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.

抗冻蛋白对冷冻猪肉品质的影响

研究抗冻蛋白(antifreeze proteins,AFP)对猪肉冷冻过程中品质变化的影响。以商业抗冻剂海藻糖(4 mg/m L)的抗冻效果为参考,以未添加AFP组为对照组,研究不同添加量(0.05、0.1、0.2、0.3 mg/mL)AFP对冷冻猪肉品质、肌原纤维蛋白理化性质、微观结构和水分分布的影响。结果表明:AFP可以减少冷冻猪肉的水分损失,有利于维持猪肉色泽和微观结构;在冷冻贮藏过程中,0.3 mg/mL AFP组猪肉的蒸煮损失、解冻损失及色泽的保护效果最好,其次是0.2 mg/mL AFP组和4 mg/mL海藻糖组;0.2、0.3 mg/mL AFP组剪切力和肌原纤维蛋白总巯基含量显著高于未添加AFP组(P<0.05);核磁共振成像验证了AFP的渗入降低了猪肉中结合水和不易流动水损失;扫描电子显微镜结果显示,0.2 mg/mL AFP使猪肉肌纤维排列整齐,更加紧凑和致密。总体而言,添加AFP对改善冷冻猪肉品质有积极作用,是一种潜在的安全、高效冷冻保护方法。

鳕鱼明胶抗冻多肽的制备及其在鱼糜冻融中的应用

为优化高活性鳕鱼明胶抗冻多肽(DcAFPs)的制备方法,明确鳕鱼明胶多肽对鱼糜的冷冻保护效果,本研究将鳕鱼明胶多肽添加至鱼糜中,以冷冻后鱼糜肌原纤维蛋白含量为考察指标,通过单因素和正交试验进行工艺优化。通过测定DcAFPs分子量分布和氨基酸组成,分析其基本性质;通过测定鱼糜肌原纤维蛋白含量,并运用内源荧光光谱和SDS-PAGE凝胶电泳技术,评估DcAFPs对反复冻融循环后鱼糜的保护效果。结果表明,DcAFPs的最优制备工艺是:使用复合蛋白酶、底物浓度为3.5%(w/v)、酶底比为5%(w/w)、水解p H为8、水解温度为55℃、水解时间为2 h。DcAFPs分子量主要集中在185~3081 Da,Gly、Ala和Glu的含量最高。DcAFPs能有效防止鱼糜在冻融过程中肌原纤维蛋白含量的减少,抑制肌原纤维蛋白结构和组成的变化,延缓蛋白变性,其总体效果优于商业抗冻剂。综上所述,DcAFPs对反复冻融的鱼糜显示出显著的冷冻保护效果。

Observation on the modifying activity of the protein from Elytrzgia repens rhizome for ice crystal

In winter, spring and summer, the rhizome of wild Elytrzgia repens of Heilongjiang Province was selected to extract the soluble which whole protein and the apoplastic protein, and analyzed by SDS-PAGE. The result indicated that there were two specific polypeptides in two types protein from winter; their relative molecular weight were identified as 52 ku and 26 ku by analyzing software; the apoplastic protein from winter had the ability of modifing the growth of ice crystal which appeared hexagonal in shape observed with the phase-contrast photomicroscope. So the apoplastic protein from winter has the antifreeze characters and the 52 ku protein is more likely the antifreeze protein

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。

燕麦抗冻蛋白对不同冻藏条件下的面筋蛋白性质的影响

为探究燕麦抗冻蛋白(AsAFPs)对不同冻藏条件下面筋蛋白品质的影响,将AsAFPs按照0.5%的质量分数添加到面团中,通过对不同冻藏温度和冻藏时间下小麦面筋蛋白的可溶性蛋白含量、持水性、持油性、乳化性和乳化稳定性、起泡性和泡沫稳定性及所含游离巯基含量的测定。结果表明:经4周的冻藏,各组小麦面筋蛋白中可溶性蛋白含量呈现下降趋势(P<0.05),而加入AsAFPs之后可以减缓冻藏过程中可溶性蛋白含量的降低;随冻藏时间的延长,各组小麦面筋蛋白持水性下降、持油性上升(P<0.05),加入AsAFPs后,可以减少疏水区域的暴露,使各组持水性下降的程度得到减弱,持油性上升的程度得到减弱;各组小麦面筋蛋白的乳化性呈现下降趋势,乳化稳定性则呈现上升趋势(P<0.05),加入AsAFPs之后,乳化性下降的程度减弱,乳化稳定性上升的程度减弱;各组小麦面筋蛋白的起泡性呈下降趋势,起泡稳定性则呈上升趋势(P<0.05),而加入AsAFPs后,小麦面筋蛋白起泡性下降程度减弱,泡沫稳定性上升的程度减弱;各组小麦面筋蛋白游离巯基含量呈现增加的趋势(P<0.05),而加入AsAFPs后小麦面筋蛋白中游离巯基含量明显下降。AsAFPs的加入可以改善小麦面筋蛋白的功能特性,减缓冻藏过程中面筋蛋白品质的劣变。研究结果为AsAFPs在冷冻面制品中的应用以及冷冻储藏温度的优化提供一定的参考。

