日本结缕草ZjNAC1的克隆、转录激活活性、亚细胞定位及表达分析

采用RACE技术从日本结缕草中克隆得到ZjNAC1转录因子(GenBank No. MH428376),其开放阅读框为945bp,编码314个氨基酸。ZjNAC1编码的蛋白在N端有1个典型的NAM保守结构域,属于NAC转录因子家族。通过染色体步移的方法,获得ZjNAC1基因ATG上游1635bp序列,分析发现其包含响应脱落酸(ABA)、茉莉酸甲酯(MeJA)及逆境胁迫的作用元件。构建pGBKT7-ZjNAC1载体,转化Y2HGold酵母感受态细胞,发现ZjNAC1具有转录激活活性。农杆菌介导的35S-ZjNAC1-YFP载体注射本生烟草叶片瞬时表达结果显示,ZjNAC1定位于细胞核。实时荧光定量结果表明,ZjNAC1在日本结缕草根中表达量最高;此外,ZjNAC1的表达受10μmol/L MeJA和20%PEG4000处理的诱导,但受200μmol/L乙烯(ET)、10μmol/L ABA和300mmol/L NaCl处理的抑制。     

OCCURRENCE OF INDUCIBLE NITRIC OXIDE SYNTHASE IMMUNOREACTIVE MATERIAL IN THE RETINA OF THE CLAWED FROG,Xenopus laevis

借助Ⅱ和Ⅲ型一氧化氮合成酶（ＮＯＳ）特异性抗体对成年爪蛙（Ｘｅｎｏｐｕｓｌａｅｖｉｓ）视网膜中含ＮＯＳ的结构进行了免疫细胞化学定位研究。发现视杆和视锥细胞的视网膜杆、少数神经节细胞和无足细胞的细胞体均含有ＮＯＳⅡ免疫反应物质,缪勒细胞呈现强的ＮＯＳⅡ免疫反应性,外神经丛层的神经突起亦含强的ＮＯＳⅡ免疫反应性。在视网膜的细胞结构中,未见ＮＯＳⅢ免疫反应性。该结果暗示由ＮＯＳⅡ产生的一氧化氮可能参与视觉传导的调节。     

动物生长发育中主基因的QTL定位法

将系统学研究动物生长发育的方法和控制数量性状主基因在遗传标记图谱上的定位相结合,提出了动物生长发育过程中任一时刻的数量性状基因座位的定位方法（ＱＴＬ）。并对控制数量性状的主基因表达的时序,传统的基因遗传参数的估计,以及系统分析在分子遗传中的应用前景进行了讨论。     

Fluorescence localization of epidermal cathepsins L and B in the Japanese eel

Histochemical localization of proteolytic activities in the dorsal epidermis of Japanese eel was demonstrated by fluorescent microscopy utilizing 4-methoxy-2-naphthylamide (4M$\beta$NA) derivatives as substrates and 5-nitrosalicylaldehyde as a trapping agent. Carbobenzoxy-L-phenylalanyl-L-arginyl-4M$\beta$NA (Cbz-Phe-Arg-4M$\beta$NA) and Cbz-Arg-Arg-4M$\beta$NA were used for direct detection of cathepsins L and B activities, respectively, in fresh frozen sections and unfixed cells of the eel epidermis. The fluorescing areas, where Cbz-Phe-Arg-4M$\beta$NA was hydrolyzed by cathepsin L, were shown in mucus secretory cells and club cells and broadly around skin surface. The fluorescing areas due to Cbz-Arg-Arg-4M$\beta$NA hydrolysis by cathepsin B were localized similarly in these tissues. The fluorescing intensity for both catheptic activities in mucus secretory cells was higher than that in club cells, where small fluorescing granules were distributed. These results indicate that eel cathepsins L and B are stored in epidermal secretory cells at different levels and probably serve as defense factors before or after secretion by these cells. Abbreviations: Cbz – carbobenzoxy; 4M$\beta$NA – 4-methoxy-2-naphthylamide; NSA – 5-nitrosalicylaldehyde.     

