Purification and Identification of Antifreeze Proteins in Ammopiptanthus mongolicus

The conventional protein chromatography technique was adopted to purify the antifreeze proteins (AFPs) from the leaves of Ammopiptanthus mongolicus (Maxim.) Cheng f. Two bands on native PAGE gel showed thermal hysteresis activity, one was band B1, whose thermal hysteresis was 0.46 ℃ at 8 g/L, which showed two bands (67 kD, 21 kD) on SDS_PAGE gel; the other was B3, whose thermal hysteresis was 0.45 ℃ at 10 g/L, and it contained only a single protein (39.8 kD). Both B1 and B3 are not glycoproteins, because neither do they interact with Shiff_reagent, nor show ultraviolet characteristics of a typical glycoprotein.

Ectopic expression of antifreeze protein gene from Ammopiptanthus nanus confers chilling tolerance in maize

Improved chilling tolerance is important for maize production. Previous efforts in transgenics and marker-assisted breeding have not achieved practical results. In this study, the antifreeze protein(AnAFP) from the super-xerophyte Ammopiptanthus nanus was aligned to KnS-type dehydrins.Phosphorylation in vitro and subcellular localization showed that AnAFP was phosphorylated by maize casein kinase II and translocated from nucleus to cytoplasm under chilling stress. AnAFP also increased lactate dehydrogenase activity. A parent line of commercial maize hybrids was transformed with the AnAFP gene. Based on thermal asymmetric interlaced PCR, one hemizygous and two homozygous integration sites were identified in one T_(1) line. Ectopic expression of AnAFP in transgenic lines was confirmed by quantitative real-time PCR, RNA-seq, and Western blotting. After chilling treatment, the transgenic lines showed significantly improved phenotype, higher kernel production, survival rate and biomass, and lower relative electrolyte leakage and superoxide dismutation than the untransformed line. Thus, ectopic expression of AnAFP gene improved chilling tolerance in the transgenic lines, which could be used to apply for further safety assessment for commercial breeding.

Expression of a Carrot 36 kD Antifreeze Protein Gene Improves ColdStress Tolerance in Transgenic Tobacco

Antifreeze proteins （AFPs） enable organisms to survive under cold conditions, and have great potential in improving cold tolerance of cold-sensitive plants, In order to determine whether expression of the carrot 36 kD antifreeze protein gene confers improved cold-resistant properties to plant tissues, we tried to obtain transgenic tobacco plants which expressed the antifreeze protein. Cold, salt, and drought induced promoter Prd29A was cloned using PCR from Arabidopsis. Two plant expression vectors based on pBI121 were constructed with CaMV35S：AFP and Prd29A：AFP. Tobacco plantlets were transformed by Agrobacterium-medicated transformation. PCR and Southern blotting demonstrated that the carrot 36 kD afp gene was successfully integrated into the genomes of transformed plantlets. The expression of the afp gene in transgenic plants led to improved tolerance to cold stress. However, the use of the strong constitutive 35S cauliflower mosaic virus （CaMV） promoter to drive expression of afp also resulted in growth retardation under normal growing conditions. In contrast, the expression of afp driven by the stress-inducible Prd29A promoter from Arabidopsis gave rise to minimal effects on plant growth while providing an increased tolerance to cold stress condition （2℃）. The results demonstrated the prospect of using Prd29A-AFP transgenic plants in cold-stressed conditions that will in turn benefit agriculture.

Heterologous expression of Lolium perenne antifreeze protein confers chilling tolerance in tomato

Antifreeze proteins（AFP） are produced by certain plants, animals, fungi and bacteria that enable them to survive upon extremely low temperature. Perennial rye grass, Lolium perenne, was reported to possess AFP which protects them from cold environments. In the present investigation, we isolated AFP gene from L. perenne and expressed it in tomato plants to elucidate its role upon chilling stress. The T1 transgenic tomato lines were selected and subjected to molecular, biochemical and physiological analyses. Stable integration and transcription of Lp AFP in transgenic tomato plants was confirmed by Southern blot hybridization and RT-PCR, respectively. Physiological analyses under chilling conditions showed that the chilling stress induced physiological damage in wild type（WT） plants, while the transgenic plants remained healthy. Total sugar content increased gradually in both WT and transgenic plants throughout the chilling treatment. Interestingly, transgenic plants exhibited remarkable alterations in terms of relative water content（RWC） and electrolyte leakage index（ELI） than those of WT. RWC increased significantly by 3-fold and the electrolyte leakage was reduced by 2.6-fold in transgenic plants comparing with WT. Overall, this report proved that Lp AFP gene confers chilling tolerance in transgenic tomato plants and it could be a potential candidate to extrapolate the chilling tolerance on other chilling-sensitive food crops.

