稻曲病菌交配型基因MAT1-2-1和MAT1-2-8的特性分析

 有性生殖在真菌的生活史和进化过程中具有重要作用,而交配型基因是控制有性生殖的关键因子。前期研究发现稻曲病菌(Villosiclava virens)MAT1-2型菌株中包含MAT1-2-1和MAT1-2-8两个交配型基因,但是它们如何调控稻曲病菌有性生殖依然不清楚。本文研究了它们在不同侵染和生长发育时期的表达模式和编码的蛋白结构特性。研究表明MAT1-2-1在侵染不同阶段一直下调表达;而MAT1-2-8在侵染早期(5 dpi)上调表达,在侵染后期下调表达。与营养菌丝阶段比较,MAT1-2-1和MAT1-2-8在有性发育过程菌核形成、菌核萌发、子座原基形成和子座成熟4个阶段的表达量都是下降的,在菌核形成阶段表达量最低。生物信息学分析显示MAT1-2-1和MAT1-2-8具有磷酸化位点,为非分泌蛋白,无明显的跨膜结构域。蛋白同源比对分析表明MAT1-2-1与香柱菌(Epichloë typhina)的MAT1-2-1同源性最高,而MAT1-2-8与绿僵菌(Metarhizium)的MBR_08192蛋白同源性最高。进一步研究发现MAT1-2-1和MAT1-2-8能够互作,并分别主要定位在细胞核和细胞基质中。通过质谱技术鉴定到MAT1-2-1的一些候选互作蛋白,如假定Ran交换因子Prp20/Pim1(KDB12229.1)、假定rRNA处理蛋白Ebp2(KDB12923.1)及组蛋白H1(KDB12711.1)等。因此,以上结果为研究稻曲病菌交配型基因MAT1-2-1和MAT1-2-8调控有性生殖的生物学功能奠定了基础。     

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

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.     

过表达ZmIBH1-1提高玉米苗期抗旱性

【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。     

Comparative effect of advanced soy products or corn protein concentrate with porcine meal on growth,body composition,and distal intestine histology of Florida pompano,Trachinotus carolinus

The present study was designed to investigate the effects of diets containing advanced soy products (enzyme‐treated soy and fermented soy) or corn protein concentrate (CPC) in combination with porcine meal (PM) to completely replace poultry byproduct meal (PBM) on growth performance, body composition, and distal intestine histology of Florida pompano, Trachinotus carolinus. Four experimental diets were formulated to be isonitrogenous and isolipidic, to contain 400 g/kg crude protein and 80 g/kg lipid. A reference diet (PBM diet [PBMD]) contained 150 g/kg PBM and 495 g/kg soybean meal (SBM), and three test diets were formulated replacing PBM with 15 g/kg of CPC (CPC diet [CPCD]) or replacing all SBM and PBM with 535 g/kg fermented soy (fermented soybean meal diet [FSBMD]) or 451.3 g/kg enzyme‐treated soy (enzyme‐treated soybean meal diet [ESBMD]). All three test diets were supplemented with 38 g/kg of PM. Diets were fed based on a percentage of bodyweight adjusted after sampling the fish every 2 weeks to triplicate groups of Florida pompano juveniles (mean weight 8.06 ± 0.22 g). After 8 weeks of feeding, fish fed CPCD and ESBMD performed equally well in terms of final body weight, thermal growth coefficient, and percentage weight gain in comparison to fish fed PBMD. In all cases, feeding FSBMD resulted in poor feed conversion and lower feed intake compared to other treatments. Protein retention efficiency, whole‐body proximate composition, phosphorus, sulfur, potassium, magnesium, calcium, sodium, and zinc contents were not significantly influenced by the dietary treatments. The results obtained in the present histological study showed no significant differences in the thickness of serous layer, muscular layer, and submucosal layer of the intestine among treatments. Fish fed CPCD showed a significant widening of the lamina propria with an increase of cellular infiltration and higher presence of goblet cells compared to other dietary treatment. Based on these results, 451 g/kg ESBM or combination of 150 g/kg of CPC and 495 g/kg SBM supplemented with 38 g/kg PM can be utilized to develop a practical diet for juvenile Florida pompano without impacting growth, nutritive parameters, and several distal intestine health parameters.     

Mapping the spatial distribution of shade provision of street trees in Boston using Google Street View panoramas

Street trees provide shade and increase human thermal comfort during hot summer. In this study, we investigated the spatial distribution of shade provision of street trees in Boston, Massachusetts. The sky view factor (SVF), which influences the solar radiation to the ground and affects human thermal comfort, was used to indicate the contribution of street trees on shade provision. Google Street View (GSV) panoramas were used to calculate the photographic method based SVF (SVF_P), with the consideration of all kinds of obstructions within street canyons. A building height model was used to calculate the simulation based SVF (SVFs), with consideration of obstruction of building blocks only. Considering the fact that street trees and building blocks are the two major obstructions of radiation within street canyons, therefore, the difference between the two SVF estimation results can be considered as the shade provision of street trees. The results show that street trees help to decrease the SVF by 24.61% in Boston, Massachusetts. The shading level varies spatially in the study area. Generally, the southwestern area has much higher shading level than the north and the east. We further explored the shading variation among different socioeconomic groups in the study area. Result shows that Hispanics tend to live in neighborhoods with lower shading level. This study can help to provide a reference for future urban greening projects for global climate change adaption.     

