基于Bt毒素的杀虫蛋白理性设计与创新应用策略

Bt毒素是源于苏云金芽孢杆菌(Bacillusthuringiensis)的具有特殊杀虫功能的大分子蛋白,其制剂和转基因作物已广泛用于害虫防治,产生了巨大的经济和社会生态效益。围绕Bt毒素挖掘和提升其应用价值是持续研究的热点,特别是随着Bt毒素结构功能和作用机制日趋明晰,为其功能修饰和创新应用创造了条件,相关研究蓬勃发展,成效显著。大量研究表明,采用定点突变、结构域替换或融合以及抗独特型抗体模拟等策略,是理性设计活性更高、稳定性更强、杀虫谱更广、非靶标生物安全性更高甚至是可用于害虫抗药性治理的有别于母体Bt毒素的突变体、结构杂合体乃至功能效应物抗体等新型杀虫蛋白的有效手段;此外,采用催化毒素活化、驱动毒素靶向受体结合、促进毒素表达以及同源或异源杀虫材料复配或共表达的协同促效等创新增效策略,也是助推Bt毒素应用价值的重要手段。本文总结了Bt毒素结构功能和作用机制,梳理了基于Bt毒素功能修饰的突变体、结构杂合体以及功能效应物抗体等新型杀虫蛋白理性设计和基于Bt毒素功能增效的创新应用策略等相关研究进展,并结合作者团队在模拟Bt毒素杀虫功能效应物抗体靶向设计研发方面的最新成果,探讨了基于Bt毒素的杀虫蛋白理性设计与创新应用策略未来发展动向及潜在可行捷径,为相关研究提供较为全面的最新有价值的文献资料和启发思路。

土壤真菌群落和潜在功能对施加外源Bt毒素的响应

转Bt(Bacillus thuringiensis)基因植物和Bt重组菌生物农药释放的Bt毒素是一类具有生物毒性的潜在环境外源污染物,Bt毒素环境行为和生态效应是转基因植物和植物用转基因微生物安全风险评价的重要内容,但是外源Bt毒素对土壤真菌群落和潜在功能的影响还不清楚。以施加不同浓度Bt毒素处理土壤和未施加Bt毒素对照土壤为研究对象,分析Bt毒素在土壤中的持留动态;同时采用真菌18S rRNA基因高通量测序技术,分析施加Bt毒素对土壤真菌群落和功能多样性的影响。结果表明,Bt毒素施加量和培养时间均可以显著影响土壤真菌群落组成,且随着Bt毒素施加量增加和土壤培养时间延长,土壤真菌群落差异性逐渐变大。施加Bt毒素提高了土壤真菌群落香农指数和关联网络的负相关性比例及模块数,因而没有对土壤真菌群落的多样性和稳定性产生负面影响。上述结果表明,评估Bt毒素的环境行为及微生态效应要关注Bt毒素施加量及其长期影响。随着Bt毒素施加量增加,Phymatotrichopsis、Homalogastra、Geosmithia和Apiotrichum等真菌以及参与蛋白质降解、碳素代谢和磷素代谢的功能基因编码酶相对丰度显著升高,推测上述真菌物种和潜在功能参与了Bt毒素在土壤中的降解和转化过程。研究结果为转Bt基因植物、Bt重组菌生物农药以及Bt毒素的生态安全风险评价提供了科学参考和理论依据。

BT135VRE页岩气开发专用套管性能研究

与常规油气开采相比,页岩气开采多为大斜度和水平井,完井射孔多达20段以上,同时套管在井下还要受到反复的拉、压、挤等交变载荷,下井深、井下温度高,工况条件苛刻。针对以上问题,设计并开发了BT135VRE高钢级页岩气开采专用套管,同时对其理化性能、组织、热稳定性、常温抗挤毁性能、高温抗挤毁性能进行了研究。结果表明,在900℃淬火+670℃回火的热处理工艺下,BT135VRE屈服强度≥965 MPa,0℃横向冲击吸收功≥100 J,高强度下匹配高的韧性,同时材料在高温下还具有良好的热稳定性和高温抗挤毁性能,满足复杂工况条件下页岩气开采需求。