Expression of a Carrot Antifreeze Protein Gene in Escherichia coli

The recombinant expression vector pET43.1b-AFP, which contains full encoding region of a carrot 36 kD antifreeze protein (AFP) gene was constructed. The recombinant was transformed into expression host carrying T7 RNA polymerase gene (DE3 lysogen) and induced by 1 mmol稬-1 IPTG (isopropyl--D-thiogalactoside) to express 110 kD polypeptide of AFP fusion protein. The analysis of product solubility revealed that pET43.1b-AFP was predominately soluble, and the expressed amount reached the maximum after the IPTG treatment for 3 h.

Thermodynamic Properties of Linear Protein Solutions:an Application to Type Ⅰ Antifreeze Protein Solutions

A statistical thermodynamic theory of linear protein solutions was proposed with the aid of a lattice model and applied to type Ⅰ antifreeze protein（AFPI） solutions.The numerical results for several AFPI solutions show that the Gibbs function of the solution has a minimum at a certain protein concentration,but the protein chemical potential increases with increasing the concentration.The influences of temperature and protein chain length on the AFPI chemical potential were also discussed.The evaluation for the colligative depression of the freezing point confirms that the antifreeze action should be recognized as non-colligative.The theoretical deduction for the concentration dependence of the thermal hysteresis activity coincides qualitatively with the previous experimental and theoretical results.

Construction of plant expression vector of Pseudopleuronectes americanus antifreeze protein gene

The Pseudopleuronectes americanus antifreeze protein gene was synthesized and control sequences were added such as 35S promoter and nos terminator that can facilitate the transcription and Ω sequence and Kozak sequence that can improve the expression in translation level, the high expression cassette of antifreeze protein was constructed. This cassette was connected to pBI121.1 and finally got the high expression vector pBRTSAFP introduced into the maize callus. The expression of gus gene that linked to the antifreeze protein gene was detected, and the results was that the gus gene can express strongly and instantaneously.

Plant Antifreeze Proteins and Their Expression Regulatory Mechanism

Low temperature is one of the major limiting environmental factors which constitutes the growth, development, productivity and distribution of plants. Over the past several years, the proteins and genes associated with freezing resistance of plants have been widely studied. The recent progress of domestic and foreign research on plant antifreeze proteins and the identifica- tion and characterization of plant antifreeze protein genes, especially on expression regulatory mechanism of plant antifreeze proteins are reviewed in this paper. Finally, some unsolved problems and the trend of research in physiological functions and gene expression regulatory mechanism of plant antifreeze proteins are discussed.

Comparative Analysis of Hydration Layer Reorientation Dynamics of Antifreeze Protein and Protein Cytochrome P450

Antifreeze proteins(AFPs)inhibit ice recrystallization by a mechanism remaining largely elusive.Dynamics of AFPs’hydration water and its involvement in the antifreeze activity have not been identified conclusively.We herein,by simulation and theory,examined the water reorientation dynamics in the first hydration layer of an AFP from the spruce budworm,Choristoneura fumiferana,compared with a protein cytochrome P450(CYP).The increase of potential acceptor water molecules around donor water molecules leads to the acceleration of hydrogen bond exchange between water molecules.Therefore,the jump reorientation of water molecules around the AFP active region is accelerated.Due to the mutual coupling and excitation of hydrogen bond exchange,with the acceleration of hydrogen bond exchange,the rearrangement of the hydrogen bond network and the frame reorientation of water are accelerated.Therefore,the water reorientation dynamics of AFP is faster than that of CYP.The results of this study provide a new physical image of antifreeze protein and a new understanding of the antifreeze mechanism of antifreeze proteins.

Effect of Plant AntifreezeProteins on Porcine Embryo to Cryopreservaton

This study was to research the cryopreservation effect of plant antifieeze proteins(AFPs) on day 7 porcine expanded and hatched blastocysts that were frozen in 1.5 M glycerol by conventional slow freezing method. The developmental rates of porcine embryos frozen with 0.5 mg/ml AFPs in freeze medium and cultured for 12 and 24 hours in vitro are 25% and 0%respectively. With the concentration of AFPs increased to 5 mg/ml, the corresponding values became 80% and 40%. The hatched rates for porcine embryos frozen with 0.5 mg/ml and 5 mg/ml of AFPs and cultured for 24 hours in vitro are 0% and 20% respectively..The developmental and hatched rate of the contnrol are all 0%(0/4). These results indicate that the survival rates of porcine expanded and hatched blastocyst can be improved by supplementing freeze medium with plant AFPs.

