马铃薯热激转录因子HsfA3基因的克隆及其耐热性功能分析     

唐锐敏  贾小云  朱文娇  印敬明  杨清 《作物学报》2021,(4):672-683

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Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity     

Anguo Li  Ruihao Huang  Chaogang Wang  Qunju Hu  Hui Li  Xiao Li 《Marine drugs》2021,19(5)

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.  相似文献   

过表达ZmIBH1-1提高玉米苗期抗旱性     

朱芳芳  董亚辉  任真真  王志勇  苏慧慧  库丽霞  陈彦惠 《中国农业科学》2021,54(21):4500-4513

【目的】干旱是严重影响玉米生长发育进程的一个重要因素。挖掘玉米抗旱相关基因,通过转基因功能验证和转录组分析,解析关键基因在响应干旱胁迫过程中的分子调控机制,为抗旱分子育种和遗传改良提供理论依据。【方法】以玉米自交系B104（WT）为背景材料,利用农杆菌介导方法构建过表达ZmIBH1-1转基因株系（ZmIBH1-1-OE）;通过对转基因植株进行草铵膦抗性筛选、标记基因和目的基因PCR检测,以及运用实时荧光定量PCR检测目的基因的表达情况,鉴定阳性植株和株系;以WT和ZmIBH1-1-OE转基因株系为材料,通过干旱处理（20% PEG6000）,进行表型鉴定和耐旱生理生化指标测定,验证ZmIBH1-1的抗旱功能;通过对干旱胁迫下玉米4叶期转录组的比较分析,鉴定出差异表达的基因（differentially expressed genes,DEGs）;结合DAP-seq（DNA affinity purification sequencing）分析,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的下游靶基因,利用基因组可视化软件IGV（integrative genomics viewer）分析ZmIBH1-1蛋白结合候选靶基因的位置,然后通过Dual-Luciferase试验验证ZmIBH1-1蛋白与靶基因的调控关系。【结果】通过玉米遗传转化获得12个转化事件;T₃代中,能同时检测到标记基因Bar和目的基因ZmIBH1-1的植株有458个,实时荧光定量PCR检测结果表明,ZmIBH1-1-OE中ZmIBH1-1的表达量显著高于WT,株系3和株系8表达量最高,将其自交获得T₄代转基因株系用于后续试验。在干旱胁迫条件下,ZmIBH1-1-OE株系存活率、叶片相对含水量、叶绿素含量、可溶性蛋白含量及其生理生化指标（超氧化物歧化酶、过氧化物酶、过氧化氢酶活性）均显著高于WT,说明玉米中过量表达ZmIBH1-1赋予玉米更高的耐旱性。转录组分析结果表明,WT与ZmIBH1-1-OE株系在干旱胁迫下有1 214个差异表达基因;Gene Ontology（GO）功能富集分析结果表明,差异表达基因主要涉及生物过程、细胞组分和分子功能,如在生物过程中主要涉及到光合作用、应激响应、脱水响应等;KEGG富集分析表明,差异表达基因主要参与植物激素信号传导、新陈代谢等过程。结合转录组显著差异表达基因和DAP-Seq分析所得到ZmIBH1-1蛋白的靶基因,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的11个候选靶基因,包括2个钙信号相关基因、3个半胱氨酸代谢相关基因、1个bHLH转录因子、1个应激响应蛋白、1个谷胱甘肽转移酶、1个氧化还原过程蛋白和2个乙烯响应因子;基因组可视化结果显示ZmIBH1-1蛋白可以结合靶基因启动子区;随后通过Dual-Luciferase试验进一步表明,ZmIBH1-1蛋白可以直接作用于11个候选靶基因,其中,ZmIBH1-1蛋白可以促进ZmCa-M、ZmSYCO、ZmbHLH54、ZmGlu-r1、ZmCLPB3和ZmP450-99A2的表达,抑制ZmAGD12、ZmCYS、ZmCYSB、ZmERF-107和ZmEIN3的表达。此外,在干旱胁迫下NAC、WRKY、MYB等转录因子在ZmIBH1-1-OE和WT株系中也存在差异表达。【结论】ZmIBH1-1的过表达可以增强玉米苗期的耐旱性;ZmIBH1-1蛋白通过直接调控乙烯信号通路中的ZmERF-107和ZmEIN3的表达提高玉米的耐旱性;ZmIBH1-1蛋白通过直接调控钙信号相关基因ZmCa-M和ZmAGD12增强玉米的耐旱性;ZmIBH1-1蛋白可能通过间接调控NAC、WRKY、MYB等转录因子响应干旱胁迫。  相似文献   

