Preliminary study of endocytosis-related gene mRSD-9

AIM: To study the function of the gene mRSD-9 . METHODS: The techniques of immunoprecipitation and immunofluorescence were applied to verify the interaction between mRSD-9 and endophlin 3. The ATP/GTP combined experiment and the clathrin releasing experiment were conducted to investigate the effect of mRSD-9 on endocytosis. RESULTS: Expressed Myc-mRSD-9 was precipitated by Flag-endophilin 3. Conversely, the expressed Flag-endophilin 3 was also precipitated by Myc-mRSD-9. Under laser scanning confocal microscope, mRSD-9-GFP fusion protein and endophilin 3-RFP fusion protein were observed to co-localize in CHO cells. The combination of mRSD-9 protein with ATP/GTP was found and was specific because no combination was detected using mutant ΔmRSD-9. In empty vector transfected group, the quantity of transferrin in red fluorescent expressed cells was roughly the same as the untransfected cells. In pDsRed1-N1-mRSD-9 transfected group, the quantity of transferrin in mRSD-9 protein expressed cells was obviously reduced. In pDsRed1-N1-ΔmRSD-9 transfected group, the quantity of transferrin in ΔmRSD-9 protein expressed cells was significantly increased. CONCLUSION: The protein coded by mRSD-9 gene can suppress endocytosis of transferrin.     

硼对平邑甜茶幼苗硝态氮吸收、利用及分配特性的影响

以平邑甜茶幼苗为试材,运用~(15)N同位素示踪和非损伤微测技术,研究了不同供硼(硼酸0、1.0、2.0、3.0、4.0、5.0和6.0 mg·L~(-1))水平对平邑甜茶根系生长及氮素吸收、利用和分配特性的影响。结果表明,3.0 mg·L~(-1)硼酸处理的幼苗根系活力及根系形态指标显著高于其他处理,幼苗的全氮量及~(15)N吸收量增幅最大,分别比对照提高了19.4%和75.0%。随供硼水平的增加,植株氮素利用率呈现先增高后降低的趋势,在3.0 mg·L~(-1)硼酸处理时最大,为14.8%,是对照的1.8倍。施硼处理对幼苗的~(15)N分配率有一定的影响,3.0 mg·L~(-1)硼酸处理的根系~(15)N分配率达到最大,且显著高于对照。非损伤微测结果显示,3.0 mg·L~(-1)硼酸处理时,平邑甜茶根系对NO_3~-有强烈吸收且内流速度达到最大,在缺硼和高硼(硼酸0和6.0 mg·L~(-1))处理时有明显外排趋势。因此,3.0 mg·L~(-1)硼酸处理最有利于平邑甜茶根系的生长、根系活力的提高和氮素的吸收利用,而低硼和过量供硼均会抑制根系生长及氮素的吸收利用。     

The Influence of Carbon Dioxide or Ozone Concentration on Growth and Assimilate Partitioning in Seedlings of Nine Conifers

Abstract

Seedlings of nine different conifers were exposed to 355 and 730 μmol mol-1 CO₂, or low (> 15 nmol mol^?1) and elevated 0₃ concentration (70 nmol mol^?1) for 81–116 days. The experiments were conducted in growth chambers placed in a greenhouse. Increased CO₂ concentration enhanced the mean relative growth rate (RGR) and total plant dry weight by 4 and 33% in Larix leptolepis, by 4 and 38% in Larix sibirica, by 7 and 47% in Picea glauca and by 3 and 16% in Picea sitchensis, respectively. The growth rates and dry weights of Pimis contorta, Pinus mugo and Pseudotsuga menziesii were not significantly affected. Carbon dioxide enrichment enhanced RGR of two provenances of Picea abies by 4 and 6%, respectively, while a third provenance was unaffected. In Pimis sylvestris, only the RGR of one of three provenances was stimulated by CO₂ enrichment (4%).

After two growth seasons CO₂ enrichment enhanced RGR and total plant dry weight by 11 and 35% in Picea abies and by 12 and 36% in Pinus sylvestris, respectively. Elevated CO₂ decreased the shoot:root ratio in Larix leptolepis, and decreased the needlerstem ratio in Picea glauca, but increased it in Pseudotsuga menziesii.

