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.

The Antifreeze Critical Strength of Low-temperature Concrete Effected by Index

The antifreeze critical strength and the pre-curing time of low-temperature concrete were studied by means of guaranteed rate of compressive strength and antifreeze performance for the structural safety requirement of concrete engineering,suffering once freeze damage under air environment.It is shown that the antifreeze critical strength is 3.7-4.4MPa,pre-curing time is 18-32 h by guaranteed rate of compressive strength,and the antifreeze critical strength is 3.7-4.4MPa,pre-curing time is 18-32 h by guaranteed rate of antifreeze performance.It can be found that the method of guaranteed rate of compressive strength is sensitive to the defect which generated by freeze damage in the concrete interior.The method is fit to evaluate the antifreeze critical strength of low-temperature concrete.

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.

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.

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.

Impact of Borate on Structure of Antifreeze Glycoproteins

Antifreeze glycoproteins(AFGPs)facilitate the survival of various organisms in the polar region by preventing internal ice accumulation via an adsorption-inhibition mechanism.Inhibition of AFGP antifreeze activity by the borate buffers has been widely acknowledged as the direct experimental evidence supporting the hydroxyl,rather than methyl,binding mechanism.On the other hand,perturbation of borate binding on the AFGP configuration,which might have considerable influence on the binding efficiency of not only the hydroxyl but also the methyl groups,has rarely been quantitatively examined.Herein we studied,using molecular dynamics simulations,the perturbation on the configuration of a solvated AFGP8 protein induced by the binding of one single borate anion.Near the freezing point,this binding not only makes the disaccharide groups adjacent to the borate-binding disaccharide close to each other but also affects the entire AFGP8 conformation.The structural changes induced by the binding of borate on different disaccharide sidechains exhibit clear site-specificities and the effect of borate binding on the structural changes is significantly reduced at higher temperatures.Our study is valuable for further understanding the relationship between the structure and antifreeze activity of these antifreeze glycoproteins.

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。

Research of Environment-friendly Low Emissivity Glass

The environment-friendly glasses which integrate function and structure were introduced, among these glasses can save energy very efficiently due to its low infrared emissivity. The fundamental principle of the low emissivity glass and the research progress of this kind glass were analyzed. Meanwhile, high performance and low applied development trend of emissivity glass were reviewed.

Research on the Eco-tourism and Environment-friendly Land Use Patterns in Shangri-La

Through the analysis of the geographical features of the Shangri-La,the eco-tourism and environment-friendly land use patterns were proposed. And the significances of the mode of economic development in Shangri-La were analyzed.

The Evaluation Principles,Indicator System and Methods for Environment-friendly Utilization of County-level Land Resources

From the perspective of environmental issues concerning land use and importance of county,we analyze the great significance of environment-friendly use of county land resources. On the basis of discussing the concept and connotation of environment-friendly utilization of land resources,we build the basic framework for the evaluation of environment-friendly utilization of land resources from environment,economy and society,and propose the evaluation methods combining composite index method and multi-angle single indicator evaluation method. At the same time,we establish the grading system and grading standards for environment-friendly county land use,to promote the qualitative and quantitative evaluation of environment-friendly county land use.

The Study of Strategy for the Development of Ying Charoen Market to Become Environment-Friendly Market

The results of using microorganism to clean appliances and buildings show that it was a highly efficient method.The researcher found that microorganism was more human and animal friendly than chemicals,and was good for environment as well.Ying Charoen Market was constructed in the year 1955,so it has existed for more than 63 years.The market has all kinds of food and has been a food center for people living in Northern Bangkok for a long time.Thus,it is not surprising that the market is also the center of people,garbage,and pollution,which produce negative effects on the environment.The research population consists of stakeholders(owner,managers,employees,suppliers,vendors,and consumers)in Ying Charoen Market.In details,there are 1,512 vendors in the market,which can be classified into 15 groups according to the main types of products.This study used mixed methods of methodology,in particular,exploratory sequential method.It was a qualitative research and an action participatory research.The objectives were to study the current condition,context,factors,and problems of Ying Charoen Market that are related with the environment,and to establish strategies for developing Ying Charoen Market to become environment-friendly.The data were analyzed by content analysis.The results expected from this research is to have a guidance for producing the strategy of environment management,which will lead to the participation of related people,lower cost in the long term,and good health for employees,vendors,and consumers in the market,as well as positive effects on the environment in general.In addition,the research is piloting the service of how to operate business in environment-friendly way.It is also a model for other markets or enterprises all over the country,presenting them how to manage their business to become sustainable and environment friendly.