低温胁迫对Bt棉纤维中杀虫蛋白含量及氮代谢的影响

本研究以常规种泗抗1号(Sikang 1,SK1)和杂交种泗抗3号(Sikang 3,SK3)为材料进行盆栽试验,研究了不同低温水平及其处理持续时间对Bt棉盛铃期纤维中杀虫蛋白含量变化及氮代谢生理特征。结果表明,纤维中的杀虫蛋白含量随着温度的降低总体呈下降趋势,同时低温处理持续期显著影响杀虫蛋白含量。与对照相比,纤维中杀虫蛋白含量的降低幅度随低温胁迫时间的延长而增大。此外,随着处理温度的降低,可溶性蛋白含量、谷丙转氨酶活性、谷草转氨酶活性呈下降趋势,游离氨基酸含量、肽酶活性、蛋白酶活性呈上升趋势,且在低温处理48 h后,均与杀虫蛋白含量呈极显著相关。因此,低温胁迫促使了蛋白质的合成功能下降,分解能力增强,导致可溶性蛋白含量下降,游离氨基酸含量升高,最终导致杀虫蛋白含量下降,且其受低温胁迫持续期显著影响。

BT@PANI核壳粒子的绿色制备及PVDF基复合材料的介电性能

聚合物薄膜介电电容器在新能源汽车、太阳能和风力并网发电与储能等领域具有广泛应用和广阔的发展前景。钛酸钡/聚合物复合材料具有介电损耗低、击穿场强高的特点,是电容器中核心部分高介电有机薄膜材料极具潜力的选择之一。目前,迫切需要解决高极化特性与低损耗难以协同改善的问题。本工作提出以苹果酸同时作为钛酸钡(BT)表面修饰剂和聚苯胺(PANI)掺杂剂,采用原位聚合法制备了BT@PANI核壳粒子,并以此为填料制备了聚偏氟乙烯(PVDF)基复合材料。结果显示,通过苹果酸与盐酸的两步表面改性法,可获得钛酸钡-苯胺阳离子(BT-An^(+))粒子,为此不同苯胺(An)与BT-An^(+)质量比条件下填料产物的核壳结构特征明显。当An与BT-An^(+)的质量比为0.5∶1时产物平均粒径最小,约为450 nm,粒径分布最窄,且电导率为1.46×10^(-3)S/cm。以此为填料,30%(质量分数)填充量的PVDF基复合材料在10^(3)~10^(6)Hz范围内介电常数保持在高值水平,由31仅下降到15,介电损耗在宽的频率范围内低于0.3,不仅表现出优良的频率稳定性,而且在获得较高的介电常数情况下,实现了介电损耗的有效抑制。此外,这项工作极大地降低了无机氧化性酸与有机试剂的使用率,为实现高性能介电复合材料的绿色制备提供了实验指导。

折叠结构的PVDF/BTO复合薄膜压电纳米发电机的制备及性能研究

为提高聚偏氟乙烯(PVDF)的压电输出性能,将钛酸钡(BTO)纳米颗粒引入到PVDF溶液中,使用流延法制备PVDF/BTO复合压电薄膜,采用高温拉伸与电极化工艺处理复合薄膜。使用扫描电子显微镜、X射线衍射仪、傅里叶变换红外光谱仪、差示扫描量热仪和准静态压电常数测试仪对复合薄膜的形貌和压电性能进行表征,探究BTO含量对PVDF/BTO复合薄膜的影响,研究BTO诱导PVDF链条偶极子重新排列形成β晶型的机理。结果表明,当BTO含量为10%(质量分数)时,PVDF/BTO复合薄膜的晶体结构中β晶型的含量最多,压电性能最强。最后设计了不同层数的折叠结构并组装制成压电纳米发电机(PENG),发现折叠层数越多,PENG的最大输出电压越高。

昆虫对杀虫剂和转Bt基因植物的抗性进化机制研究进展

为防治昆虫对农作物的危害,采取了喷施杀虫剂和种植转Bt基因抗虫植物等措施。然而,杀虫剂的大规模使用和转Bt基因抗虫植物的大面积连续种植使得一些靶标昆虫产生了抗性进化,这不仅影响防治效果,而且还影响整个农业生态系统服务功能。本文综述了昆虫对化学杀虫剂、微生物杀虫剂和转Bt基因植物产生抗性的分子机制。昆虫对化学杀虫剂的抗性进化机制主要是靶标位点敏感性下降和解毒酶系活性增强;对微生物杀虫剂的抗性进化机制主要是免疫系统激活和共生菌群变化;对转Bt基因植物的抗性进化机制主要是昆虫中肠结合受体基因突变或表达下调和中肠蛋白酶活性降低。为了减缓昆虫抗性进化和提高杀虫剂效率,未来建议减少使用化学杀虫剂,合理利用杀虫谱广和活性高的微生物杀虫剂以及抗虫植物等方法系统治理农业害虫。

A comparison study on structure-function relationship of polysaccharides obtained from sea buckthorn berries using different methods:antioxidant and bile acid-binding capacity