血清TAP、proGRP、cyfra21-1与Ⅲ~Ⅳ期NSCLC新辅助化疗疗效及预后的关系

目的探讨血清肿瘤异常蛋白(TAP)、胃泌素释放肽前体(proGRP)、细胞角蛋白19片段(cyfra21-1)与Ⅲ~Ⅳ期非小细胞肺癌(NSCLC)新辅助化疗(NCT)疗效及预后的关系。方法选取100例Ⅲ~Ⅳ期NSCLC患者为研究对象,所有患者均采用NCT治疗,根据疗效将患者分为有效组和无效组,比较不同疗效患者化疗前后TAP、proGRP、cyfra21-1水平,分析化疗后TAP、proGRP、cyfra21-1水平对NSCLC患者NCT疗效的评估价值。所有患者均随访1年,分析化疗后TAP、proGRP、cyfra21-1表达水平与预后的关系。结果化疗后,有效组TAP、proGRP、cyfra21-1水平低于无效组(P<0.05)。ROC曲线结果显示,TAP评估NSCLC患者NCT疗效的AUC和截点值分别为0.739、178.18μm 2,proGRP评估NSCLC患者NCT疗效的AUC和截点值分别为0.810、52.21 ng/L,cyfra21-1评估NSCLC患者NCT疗效的AUC和截点值分别为0.775、7.70μmol/L,联合评估NSCLC患者NCT疗效的AUC为0.913,高于单项诊断(P<0.05)。TAP、proGRP、cyfra21-1高表达患者的1年生存率均低于低表达患者(P<0.05)。结论TAP、proGRP、cyfra21-1联合评估Ⅲ~Ⅳ期NSCLC患者NCT疗效具有较高价值,且其均与NSCLC患者预后密切相关。

An enriched environment increases the expression of fibronectin type Ⅲ domain-containing protein 5 and brain-derived neurotrophic factor in the cerebral cortex of the ischemic mouse brain

Many studies have shown that fibronectin type III domain-containing protein 5(FDNC5) and brain-derived neurotrophic factor(BDNF) play vital roles in plasticity after brain injury. An enriched environment refers to an environment that provides animals with multi-sensory stimulation and movement opportunities. An enriched environment has been shown to promote the regeneration of nerve cells, synapses, and blood vessels in the animal brain after cerebral ischemia;however, the exact mechanisms have not been clarified. This study aimed to determine whether an enriched environment could improve neurobehavioral functions after the experimental inducement of cerebral ischemia and whether neurobehavioral outcomes were associated with the expression of FDNC5 and BDNF. This study established ischemic mouse models using permanent middle cerebral artery occlusion(pMCAO) on the left side. On postoperative day 1, the mice were randomly assigned to either enriched environment or standard housing condition groups. Mice in the standard housing condition group were housed and fed under standard conditions. Mice in the enriched environment group were housed in a large cage, containing various toys, and fed with a standard diet. Sham-operated mice received the same procedure, but without artery occlusion, and were housed and fed under standard conditions. On postoperative days 7 and 14, a beam-walking test was used to assess coordination, balance, and spatial learning. On postoperative days 16–20, a Morris water maze test was used to assess spatial learning and memory. On postoperative day 15, the expression levels of FDNC5 and BDNF proteins in the ipsilateral cerebral cortex were analyzed by western blot assay. The results showed that compared with the standard housing condition group, the motor balance and coordination functions(based on beam-walking test scores 7 and 14 days after operation), spatial learning abilities(based on the spatial learning scores from the Morris water maze test 16–19 days after operation), and memory abilities(based on the memory scores of the Morris water maze test 20 days after operation) of the enriched environment group improved significantly. In addition, the expression levels of FDNC5 and BDNF proteins in the ipsilateral cerebral cortex increased in the enriched environment group compared with those in the standard housing condition group. Furthermore, the Pearson correlation coefficient showed that neurobehavioral functions were positively associated with the expression levels of FDNC5 and BDNF(r = 0.587 and r = 0.840, respectively). These findings suggest that an enriched environment upregulates FDNC5 protein expression in the ipsilateral cerebral cortex after cerebral ischemia, which then activates BDNF protein expression, improving neurological function. BDNF protein expression was positively correlated with improved neurological function. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Fudan University, China(approval Nos. 20160858 A232, 20160860 A234) on February 24, 2016.