卵形鲳鲹TLR13基因的克隆、表达分析和亚细胞定位研究

Toll样受体(Toll-like receptor, TLR)是一种古老的先天性免疫受体,参与病原体相关分子模式识别,对维持免疫稳态和预防感染至关重要。本研究克隆和鉴定了卵形鲳鲹(Trachinotus ovatus) TLR13基因(命名为ToTLR13),其开放阅读框(ORF)为1 269 bp,编码422个氨基酸,等电点为8.13。保守结构域分析显示,ToTLR13含有跨膜结构域(TM)、LRR结构域和TIR结构域,符合TLR家族的典型特征。通过建立TLR13保守域三级结构发现,ToTLR13与小鼠(Mus musculus)和大黄鱼(Larimichthys crocea) TLR13功能结构域的蛋白三级结构具有较高重叠性。多序列比对显示,ToTLR13与其他硬骨鱼TLR13具有较高的相似性,与其他纲物种的序列相似性较低。系统进化树结果显示,ToTLR13与硬骨鱼TLR13聚在一起,其中与鞍带石斑鱼(Epinephelus lanceolatus)最为接近,与哺乳动物、两栖类和贝类相分离。实时荧光定量PCR (Real-time fluorescence quantitative PCR, RT-qPCR)分析显示,ToTLR13在健康卵形鲳鲹的心、鳃、肾、头肾、肝、脾、脑和肌肉中普遍表达,其中鳃的表达量最高,其次是脾。ToTLR13在其鳃、脾、肝和肾免疫相关组织中的表达情况呈现出不同程度的上调,提示其经无乳链球菌(Streptococcus agalactiae)和溶藻弧菌(Vibrio alginolyticus)免疫刺激后可能激活了炎症反应,启动了先天性免疫反应。亚细胞定位显示,ToTLR13定位于A549细胞质。本研究表明,ToTLR13在抵御病原菌免疫应答过程中可能发挥重要的作用,研究结果可为阐明脊椎动物TLRs的功能进化史提供基础资料。     

Identification of a cell-wall peptidase (NlpC/P60) from Nocardia seriolae which induces apoptosis in fathead minnow cells

Nocardia seriolae, a Gram-positive bacterium, is the main pathogen of fish nocardiosis. Protein NlpC/P60 is a cell-wall peptidase and a potential virulence factor of N. seriolae. Subcellular localization research revealed that both NlpC/P60-GFP and NlpC/P60Δsig-GFP fusion proteins were evenly distributed in the whole cell of fathead minnow (FHM) cells. Furthermore, typical apoptotic features, such as nuclear pyrosis and apoptotic bodies, were observed in the transfected FHM cells and grouper spleen cells by the overexpression of protein NlpC/P60. Then, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase-3 activity and apoptosis-related gene (Bax, BNIP3, TNF1 and TNF6) mRNA expression were conducted. The results showed that ΔΨm was decreased, caspase-3 was significantly activated, and the mRNA expression of pro-apoptotic genes (Bax and BNIP3) and tumour necrosis factors (TNF1 and TNF6) was up-regulated in NlpC/P60-overexpressed cells. Taken together, the results indicated that the protein NlpC/P60 of N. seriolae might involve in apoptosis regulation. This study may lay the foundation for further study on the function of N. seriolae NlpC/P60 and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae.     