Analysis of Factors Influencing Development of Resource-saving and Environment-friendly Agriculture in China

By selecting the data from 1989 to 2009 in China, we conduct regression analysis on the total output value of agriculture, the usage amount of chemical fertilizer, total power of agricultural machinery, effective irrigation area, and the damage area. The results show that the usage amount of chemical fertilizer and the damage area, the great obstacle to the construction of resource-saving and environment-friendly agriculture, have significant impact on the output value of agriculture; total power of agricultural machinery and effective irrigation area have insignificant impact on the output value of agriculture. Based on these, the suggestions for promoting the development of resource-saving and environment-friendly agriculture are put forward as follows: transform agricultural production mode, and optimize the industrial structure of agriculture; promote scientific use of chemical fertilizer, and promote the ability of agriculture to resist risks; promote the development of biogas in rural areas, and carry out the comprehensive use of biological material resources in rural areas.

列车活塞风对寒区铁路隧道温度场的影响

研究目的:列车通过隧道会产生活塞风,活塞风会将寒区隧道洞外冷空气带入洞内,导致洞内温度场分布发生变化,若对这种影响认识不到位,会导致洞内抗防冻措施不合理造成冻害发生,影响隧道运营安全。鉴于此,采用理论分析和数值模拟等方法,分析列车活塞风对隧道洞内温度场的影响机制,研究列车活塞风对寒区特长铁路隧道洞内温度场的影响规律,为寒区隧道抗防冻设计提供技术支撑。研究结论:(1)冬季列车通过隧道后,活塞风会引起洞内温度场在短时间内显著变化,列车活塞风对洞内温度场持续影响时间较短,列车通过后,洞内温度场会逐渐恢复,特别是当列车频次较小时,基本可恢复到自然通风条件下状态;(2)行车频次导致洞内纵向负温长度增大或减小率均在1%左右;随着围岩温度的增大,隧道洞内纵向负温长度显著降低,且围岩温度越高,活塞风作用方向对洞内温度场影响越小;(3)考虑活塞风对寒区隧道防寒抗冻影响时,应重点考虑其对两侧水沟抗防冻的影响;由于局部衬砌会出现交替冻融现象,隧道结构设计时,应对衬砌耐久性做一定的考虑;(4)本研究成果可供寒区特长铁路隧道建设参考,特别是单向自然风条件下铁路隧道抗防冻设计。

Design of Multifunctional Milk Powder Spoon Based on Environment-friendly and Developmental Concept

Recycling of junks is positive for the environmental protection.Along with the proposal of three-child policy,purchase volume of milk powder surely will experience a drastic growth,and the milk powder spoon as an affiliated product will win the attention of parents.Against the background of consumption upgrading,consumers have had higher requirements on the humanization and safety design of products.Most of milk powder spoons on the market are plastic,and the disposal of waste milk powder spoon has gradually become a burden for the environment.Based on the environment-friendly and developmental concept,and also the characteristics of infant development,this paper proposed the“in-one”design,applied color and tactile designs into the milk powder design,chose food grade silicone to make the milk powder spoon not only a spoon but also a baby teether toy,so as to expand the service life and functions of the product,meet the requirements of saving energy,protecting environment and developing circular economy,which also to some extent reduces the burden on environment brought by plastics,improves the efficiency of junk recycling,and meet the individualized needs of consumers.