Elevated O₃ significantly decreased the plant dry weight in Picea sitchensis, Pseudotsuga menziesii and in one of three provenances of Pinus sylvestris, while the other species and provenances were unaffected. Increased O₃ concentration increased the shoot:root dry weight ratio in one of three Picea abies provenances, in all three Pinus sylvestris provenances and in Pinus contorta. The needle:stem ratio was enhanced by O₃ in seven of the nine species. The O₃ exposure caused chlorosis of needles in all species except Pseudotsuga menziesii.     

交叉中和血清1型和血清3型鸭甲肝病毒单克隆抗体的制备与鉴定

鸭甲型肝炎（duck hepatitis A,DHA）是危害养鸭业的主要疾病之一,是一种雏鸭的急性、高度致死性传染病。本研究旨在研制一种对我国流行的鸭甲型肝炎血清1型（DHAV-1）和血清3型（DHAV-3）病毒具有交叉中和作用的单克隆抗体（monoclonal antibody,McAb）。人工合成12条DHAV-1和DHAV-3的VP1蛋白共有的抗原表位肽,分别与钥孔血蓝蛋白（keyhole limpet hemocyanin,KLH）载体偶联作为免疫抗原,免疫BALB/c小鼠制备McAb。通过检测McAb与DHAV-1和DHAV-3的交叉反应性,测定McAb对病毒增殖的抑制效率、中和效价以及攻毒保护率等来筛选McAb。本研究共获得了12株稳定分泌McAb的杂交瘤细胞株,其中6株（C1、C4、C7、C12、C13、C16）分泌的McAb同时与DHAV-1和DHAV-3发生特异性交叉反应; C1、C4、C7、C16可以抑制DHAV-1和DHAV-3在鸭胚中的增殖,抑制率在75.34%～100.00%不等;对DHAV-1和DHAV-3的中和效价：C1（1：3&1：5）、C4（1：3&1：3）、C7（1：10&1：11）和C16（1：9&1：9）;C7和C16对DHAV-1和DHAV-3攻毒雏鸭的保护率较高,分别为“70%、80%”和“100%、60%”。本研究成功研制出对DHAV-1和DHAV-3具有交叉中和活性的McAb 4株,其中2株对病毒的感染具有良好的预防效果,为DHA的防控提供了新材料和新思路。     

禽腺病毒血清4型感染鸡组织中NLRP3基因转录水平的实时荧光定量PCR分析

旨在分析禽腺病毒血清4型（FAdV-4）感染鸡组织中NLRP3基因的转录水平,本研究设计鸡NLRP3特异性引物,利用RT-PCR扩增NLRP3基因180 bp片段并克隆至pMD-18T载体,制备重组质粒pMD-18T-NLRP3。以pMD-18T-NLRP3质粒作为标准品进行荧光定量PCR并建立标准曲线。通过反应条件优化,成功建立了检测NLRP3基因的实时荧光定量PCR方法,并利用该方法对致病性FAdV-4感染鸡组织中NLRP3基因的转录水平进行了分析。结果显示,所设计的NLRP3引物可特异性扩增鸡NLRP3基因,建立的实时荧光定量PCR对鸡NLRP3标准质粒的扩增曲线良好,标准品的拷贝数与Cq值呈现良好的线性关系。与对照组相比,NLRP3分子在FAdV-4感染鸡肝和脾中的转录水平极显著高于对照组（P<0.001）,在盲肠扁桃体和法氏囊的表达显著高于对照组（P<0.01）。本研究所建立的鸡NLRP3基因SYBR Green Ⅰ实时荧光定量PCR可以检测FAdV-4感染鸡不同组织中NLRP3的转录水平;致病性FAdV-4感染所造成的组织炎症损伤与NLRP3分子密切相关。     