Identification of a mitochondrial‐targeting secretory protein from Nocardia seriolae which induces apoptosis in fathead minnow cells

Nocardia seriolae is the main pathogen responsible for fish nocardiosis. A mitochondrial‐targeting secretory protein (MTSP) 3141 with an N‐terminal transit peptide (TP) from N. seriolae was predicted by bioinformatic analysis based on the genomic sequence of the N. seriolae strain ZJ0503. However, the function of the MTSP3141 and its homologs remains totally unknown. In this study, mass spectrometry analysis of the extracellular products from N. seriolae proved that MTSP3141 was a secretory protein, subcellular localization research showed the MTSP3141‐GFP fusion protein co‐localized with mitochondria in fathead minnow (FHM) cells, the TP played an important role in mitochondria targeting, and only the TP located at N‐terminus but not C‐terminus can lead to mitochondria directing. Moreover, quantitative assays of mitochondrial membrane potential (ΔΨm) value, caspase‐3 activity and apoptosis‐related gene (Bcl‐2, Bax, Bad, Bid and p53) mRNA expression suggested that cell apoptosis was induced in FHM cells by the overexpression of both MTSP3141 and MTSP3141ΔTP (with the N‐terminal TP deleted) proteins. Taken together, the results of this study indicated that the MTSP3141 of N. seriolae was a secretory protein, might target mitochondria, induce apoptosis in host cells and function as a virulence factor.     

鸡Toll样受体15单克隆抗体的制备及其初步应用

旨在制备鸡Toll样受体15 (ChTLR15)的特异性单克隆抗体(mAb),并初步应用.利用PCR技术扩增ChTLR15第162-386位氨基酸,克隆于载体pET-30a中进行诱导表达,获得高纯度的重组ChTLR15 (162-386 aa)蛋白.然后采用皮下多点注射方式将ChTLR15免疫6周龄的雌性BALB/c小...     

基于探地雷达杂波抑制与偏移成像的树木根系定位方法

探地雷达在果树和古树名木养护管理领域具有广阔的应用前景。针对探地雷达采集的B-scan图像中杂波影响根系定位精度的问题,提出了基于鲁棒深度自动编码器(RDAE)、直接最小二乘法(DLS)和频率-波数域偏移(FKM)相结合的树木根系定位方法。首先,通过RDAE将零点校正后的B-scan图像分解为表示杂波的低秩分量和表示根系目标回波的稀疏分量,保留稀疏分量完成杂波抑制;然后使用DLS拟合目标回波形成的双曲线估算土壤的相对介电常数;最后,根据土壤的相对介电常数计算得出偏移速度作为频率-波数域偏移的输入进行偏移成像,获取根系的半径和深度信息从而完成根系定位。实验结果表明：RDAE方法在仿真和实测数据上的杂波抑制效果对比均值减法(MS)、奇异值分解(SVD)和鲁棒主成分分析(RPCA)有着更高的信杂比和改善因子,通过DLS估计的土壤相对介电常数均方根相对误差为3.84%,根系定位的最大半径相对误差和最大深度相对误差分别为8.5%、8.7%,能够完成根系位置标定,满足根系无损检测的需求,可为树木健康管理和移植提供决策支持。     

Yield-height correlation and QTL localization for plant height in two lowland switchgrass populations

Switchgrass (Panicum virgatum L.), as a model herbaceous crop species for bioenergy production, is targeted to improve biomass yield and feedstock quality. Plant height is a major component contributing to biomass yield. Accordingly, the objectives of this research were to analyze phenotypic variation for biomass and plant height and the association between them and to localize associated plant height QTLs. Two lowland switchgrass mapping populations, one selfed and another hybrid population established in the field at Perkins and Stillwater, Oklahoma, were deployed in the experiment for two years post establishment. Large genetic variation existed for plant biomass and height within the two populations. Plant height was positively correlated with biomass yield in the selfed population (r = 0.39, P<0.0001) and the hybrid population (r = 0.41, P<0.0001). In the selfed population, a joint analysis across all environments revealed 10 QTLs and separate analysis for each environment, collectively revealed 39 QTLs related to plant height. In the hybrid population, the joint analysis across overall environments revealed 35 QTLs and the separate analysis for each environment revealed 38 QTLs. The findings of this research contribute new information about the genetic control for plant height and will be useful for future plant breeding and genetic improvement programs in lowland switchgrass.