Bxy‐fuca encoding α‐L‐fucosidase plays crucial roles in development and reproduction of the pathogenic pinewood nematode,Bursaphelenchus xylophilus     

Jia Tang  Ruoqing Ma  Najie Zhu  Kai Guo  Yiqing Guo  Liqun Bai  Hongshi Yu  Jiafu Hu  Xingyao Zhang 《Pest management science》2020,76(1):205-214

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miR-140-3p inhibits viability,invasion and migration of non-small-cell lung cancer A549 cells by targeting PD-L1     

ZHANG Yu-xuan  LI Chun-wei  MAO Wen-hao  ZHU Ke-yan  SHAO Yang-qian  DENG Xiao-ming 《园艺学报》2019,35(1):8-14

AIM: To explore the target relationship between microRNA-140-3p (miR-140-3p) and programmed cell death ligand 1 (PD-L1) and their effect on the viability, migration and invasion of non-small-cell lung cancer A549 cells.METHODS: RT-qPCR was used to detect the miR-140-3p expression in HLF-1, A549 and H1299 cells, and then the A549 cells with the most significant difference were selected as the subsequent research object. TargetScan software and dual-luciferase reporter assay were performed to predict and confirm the target relationship between miR-140-3p and PD-L1. RT-qPCR and Western blot were used to determine the effects of miR-140-3p mimic and inhibitor on PD-L1 expression level. MTT assay was used to detect the viability of A549 cells. Transwell assay was performed to detect the migration and invasion abilities of the A549 cells.RESULTS: miR-140-3p was significantly down-regulated in the A549 cells and H1299 cells (P<0.05). Transfection with miR-140-3p mimic decreased the expression of PD-L1 and inhibited the viability, migration and invasion of the A549 cells. Transfection with pcDNA3.0-PD-L1 reversed the inhibitory effect of miR-140-3p on the viability, migration and invasion of the A549 cells.CONCLUSION: miR-140-3p inhibits the viability, migration and invasion of A549 cells by targeting PD-L1.  相似文献   

Altering of host larval (Spodoptera exigua) calcineurin activity in response to ascovirus infection     

Huan Yu  Lei He  Zi‐Qi Li  Ni Li  Yi‐Yi Ou‐Yang  Guo‐Hua Huang 《Pest management science》2020,76(3):1048-1059

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miR-204 inhibits proliferation of Hodgkin lymphoma cells by targeting Sirt1/p53 signaling pathway     

ZHENG Xiao-qiang  RUI Hong-bing  ZHANG Guang-hui 《园艺学报》2019,35(9):1557-1564

AIM: To investigate the effect of microRNA-204 (miR-204) on the proliferation of Hodgkin lymphoma cells and the underlying mechanism. METHODS: The expression of miR-204 and Sirt1 mRNA in Hodgkin lymphoma tissues was detected by RT-qPCR. After transfection with miR-204 mimic, Sirt1 siRNA and miR-204 mimic+pcDNA3.1-Sirt1 into the L428 cells, the cell viability and BrdU incorporation were measured by CCK-8 assay and BrdU assay, respectively. The protein levels of Sirt1 and acetylated p53 (ac-p53) were determined by Western blot.The targeting relationship between miR-204 and Sirt1 was verified by double luciferase reporter assay. RESULTS: The low expression of miR-204 and the high mRNA expression of Sirt1 were found in the Hodgkin lymphoma tissues. Compared with control group, the cell viability, BrdU incorporation and the protein levels of Sirt1 and ac-p53 were significantly decreased after L428 cells were transfected with miR-204 mimic or Sirt1 siRNA (P<0.05). Compared with miR-204 mimic alone group, the cell viability, BrdU incorporation and the protein levels of Sirt1 and ac-p53 were increased after L428 cells were co-transfected with miR-204 mimic and pcDNA3.1-Sirt1 (P<0.05). The results of double luciferase reporter assay confiermed that Sirt1 was the target gene of miR-204. CONCLUSION: The inhibitory effect of miR-204 on the proliferation of L428 cells may be achieved by inhibiting the expression of Sirt1 and promoting the up-regulation of ac-p53.  相似文献   

Identification of an H1N1 subtype of swine influenza virus and serological analysis     

Fa-chao SUN  Min TAN  Yuan-chao ZHANG  Yu-chao WANG  Sheng-liang CAO  Guo-fei DING  Fang-yuan CONG  Li-hong GUO  Si-dang LIU  Yi-hong XIAO 《农业科学学报》2019,18(7):1436-1442