猪C1QTNF3基因可变剪接体的鉴定及介导miR-101调控成脂分化的研究

旨在研究猪C1QTNF3基因可变剪接体的特性及miR-101通过C1QTNF3基因促进猪脂肪SV细胞成脂分化的机制。本研究采集3头30日龄健康马身猪仔公猪心、肝、脾、肺、肾、胃、股二头肌、腰大肌、皮下脂肪和背部脂肪组织样品,首先利用RT-PCR技术和生物信息学方法对C1QTNF3基因的不同转录本进行扩增和生物学特性分析,采用qRT-PCR技术检测C1QTNF3基因不同转录本在猪组织中的表达变化。随后,利用生物信息学预测发现,C1QTNF3上游的调控因子是miR-101,采用双荧光素酶报告试验验证miR-101对C1QTNF3的调控作用。最后,利用油红O染色和qRT-PCR技术检测过表达miR-101对猪脂肪SV细胞成脂分化的影响。基因克隆得到猪C1QTNF3存在两个转录本C1QTNF3和C1QTNF3-1,其中C1QTNF3-1为新鉴定的转录本;测序分析显示,与C1QTNF3相比,C1QTNF3-1的第1外显子区域缺失了219个碱基,少编码73个氨基酸。进化树分析显示,猪C1QTNF3和C1QTNF3-1蛋白序列与人和马来亚穿山甲等物种的亲缘关系较近。C1QTNF3和C1QTNF3-1在猪各组织中均有表达,且在各组织中C1QTNF3-1的表达量均极显著高于C1QTNF3（P<0.01）。过表达miR-101极显著下调C1QTNF3 3'UTR区域的荧光素酶活性（P<0.01）和C1QTNF3 mRNA的表达（P<0.01）,同时增加了脂肪细胞成脂分化关键基因PPARγ、C/EBPβ、SREBP-1c和FABP4的mRNA表达量（P<0.01）。本试验成功克隆了猪C1QTNF3基因的两个可变剪接体,其中C1QTNF3-1在猪不同组织中均高表达,推测该转录本为基因发挥功能的主要亚型。并深入研究了C1QTNF3基因的上游调控因子,揭示了miR-101靶向C1QTNF3促进成脂分化的机制,丰富了C1QTNF3的生物学作用和调控网络。     

外源海藻糖对NaHCO3胁迫下甘草幼苗生长调节及总黄酮含量的影响

本研究以甘草无菌苗为试验材料,采用植物组织培养的方法,分析50 mmol·L^-1 NaHCO₃ 胁迫下外源海藻糖对甘草幼苗生长量、叶绿素含量、渗透调节物质含量、抗氧化保护酶活性和总黄酮含量的影响。结果表明： 50 mmol·L^-1 NaHCO₃ 胁迫显著降低了甘草幼苗的生长量、叶绿体色素含量、K⁺ 浓度、抗氧化保护酶（SOD、POD、CAT）活性和总黄酮含量,显著提高了丙二醛、脯氨酸和可溶性糖含量以及Na⁺浓度;施加15 mmol·L^-1 海藻糖可显著提高甘草幼苗的生长量,提高叶绿素含量、K⁺ 浓度和总黄酮含量,降低丙二酸含量、脯氨酸含量、可溶性糖含量和Na⁺ 浓度,并且提高抗氧化保护酶活性。因此,NaHCO₃胁迫下施加外源海藻糖对甘草幼苗生长具有良好的调节作用,可以增强甘草的抗碱能力,促进甘草幼苗生长。本研究为外源海藻糖提高甘草耐碱性和揭示其调控机制提供理论依据。     

黄芪多糖在LPS诱导的DF-1细胞炎症反应中的抗炎作用及其调节机制

为探讨黄芪多糖(Astragalus polysaccharide,APS)在LPS(lipopolysaccharide,LPS)诱导的鸡胚成纤维细胞系DF-1(chicken embryonic fibroblast cell line,DF-1)炎症模型中对IL-1β和TNF-α表达的影响,以及细胞因子信号转导抑制因子3(suppressers of cytokine signaling 3,SOCS3)对炎症信号通路NF-κBp65和p38MAPK的调节机制。将DF-1细胞分为对照组(C)、LPS组(L)、APS组(A)以及APS+LPS组(A+L),分别在LPS刺激后6,12,24,48,72 h检测各组细胞IL-1β、TNF-α、NF-κBp65、p38MAPK和SOCS3 mRNA和蛋白水平的变化。研究发现,当LPS终质量浓度为2 mg/L时可诱导DF-1细胞出现明显的炎症反应,而APS终质量浓度为100 mg/L时为本试验最佳抗炎浓度。与对照组相比,APS组炎性因子IL-1β和TNF-αmRNA表达及蛋白含量在6~72 h均显著升高(P<0.05);与LPS组相比,APS+LPS组SOCS3 mRNA表达在6~72 h均显著升高(P<0.05),NF-κBp65、IL-1β和TNF-αmRNA表达在6~72 h均显著降低(P<0.05),而p38MAPK变化不显著(P>0.05)。在DF-1细胞中,APS单独处理可以促进IL-1β和TNF-α等细胞因子释放而增强免疫;APS和LPS共处理时,APS通过抑制炎性因子IL-1β和TNF-α的释放发挥抗炎作用,这种抑制作用与高表达的SOCS3对NF-κBp65过度激活密切相关。