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.

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

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.

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.

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.

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.

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.

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.

Advances in Cryopreservation of Organs

Organ transplantation is an effective approach for the treatment of end-stage organ failures. Currently, the donor organs used for clinical transplantation are all preserved at above-zero temperatures. These preservation methods are well-established and simple but the storage time lasts for only 4–12 h. Some researchers tried to extend the organ storage time by improving protectant and HLA matching to raise the use of stored organs and prolong the long-term survival of organs. These efforts still fall short of the clinical demand for organ transplantation. Moreover, a great many organs were wasted due to limited storage time, HLA mismatch, patients＇ conditions or distance involved. Therefore, preserving organs for several weeks or even months and establishing Organ Bank are the tough challenges and have become a shared goal of global scholars. This article reviews some issues involved in the cryopreservation of organs, such as use of cryoprotecting agents, freezing and thawing methods in the cryopreservation of hearts, kidneys and other organs.

Transcriptomic and proteomic analyses on the supercooling ability and mining of antifreeze proteins of the Chinese white wax scale insect

The Chinese white wax scale insect, Ericerus pela, can survive at extremely low temperatures, and some overwintering individuals exhibit supercooling at tempera- tures below -30℃. To investigate the deep supercooling ability ofE. pela, transcriptomic and proteomic analyses were performed to delineate the major gene and protein families responsible for the deep supercooling ability of overwintering females. Gene Ontology （GO） classification and Kyoto Encyclopedia of Genes and Genomes （KEGG） analysis indicated that genes involved in the mitogen-activated protein kinase, calcium, and PI3K-Akt signaling pathways and pathways associated with the biosynthesis of soluble sugars, sugar alcohols and free amino acids were dominant. Proteins responsible for low-temperature stress, such as cold acclimation proteins, glycerol biosynthesis-related enzymes and heat shock proteins （HSPs） were identified. However, no antifreeze proteins （AFPs） were identified through sequence similarity search methods. A random forest approach identified 388 putative AFPs in the proteome. The AFP gene ep-afp was expressed in Escherichia coli, and the expressed protein exhibited a thermal hysteresis activity of 0.97℃, suggesting its potential role in the deep supercooling ability ofE. pela.

Expression of multi-domain type III antifreeze proteins from the Antarctic eelpout (Lycodichths dearborni) in transgenic tobacco plants improves cold resistance

Type III antifreeze proteins(AFPIIIs)are a group of small globular proteins found in some polar fishes to protect them against freezing damage.Transgenic expression of AFPs has been shown to confer cold tolerance to commercially important plants and animals.We have previously isolated multiple AFPIII genes in the Antarctic eelpout(Lycodichthys dearborni)that encode larger AFPIII isoforms with up to 12 of the conventional domains.Here we have introduced the fish AFPIII genes that encode for the monomer(ld1),dimer(ld2),trimer(ld3)and tetramer(ld4)AFPIII isoforms in tobacco plants.Pot-grown 4-week-old transgenic tobacco plants were exposed to cold stress at 4◦C for 30 days and the results show that ld1,ld2,ld3 and ld4 transgenic plants present relatively lower electrolyte leakage and lower content of malondialdehyde(MDA),but accumulated higher content of proline when compared to control plants.This indicates considerable improved membrane integrity under low temperature stress and improvement of the plant cold resistance.The plants transformed with the AFPIII tetramer-and trimer-domains demonstrated a higher cold-tolerant levels when compared with plants transformed with the dimer-and monomer AFPIII domains.Our study further supports that fish AFPIIIs,especially the multidomain proteins,protect cells from non-freezing hypothermic stresses,apart from there well-known function as ice inhibitors molecules at freezing temperature.

Cloning,sequencing and prokaryotic expression of cDNAs for antifreeze protein family from Beetle Tenebrio molitor

Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR.Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank.The recombinant pGEX-4T-1-tmafp-XJ430 was introduced into E.coli BL21 to induce a GST fusion protein by IPTG.SDS-PAGE analysis for the fusion protein shows a band of 38 kDa.pCDNA3-tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA.The titer of the antibody was 1:2000.Western blotting analysis shows that the antiserum was specifically against the antifreeze protein.Our results laid the foundation for further studies on the properties and functions of insect antifreeze proteins.