Effects of gentiana scabra bage on expression of hepatic type Ⅰ,Ⅲ collagen proteins in Paragonimus skrjabini rats with liver fibrosis

Objective:To exploit- the effects of gentiana scabra bage on the expression of hepatic collagen proteins in Paragonimus skrjabini rats with liver fibrosis.Methods:Immunohistochemical technique was used to observe the changes of content of hepatic type Ⅰ,Ⅲ collagen proteins in Paragonimus skrjabini rats with Liver fibrosis before and after the gentiana scabra bage treatmeat.Results:Comparing with the model group,changes of hepatic tvpe Ⅰ and type Ⅲ collagen proteins in gentiana scabra bage treated group were significantly weakened.Conclusions:Gentiana scabra bage treatment can reduce the content of hepatic type Ⅲ and type Ⅰ collagen protein significantly in Paragonimus skrjabini rats with liver fibrosis,thereby,playing the role against hepatic fibrosis.

Molecular Cloning and Bioinformatics Analysis of TypeⅢSecretion System Effector Protein Va1686 Gene of Vibrio alginolyticus

In this study, Va 1686 gene was cloned from Vibrio alginolyticus . The total length of the gene is 1 164 bp, and it could encode 387 amino acids. The physicochemical properties, protein structure, genetic evolutionary relationship and antigenic characteristics of the effect protein Va1686 of V. alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools. The results showed that Va1686 is a stable hydrophilic and acidic protein without a transmembrane region and a signal peptide, and secondary structure to α-helix. The evolutionary analysis showed that V. alginolyticus HY9901 and V. harveyi were clustered together, which indicated that the genetic relationship between the two species was the closest. Va1686 contains a Fic superfamily conserved domain associated with cell division. Bioinformatics analysis showed that the B-cell preponderant epitopes of Va1686 might be localized in the regions of 48-49, 82-85, 125-126, 150-153, 185-186, 236-237 and so on. The 3D structure model of Va1686 subunit was simulated by SWISS-MODEL software and it was found that the vopS of V. parahaemolyticus was similar and the similarity was 89.46%. In this study, the feasibility of Va1686 as a common antigen of Vibrio was verified from the perspective of bioinformatics, which laid the foundation for the next step in vaccine development.

Molecular Cloning and Bioinformatics Analysis of TypeⅢSecretion System Effector Protein HY 322 Gene of Vibrio alginolyticus

In this study,Hy322 gene was cloned from Vibrio alginolyticus.The total length of its gene was 969 bp,and it could encode 322 amino acids.The physicochemical properties,protein structure,genetic evolutionary relationship and antigenic characteristics of the effector protein Hy322 of V.alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools.The results showed that Hy322 is an unstable hydrophilic and acidic protein without a transmembrane region and a signal peptide,and secondary structure to α-helix.The evolutionary analysis showed that V.alginolyticus HY9901 and V.harveyi were clustered together,which indicated that the genetic relationship between the two species was closest.HY322 contains a FliN super family conserved domain associated with Flagellar motor switch.Bioinformatics analysis showed that the B-cell preponderant epitopes of Hy322 might be localized in the regions of 32-33,100-102,138-140,215-216,235-238 and 246-249.The 3D structure model of Hy322 subunit was simulated by SWISS-MODEL software and itwas found that the yscQ of Yersinia were similar and the similarity was 42.25%.In this study,the feasibility of Hy322 as a common antigen of Vibrio was verified from the perspective of bioinformatics,which laid the foundation for the next step in vaccine development.

低温对菲律宾蛤仔热休克蛋白和抗冻蛋白基因表达的影响

为研究菲律宾蛤仔(Ruditapes philippinarum)“斑马蛤2号”在低温胁迫下热休克蛋白和抗冻蛋白基因表达的变化,将菲律宾蛤仔“斑马蛤2号”和南北方野生群体蛤仔急性低温(−1℃)胁迫9 d,存活率大小为斑马蛤2号(78.08%±2.14%)>大连群体(70.37%±3.70%)>北海群体(48.15%±7.71%)。低温胁迫下,挑选5个蛤仔抗低温相关的基因,热休克蛋白70基因(HSP70),HSC70,CSDE1,Y-box,抗冻蛋白Ⅱ型基因(AFPⅡ),研究这些基因在3个群体菲律宾蛤仔(斑马蛤2号、大连群体和北海群体)急性低温胁迫(−1℃)下鳃组织的表达变化。结果显示,5个基因在菲律宾蛤仔鳃组织中的时序表达与温度显著相关,其中HSP70、HSC70在3个群体中显著高表达。冷休克蛋白家族的基因CSDE1、Y-box基因只在低温处理的广西群体鳃组织中显著高表达。3个群体AFPⅡ基因相对表达量在12 h显著升高,达到峰值,相对表达量斑马蛤2号>大连群体>北海群体。不同群体蛤仔在低温胁迫时抗低温相关基因表达模式不同,斑马蛤2号的HSP70、HSC70和AFPⅡ表达量及存活率高于2个野生群体。

