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.

AFP联合热休克蛋白90α在肝癌早期诊断中的研究

目的评价甲胎蛋白(AFP)联合热休克蛋白90α(Hsp90α)在原发性肝癌早期诊断中的诊断效能。方法选取2021年8月—2022年8月因肝病在齐齐哈尔医学院附属第三医院住院的65例原发性肝癌患者(肝癌组)和54例肝硬化患者(良性肝病组),再选取同时期的60名健康体检者作为对照组,通过ELISA法测定所有研究对象的血浆Hsp90α和AFP水平,比较3组血浆Hsp90α、AFP水平,并比较Hsp90α、AFP单项及联合检测原发性肝癌的诊断效能。结果肝癌组患者血浆Hsp90α、AFP水平均显著高于良性肝病组、对照组,且良性肝病组患者Hsp90α、AFP水平均显著高于对照组,差异有统计学意义(P<0.05)。受试者工作特征(ROC)曲线分析结果显示,Hsp90α、AFP单独检测的AUC分别为0.890、0.826,均明显低于联合检测的0.951,差异有统计学意义(Z=2.155,P=0.031;Z=3.727,P=0.001)。联合检测诊断的敏感度为93.80%,特异性为85.10%,准确度为88.27%。结论血浆Hsp90α水平联合AFP可作为原发性肝癌的早期诊断生物标志物。

昆虫抗冻蛋白DAFP的原核表达、纯化和抗血清制备

目的:制备昆虫抗冻蛋白DAFP特异性的鼠抗血清。方法:根据GenBank已发表的Dendroides canadensis抗冻蛋白基因序列(gi:2737939),人工合成加拿大拟步甲虫(Dendroides canadensis)的抗冻蛋白基因(dafp),并克隆到原核表达载体pGEX-4T-1和pMAL-p2x中,在大肠杆菌中进行表达。表达的融合蛋白经Glutathione Sepharose4B纯化后,免疫小鼠制备抗DAFP的抗血清,抗体的效价及特异性分别采用ELISA和Western blot进行检测。结果:SDS-PAGE检测表明,人工合成的dafp基因在大肠杆菌中可表达相对分子质量(Mr)为38000的可溶性融合蛋白。以该融合蛋白免疫小鼠所制备的抗体,其ELISA滴度为1∶5000,Western blot证实BL21/pGEX-4T-1-dafp菌和TB1/pMAL-p2x-dafp菌的表达产物可特异性地与该抗体结合。结论:在大肠杆菌中成功地表达了DAFP的融合蛋白并制备出抗融合蛋白的鼠抗血清,为进一步研究DAFP的特性奠定了基础。

美洲拟鲽抗冻蛋白基因(afp)导入番茄的研究

将整合在Ｔｉ质粒上的ａｆｐ基因作为供体ＤＮＡ，用花粉管通道和子房注射方法导入番茄品种“中蔬四号”中，对Ｄ１、Ｄ２代进行Ｓｏｕｔｈｅｒｎ杂交获得杂交带；田间抗寒性试验表明，在春季平均气温低于正常年份４·４℃条件下，转基因组植株生长势优于对照；致死温度也比对照降低２℃．

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。

花粉管介导的转抗冻蛋白基因(AFP)水稻

低温和冷害在世界范围内广泛发生,对水稻的产量和品质造成严重影响。创造和培育具有强耐低温特性的水稻品种,对提高水稻产量具有重要的意义。本研究将从胡萝卜中克隆的抗冻蛋白基因(AFP)构建到植物表达载体pB I121(pB I121∶AFP)后,利用花粉管通道法将其转入籼稻品种NR158,获得了转基因后代。对T3代和T4代转基因植株的苗期耐冷性鉴定和PCR鉴定结果表明,部分转基因植株后代已经整合并且AFP基因得到了表达,获得了不同程度的耐低温性。

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。

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。

血清GPC3、GP73、AFP-L3和AFP检测对原发性肝癌诊断的价值

目的探讨血清磷脂酰肌醇蛋白多糖-3(glypican-3,GPC3)、高尔基体蛋白73(Golgi protein-73,GP73)、甲胎蛋白异质体AFP-L3以及AFP单独及联合检测在原发性肝癌(primary hepatic carcinoma,PHC)中的诊断价值。方法 GPC3、GP73、AFP-L3检测采用ELISA法,AFP检测采用化学发光法,分别检测原发性肝癌组(43例)、肝硬化组(43例)、慢性肝炎组(43例)、和健康对照组(43例)血清GPC3、GP73、AFP-L3、AFP水平,应用SPSS统计软件对结果进行统计学分析。结果 PHC组GP73、AFP-L3和AFP水平高于其他各组,差异均有显著性(P=0.000)。GPC3、GP73、AFP-L3和AFP的ROC曲线下面积(AUCROC)分别为0.744、0.910、0.903、0.867,灵敏度分别为53.5%、86.0%、81.4%、76.7%,特异度分别为94.6%、93.8%、95.3%、86.0%。系列试验以GP73+AFP-L3为最佳组合,灵敏度和特异度分别为70.0%和99.7%,平行试验以AFP/AFP-L3/GP73为最佳组合,灵敏度特异度分别为99.4%和76.9%。结论 GP73和AFP-L3有望成为新的诊断原发性肝癌的血清标志物。临床应用中,可用GP73+AFP-L3作为诊断PHC的最佳组合,GP73/AFP-L3/AFP作为排除PHC较好的组合。

基因枪法介导转人工合成Rs-AFP2基因小麦的获得和检测

萝卜（Raphanus sativus）抗菌肽Rs—AFP2在体外强烈抑制小麦赤霉病菌（Fusarium graminenrum）、黄色镰孢菌（Fusarium culmorum）、烟草赤星病（Alternaria longipes）的菌丝生长。为了研究Rs-AFP2在小麦抗病育种上的应用潜力，人工合成了Rs-AFP2基因。通过基因重组技术（包括限制性内切酶酶切和连接），将人工合成的R以FP2基因替代单子叶高效组成型表达栽体pAHC25中的GUS基因，构建了R￡AFP2基因的单子叶高效表达栽体pUAFP2。该栽体除携带受Ubiquitin启动子控制的Rs-AFP2基因表达盒外，还具有1个受Ubiqutin启动子控制的Bar基因表达盒，后者可为后续利用除草剂Bialaphos筛选转化再生植株提供抗性；采用基因枪法轰击小麦品种扬麦12、济麦19、晋麦47和豫麦34幼胚共4042个，经过2～3次Bialaphos筛选，最终获得扬麦12再生植株316株；利用高效表达栽体pUAFP2的Bar和Rs-AFP2基因的特异引物对上述成活的转化植株进行PCR检测，获得Bar和Rs-AFP2基因均为阳性的植株58株，转化率为1．43％。

抗冻蛋白基因AFP表达载体构建及转化紫花苜蓿初报

以胡萝卜基因组DNA为模板,用PCR方法扩增目的基因,连接到PGEM-T Easy Vector载体上,经XbalⅠ和SacⅠ双酶切,将目的片段连接到PBI121工程质粒上,通过农杆菌介导法转化和田苜蓿,为提高紫花苜蓿的抗冻性奠定基础。结果表明,成功克隆出抗冻蛋白基因AFP,并构建成AFP植物表达载体。获得了具有卡那霉素抗性的苜蓿愈伤组织,和田苜蓿愈伤组织的最佳Kan筛选浓度为60 mg/L。经PCR技术鉴定,AFP抗冻蛋白基因已整合到苜蓿基因组中。