine diet for 8 weeks. HL-7702 hepatic cells were routinely cultured in vitro and divided into control group, homocysteine (Hcy) group and Hcy+5-azacytidne (AZC) group. Serum Hcy, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by automatic biochemical analyzer. The levels of ALT and AST in the cells culture medium were determined by the microplate method. Hepatic injury in the mice were observed with HE staining. Cell viability staining was used to measure the viability of hepatocytes. RT-qPCR was used to detect the expression of miR-30a-5p in the liver tissues and hepatocytes. The correlation between the expression of miR-30a-5p and serum ALT and AST levels was analyzed by Pearson correlation analysis. DNA methylation level of miR-30a-5p promoter region in the liver tissues and hepatocytes was detected by nested landing methylation-specific PCR (nMS-PCR). RESULTS: Compared with the CBS^+/+ mice, the serum levels of Hcy, ALT and AST in the CBS^+/- mice were significantly increased (P < 0.05). HE staining showed the hepatocyte swelling and nuclear fragmentation and dissolution. The expression level of miR-30a-5p in the liver tissues was decreased (P < 0.01). Besides, the expression level of miR-30a-5p in the mice was negatively correlated with serum ALT and AST levels (r²=0.4557, P=0.0003, r²=0.4626, P=0.0003), and the DNA methylation of miR-30a-5p promoter region was increased (P < 0.01). In the HL-7702 cells, compared with control group,the ALT and AST levels were increased in Hcy group (P < 0.05, P < 0.01), and the cell viability was remarkablely decreased. DNA methylation of miR-30a-5p promoter region was increased (P < 0.01), which decreased after treated the cells with AZC (P < 0.05), while the expression level of miR-30a-5p in the cells was increased (P < 0.05). CONCLUSION: Hypermethylation of miR-30a-5p promoter region may play an important role in hepatic injury.     

上海市农民宅基地置换思考与探索——以奉贤区四团镇为例

通过对上海市奉贤区四团镇农民宅基地置换案例的剖析,指出了安置方式单一、宅基地置换资金收支不平衡、置换后农民权益保障不足等问题,给出了探寻多元化安置方式、搭建资金平台、完善后续保障体系等有效的解决方法及途径。     

“一分钟游程”构园法则的适用性研究

“一分钟游程”构园法则将人的视觉感受与园林组景共同考虑,以25~30 m作为组景转换的理想距离,对游路结构进行反复烘托与不断强化,从而形成柔和、丰富的“构景曲线”。选择南北方各具代表性的苏州沧浪亭和北京恭王府花园,分析其园林的景观布局,对比园林空间序列的“起”“承”“转”“合”间距。结果表明,“一分钟游程”构园法则在南方私家园林及北方皇家园林中同样适用,为今后的游线组织与组景布设提供了设计参考。     

Outer membrane protein A of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in RAW264.7 cells

AIM:To analyze the effects of outer membrane protein A (OmpA) from Acinetobacter baumannii ATCC 19606 on the autophagy of RAW264.7 cells. METHODS:The RAW264.7 cell model stimulated by OmpA was established. The effects of OmpA on the autophagy of RAW264.7 cells were detected by immunofluorescence, Western blot and transmission electron microscopy. RESULTS:The OmpA increased the expression of LC3B-Ⅱ and reduced the phosphorylation levels of Akt, mTOR and p70S6K. Rapamycin further reduced the phosphorylation levels of mTOR and p-70S6K, and increased the expression of LC3B-Ⅱ induced by OmpA. CONCLUSION:The OmpA of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in the RAW264.7 cells. This work provides a basis for further research on the molecular mechanism of autophagy induced by Acinetobacter baumannii to find a new method against the infection of Acinetobacter baumannii.     

大兴安岭北部林区森林防火基础设施建设成效及对策

大兴安岭北部林区森林防火基础设施建设项目是建国后国家一次性用于国有林区森林防火投资最大的基础设施建设工程。介绍了该项目建设的基本情况,总结了主要建设成效,分析了存在问题,提出了相关对策建议。