To investigate the epizootic of swine influenza virus(SIV), 60 nasal swabs were collected from a clinical cases of pig farm in Tai'an City, Shandong Province of China in April 2017. SIV was isolated by inoculating into 10-day-old Special Pathogen Free embryonated eggs and the whole genome was sequenced. An H1N1 subtype SIV was isolated and designated as A/swine/Shandong/TA04/2017(H1N1). Phylogenetic analysis showed that apart from the polymerase A(PA) fragment belonging to the 2009 pandemic H1N1 branch, seven genome segments belonged to avian-like H1N1 influenza virus lineage. The cleavage site sequence of the hemagglutinin(HA) protein was PSIQSR↓G, which is a typical molecular biological characteristic. Five potential N-glycosylation sites(N14, N26, N277, N484 and N543) were found in the HA gene. To further investigate the epidemiology of SIV in this farm, the 995 serum samples were assessed with EAH1N1 2009 pandemic H1N1 and H3 N2 antigens. The results showed that the total positive rate was 65.43%. The positive rates of single virus infection detected by EAH1N1, 2009 pdmH1N1 and H3 N2 for serum HI(Hemagglutination inhibition) were 48.35, 30.85 and 7.47%, respectively. The results showed that SIV in Shandong Province has been reassorted in some segments and the SIV-positive rate was high on the SIV outbreak farm. These data provide evidence of an epizootic of SIV.  相似文献   

Cepharanthine induces autophagy via PI3K/AKT/mTOR signaling pathway in ovarian cancer SKOV3 cells     

XIANG Fu-ying  JIANG Sheng-lin  CHENG Xian-ying 《园艺学报》2019,35(5):940

AIM: To investigate the autophagy of human ovarian cancer SKOV3 cells induced by cepharanthine and to explore its mechanism. METHODS: The effect of cepharanthine on the viability of ovarian cancer SKOV3 cells was measured by CCK-8 assay. The SKOV3 cells were treated with cepharanthine, and then the formation of autophagosome was observed with acridine orange staining under fluorescence microscope. The protein levels of LC3, AKT, p-AKT, mTOR, p-mTOR and GAPDH in the SKOV3 cells treated with cepharanthine were determined by Western blot.RESULTS: Cepharanthine significantly inhibited the viability of ovarian cancer SKOV3 cells in a dose-dependent manner (P<0.05). The number of the intracellular acidic autophagosomes with bright red fluorescence was significantly increased after cepharanthine treatment in the SKOV3 cells. The expression of LC3-Ⅱ in SKOV3 cells was significantly enhanced after cepharanthine treatment. Furthermore, treatment with cepharanthine in the SKOV3 cells also resulted in a significant down-regulation of phosphorylated form of AKT and mTOR (P<0.01), while the total protein level was not changed. Combination of cepharanthine and 3-methyladenine resulted in a substantial decrease in the cell viability compared with using cepharanthine alone.CONCLUSION: Cepharanthine significantly inhibits the growth of human ovarian cancer SKOV3 cells and induces the autophagy, which may be correlated with down-regulation of PI3K/AKT/mTOR signaling pathway.  相似文献   

NO对大鼠中脑腹侧被盖区DA神经元的调节     

孙碧英  张文慧 《动物医学进展》2003,24(4):66-68

实验观察了微量注射 NOS抑制剂 L -NAME及微量联合注射 NO的前体 L-精氨酸( L-Arg) L-NAME于大鼠中脑腹侧被盖区( VTA)对该部位多巴胺神经元的调节。发现VTA注射 L -NAME( 1 mg/ 5μL )后 ,伏隔核( Acb)多巴胺 ( DA )代谢产物—双羟苯乙酸( DOPAC)水平升高到注射前的 1 2 2 .5% ( P <0 .0 0 1 ) ,小剂量注射 L-NAME( 0 .2 mg/ 5μL)对伏隔核 DOPAC水平无明显影响 ;同样方法联合注射 L-Arg( 3 0 0 μg/ 5μL) L-NAME( 1 mg/5μL)后 ,伏隔核 DOPAC水平无明显变化。结论 :VTA微量注射 L-NAME兴奋了该部位的DA神经元 ,而 L -Arg L -NAME联合注射 ,却不能影响 DA神经元的活动 ,说明 NO可以通过L-Arg-NOS-NO途径参与 VTA多巴胺神经元的调节  相似文献