鱼类低温适应性机制与抗冻蛋白的研究进展

温度对调节鱼类的生物功能至关重要,长期生活在极端寒冷环境中的鱼类通过发生适应性进化而存活下来,抗冻蛋白的产生是其中最重要的适应性特征之一。为全面了解鱼类的低温适应性机制研究进展,首先从内分泌系统、代谢系统和免疫系统3个方面阐述了低温水环境对鱼类的基因表达和激素分泌的影响,以及对机体代谢过程的整体影响;其次,从鱼类的生理适应和进化适应两部分综述了鱼类的低温适应性机制;最后,详细阐述了抗冻蛋白的研究起源与结构特性、克隆与功能鉴定等方面的研究进展。相关研究现状表明,利用组学方法可以研究低温胁迫下鱼类的代谢途径和分子信号通路,在生物整体水平上分析低温适应机制;利用该方法,已经挖掘了多种低温耐受功能基因。由于不同鱼类及其温度适应性存在差异,未来还需要开展更多基础性研究工作。

钡A-偶氮氯膦Ⅲ络合物探针分光光度法测定蛋白质

在pH 1.0～ 1.5的酸性介质中 ,蛋白质与Ba 偶氮氯膦Ⅲ络合物发生作用 ,导致络合物溶液褪色 ,其最大吸收波长处吸光度的降低值与蛋白质的浓度成正比。基于此建立了以Ba 偶氮氯膦Ⅲ络合物为光谱探针 ,分光光度测定蛋白质含量的新方法。该法灵敏度高 ,选择性好。蛋白质 (以牛血清白蛋白为例 )的浓度在 0～ 4 0mg/L范围内服从比尔定律 ,表观摩尔吸光系数ε656=6 .4 7× 10 5L·mol-1·cm-1。生物体内常见物质均不产生干扰 ,直接应用于人血清样品中蛋白质总量的测定 。

抗冻蛋白AFPⅢ的原核表达、纯化及多克隆抗体的制备

采用基因重组技术将AFPⅢ基因与原核表达载体pET32a(+)连接,转化大肠杆菌BL21(DE3),通过PCR、单双酶切及测序鉴定构建结果。用IPTG诱导蛋白表达,将融合蛋白纯化后,免疫6-7周龄的小白鼠,制备AFPⅢ多克隆抗体,采用Western印迹法检验抗体特异性。通过酶联免疫吸附试验(ELISA)测定多克隆抗体滴度。成功地构建了AFPⅢ的原核表达载体,经在大肠杆菌中诱导表达、镍柱亲和层析纯化,得到较纯的相对分子质量约26 000 Da的融合蛋白,免疫小白鼠后得到多抗血清,Western印迹结果显示此多克隆抗体与AFPⅢ蛋白特异性结合。该试验为进一步研究AFPⅢ在转基因鱼中的定点表达以及作用机制奠定了基础。

瑞氏木霉内切葡聚糖酶Ⅲ基因的克隆及在酿酒酵母中的表达

采用刚果红染色法从瑞氏木霉cDNA文库中分离到一株具有CMCase活性的阳性克隆 ,测序结果显示该基因所编码的蛋白质为瑞氏木霉内切葡聚糖酶Ⅲ (EGⅢ )。对重组酿酒酵母所产生的EGⅢ进行了酶学性质分析 ,其最适pH为 5 0 ,最适温度为 60℃。检测了酿酒酵母蛋白质分泌系统组分SSO2和SEB1对EGⅢ分泌的影响。结果表明 ,在可过量表达蛋白质分泌系统组分SSO2的酿酒酵母H837中 ,EGⅢ的分泌量最高。由此分析 ,酿酒酵母SSO2蛋白可能在EGⅢ的分泌中 ,是一个限速步骤。通过PCR方法删除EGⅢ基因 5′端非翻译区的 98bp核苷酸序列使EGⅢ表达量提高了 5 3倍。这提示我们 ,瑞氏木霉的EGⅢ基因在酿酒酵母细胞中表达时 ,其mRNA 5′端先导序列中可能存在影响该基因表达水平的调控序列。