Zerumbone attenuates MPP+-induced cytotoxicity in human neuroblastoma SH-SY5Y cells by inhibition of oxidative stress

AIM: To investigate the role of zerumbone (ZER) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity of human neuroblastoma SH-SY5Y cells. METHODS: Human neuroblastoma SH-SY5Y cells were cultured in vitro and the protective effect of ZER against MPP⁺-induced cytotoxicity was measured by CCK-8 assay. Flow cytometry was used to determine the apoptosis and reactive oxygen species (ROS). The expression of Parkinson disease protein 7 (PARK7) was knocked-down by using PARK7-specific short hairpin RNA (shRNA). The protein levels of PARK7, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were determined by Western blot. RESULTS: MMP⁺ remarkably reduced the cell viability in a dose-dependent and time-dependent manner. The SH-SY5Y cell injury model was established by treatment with MPP⁺ at 600 μmol/L for 24 h. ZER up-regulated the protein levels of PARK7 and Nrf2 (P<0.05), alleviated apoptosis (P<0.05), and reduced ROS production (P<0.05) in the SH-SY5Y cell injury model. Meanwhile, N-acetyl-L-cysteine (NAC) had the similar functions. Moreover, significant reductions in the protein levels of Nrf2 and HO-1 (P<0.05), and obvious increases in apoptosis (P<0.05) and ROS level (P<0.05) were demonstrated in PARK7-knockdown cells. CONCLUSION: ZER protects SH-SY5Y cells against MPP⁺-induced cytotoxi-city, which may be related to activation of PARK7/Nrf2/HO-1 pathway, and subsequent attenuation of oxidative stress and apoptosis.     

品种间大豆脂肪氧化酶活性差异及β-胡萝卜素漂白性研究

为了解东北地区大豆品种间大豆脂肪氧化酶(LOX)活性差异规律,整理适于东北地区种植的大豆种质资源307份(6份半野生品种,17份地方品种及4份国外品种,其余为栽培品种),统一种植,秋季收获后,测定各品种成熟籽粒脂肪氧化酶总活性。分析品种间脂肪氧化酶总活性分布规律、大豆脂肪氧化酶总活性与β-胡萝卜素漂白性之间关系。结果表明,307份大豆品种间脂肪氧化酶总活性差异极显著,平均值为21.68 U·mL~(-1),最小值为2.51 U·mL~(-1),最大值为42.38 U·mL~(-1)。根据脂肪氧化酶总活性差异,通过聚类分析将307份大豆品种分为3类。低活性品种占总量43.65%,脂肪氧化酶总活性平均值为15.25 U·mL~(-1);中等活性品种占总量43.97%,脂肪氧化酶活性平均值为24.64 U·mL~(-1);高活性品种占总量12.38%,脂肪氧化酶总活性平均值为33.87 U·mL~(-1)。脂肪氧化酶总活性与油分含量呈正相关,与蛋白含量呈负相关,与漂白性呈正相关。     

欧洲葡萄‘粉红亚都蜜’NAC基因DRL1负向调节植物抗旱性

以欧洲葡萄‘粉红亚都蜜’（Vitis vinifera‘Yatomo Rose’）为材料，利用荧光定量PCR技术和转基因技术研究葡萄NAC转录因子DRL1基因对逆境的响应。欧洲葡萄‘粉红亚都蜜’在激素和逆境胁迫下，DRL1表达呈下降趋势，其中以ABA和干旱胁迫处理最为显著。在ABA处理下DRL1转基因烟草株系种子萌发率和根长均高于野生型。干旱处理下，转基因植株对干旱的耐受性降低，同时胁迫相关基因NtLEA5、NtP5CR1、NtPSCS1、NtERD10C和NtDREB3的表达水平比野生型显著下降。此外，DRL1转基因烟草茎中柱发育受到抑制，尤其是导管横切面积仅为野生型的58%。以上结果表明，DRL1基因可能作为1个负向调节子参与植物的干旱胁迫。     

异硫氰酸丙烯酯毒杀松材线虫作用机制初步研究

对异硫氰酸丙烯酯毒杀松材线虫作用机制进行了初步研究。测定了其对线虫虫体形态、运动行为、体内总糖和蛋白质含量的影响,以及处理前后线虫体内过氧化氢酶(CAT)、乙酰胆碱酯酶(AChE)和谷胱甘肽-S-转移酶(GST)活性变化。结果表明:经异硫氰酸丙烯酯处理后的松材线虫虫体形态严重扭曲,运动形态出现由正常的S形变化为多节弯曲和螺旋状卷曲等异常状况,线虫行动随时间的推移变得迟缓,最终呈C形、新月形或直接死亡。随着异硫氰酸丙烯酯处理质量浓度的增大,线虫体内总糖和蛋白质含量显著上升,糖和蛋白质的代谢发生紊乱;线虫体内CAT、AChE和GST活性显著降低,且均低于对照组,表明线虫体内氧化和抗氧化作用失衡;细胞受到极大的影响,神经系统遭到破坏,解毒能力明显降低。研究结果可为研究利用异硫氰酸丙烯酯作为植物源杀线虫剂提供依据。