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。

复合抗冻剂的研制及对鮰鱼肌原纤维蛋白的抗冻效果

以鲫鱼鱼鳞为原料,用胰蛋白酶酶解法获得抗冻粗肽。以抗冻粗肽、海藻糖和褐藻寡糖为因素,运用{3,3}单纯形格子法进行配方设计,以冷冻24 h后嗜热链球菌的存活率为抗冻活性筛选指标,获得复合抗冻剂理论模型配方。选用复合抗冻剂最优组(K1、K2)和理论模型配方组(K3)进行冷冻鮰鱼肌原纤维蛋白试验,验证复合抗冻剂的抗冻效果。结果表明:抗冻粗肽组嗜热链球菌的存活率达57.11%。与大豆卵磷脂互作的红外光谱显示:抗冻粗肽可能与磷脂亲水头部和水之间发生相互作用,有利于稳定膜结构。K1和K2嗜热链球菌的存活率在80%以上,K3嗜热链球菌的存活率高达92%。添加K1、K2和K3复合抗冻剂后蛋白质表面疏水性显著降低(P<0.05),蛋白质溶解度显著升高(P<0.05)。K1和K2的α-螺旋和β-折叠含量显著下降(P<0.05),K2和K3的r值显著降低(P<0.05)。结论:理论模型K3是具有高抗冻活性,可抑制蛋白质聚集和维持蛋白质结构稳定的一种良好的水产品复合抗冻剂。本研究为复合抗冻剂的开发提供新思路,为后续开发鮰鱼预制菜冷冻产品提供了理论参考。

3种新型抗冻材料应用于配子及胚胎冷冻保存的研究进展

配子及胚胎的冷冻保存是将配子及胚胎在超低温条件下冷冻,达到可以长期保存的目的。冷冻保存技术在种质资源保护、保存和改良动物基因等方面发挥重要作用。由于传统冷冻保护剂具有毒性,目前研究人员正致力于开发毒性低、抗冻效果显著的新型抗冻材料,如抗冻蛋白、纳米材料和聚合物。本文简述了冷冻保存技术的原理及存在问题,综述了3种新型抗冻材料的抗冻机制及应用进展,并从材料工程的角度为冷冻保存配子及胚胎提供一个新的视角,为更多新型材料的开发拓展新思路。

抗冻剂提高肉糜类制品冻融稳定性的研究进展及展望

冻藏过程中温度变化导致的冻融循环会使肉糜类制品出现汁液流失、蛋白质变性、营养价值损失等问题,严重影响产品的风味、口感等食用特性.抗冻剂是一类能够有效提高抗冻能力,保证产品品质稳定的食品添加剂.文章综述了磷酸盐类、糖和糖醇类、抗冻蛋白三种抗冻剂的抗冻机理以及在肉糜类制品中的应用,并对一种同样具有抗冻作用的蛋白类物质——蛋白基Pickering乳液进行了展望,希望为改善肉糜类凝胶制品冻融稳定性而开发出符合人们健康需求且成本低廉、适合工业化生产的新型抗冻剂提供思路.

黄粉虫抗冻蛋白吸附天然气水合物过程中的行为研究

采用分子动力学模拟的方法,计算并分析了黄粉虫抗冻蛋白吸附甲烷水合物过程中局部结构的变化。研究表明,水合物结合位点上4号苏氨酸Thr4在靠近甲烷水合物表面时,其位置会发生变化以适应水合物的笼状结构,通过取代甲烷分子与水合物表面结合,并促使其他苏氨酸靠近水合物,最终黄粉虫抗冻蛋白吸附在水合物上,抑制了冰固体生长。本研究结果对研发可降解的天然产物类水合物抑制剂具有参考意义。