拟南芥γ-生育酚甲基转移酶启动子在转基因烟草中的表达特性分析

γ-生育酚甲基转移酶(γ-TMT)是维生素E生物合成途径中的关键酶之一,催化γ、δ-生育酚甲基化,生成活性较高的α、β-生育酚。研究构建了含有γ-TMT启动子和GUS报告基因的植物表达载体,通过农杆菌介导转化烟草,筛选出PCR阳性植株,进行GUS组织化学染色,结果表明,在γ-TMT启动子的驱动下,报告基因GUS在烟草的根、茎、叶中均有表达。     

ASH1L甲基转移酶在牛卵丘细胞中的表达与功能研究

旨在探究缺失、小的、同源异形1（absent，small，or homeotic 1-like，ASH1L）甲基转移酶在牛卵丘细胞中的表达与功能。本研究通过免疫荧光染色在健康母牛卵丘细胞中对ASH1L甲基转移酶进行定位，并分析细胞的组蛋白H3第36位赖氨酸（histone H3 lysine36，H3K36）甲基化修饰模式；合成靶向Ash1L基因的siRNA，对siRNA-1、siRNA-2、siRNA-3及对照组进行荧光定量PCR和蛋白质免疫印迹，筛选有效siRNA；采用荧光定量PCR分析干扰Ash1L表达对处理组及对照组中凋亡相关基因及多梳抑制复合体（polycomb repressive complex 2，PRC2）组成基因的表达水平的影响。结果显示，ASH1L甲基转移酶位于牛卵丘细胞的细胞核中，呈点状分布。成功筛选到能有效干扰牛Ash1L基因的siRNA-2，其干扰效率为60%~70%。将siRNA-2转染卵丘细胞后，该干扰组细胞中H3K36的单甲基化、二甲基化及三甲基化3种甲基化水平均显著低于对照组（P<0.05）；干扰Ash1L导致凋亡相关基因Bax、Bcl-2及caspase-3表达水平显著上调，凋亡基因Bax和caspase-3表达量高于抗凋亡相关基因Bcl-2（1.311和1.179 vs 1.146）；同时，干扰Ash1L基因表达也引起PRC2蛋白亚基EZH2和Suz12基因的mRNA表达量显著升高（P<0.05）。综上所述，本研究探讨了ASH1L甲基转移酶在牛卵丘细胞中的表达和功能，ASH1L在牛卵丘细胞中呈点状分布，且Ash1L基因的抑制导致H3K36me1/2/3水平均显著下降及凋亡基因和PRC2蛋白相关亚基EZH2和Suz12基因表达的升高，为进一步研究其对家畜胚胎的调控作用提供技术和理论基础。     

水稻果胶甲基转移酶OsTSD2基因突变体的分析

探讨水稻果胶甲基转移酶OsTSD2基因的功能,尤其是对生长发育的影响,筛选并鉴定了OsTSD2基因突变的3个突变体tsd2a、tsd2b和tsd2c。表型观察显示:突变体水稻结实率下降、种子粒长变短、粒宽变窄、千粒重下降;突变体的颖壳颜色变暗,颖壳的破裂程度加重并出现新的背部破裂;荧光免疫标记试验研究发现,突变体种子萌发率下降可能是由于突变体种子盾片中的甲基化果胶含量低所致。结果表明,OsTSD2基因的下调导致水稻的种子在发育和萌发过程中存在明显的缺陷。     

广西麻鸡m6A甲基转移酶基因表达与肌纤维类型及成肌分化的关系

[目的]肌纤维类型及其转化的调节是改善肉质的重要途径,最新研究表明,m6A RNA甲基化修饰在肌肉的分化中发挥着重要作用.以优良的地方品种——广西麻鸡作为研究对象,研究其性成熟日龄不同部位肌纤维类型的组成差异,探究m6A甲基化转移酶相关基因甲基化转移酶样蛋白3/14(methyltransferase like 3/1...     

Bioactivity Potential of Marine Natural Products from Scleractinia-Associated Microbes and In Silico Anti-SARS-COV-2 Evaluation

Marine organisms and their associated microbes are rich in diverse chemical leads. With the development of marine biotechnology, a considerable number of research activities are focused on marine bacteria and fungi-derived bioactive compounds. Marine bacteria and fungi are ranked on the top of the hierarchy of all organisms, as they are responsible for producing a wide range of bioactive secondary metabolites with possible pharmaceutical applications. Thus, they have the potential to provide future drugs against challenging diseases, such as cancer, a range of viral diseases, malaria, and inflammation. This review aims at describing the literature on secondary metabolites that have been obtained from Scleractinian-associated organisms including bacteria, fungi, and zooxanthellae, with full coverage of the period from 1982 to 2020, as well as illustrating their biological activities and structure activity relationship (SAR). Moreover, all these compounds were filtered based on ADME analysis to determine their physicochemical properties, and 15 compounds were selected. The selected compounds were virtually investigated for potential inhibition for SARS-CoV-2 targets using molecular docking studies. Promising potential results against SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and methyltransferase (nsp16) are presented.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells

AIM:To explore the effect of pyrrolidine dithiocarbamate (PDTC),an NF-κB inhibitor,on the proliferation and apoptosis of human multiple myeloma U266 cells and its mechanisms.METHODS:The U266 cells were treated with PDTC at different concentrations (0,25,50,100 and 200 μmol/L)in vitro.The growth inhibitory rate of the U266 cells was detected by CCK-8 assay and cell counting.The cell cycle of the U266 cells was determined by flow cyto-metry,and the apoptosis was examined by flow cytometry with Annexin V-FITC/PI staining.The effect of PDTC on the expression of DNA methyltransferase 1(DNMT1) at mRNA and protein levels was measured by RT-qPCR and Western blot,respectively.The effects of PDTC on the protein levels of NF-κB (P65),DNMT1,Bcl-2,cyclin D1,cleaved caspase-3 and cleaved caspase-8 were determined by Western blot.RESULTS:The protein level of NF-κB (P65) was decreased after treatment with PDTC for 48 h or 72 h.PDTC inhibited the proliferation of U266 cells in both dose-and time-dependent manners.After treatment with PDTC for 48 h,the percentage of U266 cells in G₂ phase increased compared with control group (P<0.05).PDTC induced the apoptosis of U266 cells in a dose-dependent manner.The expression of DNMT1 at mRNA and protein levels decreased (P<0.05).The results of Western blot showed that the expression of Bcl-2 in PDTC groups decreased,while the protein levels of cyclin D1,cleaved caspase-3 and cleaved caspase-8 were higher than those in control group (P<0.05).CONCLUSION:The NF-κB inhibitor PDTC inhibits the proliferation of U266 cells by inducing cell apoptosis.It may be related to the down-regulated expression of DNMT1,cell cycle arrest and activation of the apoptotic pathways.     

蓖麻DNA甲基转移酶序列与基因表达分析

植物DNA甲基化是与调控基因表达、保护基因组免受逆境胁迫等相关的表观遗传现象,其中关键酶DNA甲基转移酶催化DNA序列维持甲基化和从头甲基化。本研究基于蓖麻全基因组、利用生物信息学分析方法、结合转录组数据和RT-PCR半定量表达分析,鉴别出蓖麻DNA甲基转移酶氨基酸序列的结构特征、系统发生和表达形式。结果如下：通过同源序列BLAST从蓖麻基因组数据库中获得8个DNA甲基转移酶基因;系统进化分析将8个蛋白分为4个亚家族：2个MET同源蛋白、2个CMT同源蛋白、3个DRM同源蛋白和1个DNMT2同源蛋白,进一步发现DNA甲基转移酶在系统进化上较为保守,分化发生在单双子叶植物分化之后;亚细胞定位分析发现8个DNA甲基转移酶都定位在细胞核中;蛋白结构域比对结果显示8个蛋白的C端催化结构域都有6个保守motif参与甲基化修饰催化功能,表明蛋白具有基本的催化甲基化功能,而N端调节功能域的差异将蛋白分为四类,其中RcDNMT2缺少N端调节结构域;结合转录组数据和RT-PCR半定量表达分析发现蓖麻DNA甲基转移酶基因（除RcDRM2外）在发育胚乳中相对其他检测组织高表达,推测是该时期的胚乳低甲基化诱导甲基化基因高表达。该结果有助于全面了解蓖麻DNA甲基转移酶特征及为从表观遗传深入开展蓖麻品种改良具有重要理论指导作用。     

森林草莓6mA甲基转移酶基因鉴定及功能分析

通过生物信息学手段，鉴定出森林草莓（Fragaria vesca）基因组中两类可能的6mA甲基转移酶基因5个（3个MT-A70、2个DAMT基因）。对细菌、真菌、动物和植物中的6mA甲基转移酶基因的系统进化分析及染色体定位表明，森林草莓MT-A70基因可分为3个亚家族，且在动、植物分化前就已经存在：两个DAMT基因由物种特异的串联重复产生，且结构变化快，推测其在进化历程中功能发生了分化。转录组数据及实时定量荧光PCR分析显示，各个基因均具有时空表达特异性和非生物胁迫响应。其中1个DAMT基因表达量在果实中较高且随果实成熟上调，推测可能参与调控果实成熟。此外，这5个基因在冷胁迫下表达水平有不同程度下调：而在热胁迫下各基因表达量变化显著不同。由此推测6mA甲基转移酶基因可能参与了森林草莓的生长发育和对冷、热胁迫的响应。     

Effect of SMYD3 over-expression on DNMT3B levels and proliferation ability in human cholangiocarcinoma cell line FRH0201

AIM: To explore the effect of SET and MYND domain-containing protein 3 (SMYD3) over-expression on the expression of DNA methyltransferase 3B (DNMT3B) and the proliferation ability in human cholangiocarcinoma cell line FRH0201. METHODS: Transient transfection of SMYD3 eukaryotic expression plasmid pEGFP-C3-SMYD3 into human cholangiocarcinoma cell line FRH0201 was performed. The expression of DNMT3B at mRNA and protein levels was detected by RT-PCR and Western blotting,respectively. Cell proliferation was examined by CCK-8 method and cell cycle situation was checked by flow cytometry. RESULTS: After transfected with SMYD3 eukaryotic expression plasmid pEGFP-C3-SMYD3, the over-expression of SMYD3 in FRH0201 cells was observed. Compared with the untransfected cells, the expression of DNMT3B was significantly increased (P<0.01), the proliferation rate was obviously accelerated (P<0.05) and the number of the cells in G₂/M phase was significantly increased (P<0.05) in FRH0201 cells transiently transfected with pEGFP-C3-SMYD3 plasmid. CONCLUSION: The transient transfection of pEGFP-C3-SMYD3 plasmid induces over-expression of DNMT3B and promotes the proliferation of human cholangiocarcinoma cell line FRH0201.