In this study,the structural characters,antioxidant activities and bile acid-binding ability of sea buckthorn polysaccharides(HRPs)obtained by the commonly used hot water(HRP-W),pressurized hot water(HRP-H),ultrasonic(HRP-U),acid(HRP-C)and alkali(HRP-A)assisted extraction methods were investigated.The results demonstrated that extraction methods had significant effects on extraction yield,monosaccharide composition,molecular weight,particle size,triple-helical structure,and surface morphology of HRPs except for the major linkage bands.Thermogravimetric analysis showed that HRP-U with filamentous reticular microstructure exhibited better thermal stability.The HRP-A with the lowest molecular weight and highest arabinose content possessed the best antioxidant activities.Moreover,the rheological analysis indicated that HRPs with higher galacturonic acid content and molecular weight showed higher viscosity and stronger crosslinking network(HRP-C,HRP-W and HRP-U),which exhibited stronger bile acid binding capacity.The present findings provide scientific evidence in the preparation technology of sea buckthorn polysaccharides with good antioxidant and bile acid binding capacity which are related to the structure affected by the extraction methods.

TiVNbTa高熵合金球形粉末制备及选区激光熔化成形

利用氢化脱氢、机械球磨、等离子体球化技术成功制备了TiVNbTa难熔高熵合金球形粉末,并采用选区激光熔化(SLM)技术将此球形粉末制备成TiVNbTa难熔高熵合金块体试样,研究不同SLM工艺参数对合金显微组织和力学性能的影响。结果表明:所制备的粉末球形度高、分散良好,平均粒度为48μm;SLM成形的TiVNbTa难熔高熵合金具有超细晶组织,其熔池心部为胞状晶区,熔池边缘为柱状晶区。随着激光功率的增加,合金的压缩屈服强度整体变化不大,但塑性应变呈下降趋势;然而,随着扫描速率的增加,合金的压缩屈服强度和塑性应变持续下降。当激光功率为175 W、扫描速率为400 mm/s时,沉积态合金具有最低的孔隙率,XOY面和YOZ面的孔隙率分别降低至0.26%和1.42%,压缩屈服强度σ_(0.2)为1001 MPa,最大抗压强度σ_(bc)为1741 MPa,塑性应变ε_(p)为10.0%。

深入了解目的基因(Bt基因)的检测与鉴定

目的基因的检测与鉴定是基因工程基本操作程序的最后一步,用以确定目的基因是否真正在受体细胞中稳定表达其遗传特性。本文结合实际教学案例,介绍目的基因的检测和鉴定方法,为理解基因工程提供一定的参考。

Inhibition of protein degradation increases the Bt protein concentration in Bt cotton

Bacillus thuringiensis(Bt)cotton production is challenged by two main problems,i.e.,the low concentration of Bt protein at the boll setting stage and the lowest insect resistance in bolls among all the cotton plant’s organs.Therefore,increasing the Bt protein concentration at the boll stage,especially in bolls,has become the main goal for increasing insect resistance in cotton.In this study,two protein degradation inhibitors(ethylene diamine tetra acetic acid(EDTA)and leupeptin)were sprayed on the bolls,subtending leaves,and whole cotton plants at the peak flowering stage of two Bt cultivars(medium maturation Sikang 1(SK1))and early maturation Zhongmian 425(ZM425)in 2019 and 2020.The Bt protein content and protein degradation metabolism were assessed.The results showed that the Bt protein concentrations were enhanced by 21.3 to 38.8%and 25.0 to 38.6%in the treated bolls of SK1 and ZM425 respectively,while they were decreased in the subtending leaves of these treated bolls.In the treated leaves,the Bt protein concentrations increased by 7.6 to 23.5%and 11.2 to 14.9%in SK1 and ZM425,respectively.The combined application of EDTA and leupeptin to the whole cotton plant increased the Bt protein concentrations in both bolls and subtending leaves.The Bt protein concentrations in bolls were higher,increasing by 22.5 to 31.0%and 19.6 to 32.5%for SK1 and ZM425,respectively.The organs treated with EDTA or/and leupeptin showed reduced free amino acid contents,protease and peptidase activities and significant enhancements in soluble protein contents.These results indicated that inhibiting protein degradation could improve the protein content,thus increasing the Bt protein concentrations in the bolls or/and leaves of cotton plants.Therefore,the increase in the Bt protein concentration without yield reduction suggested that these two protein degradation inhibitors may be applicable for improving insect resistance in cotton production.

The Economics of Competing Water Uses under a FERC Licensing Agreement: Estimation of Property Value, Recreation, and Hydroelectric Impacts

Reservoirs provide a variety of services with economic values across multiple sectors. As demands for reservoir services continue to grow and precipitation patterns evolve, it becomes ever more important to consider the integrated suite of values and tradeoffs that attend changes in water uses and availability. Section 316 (b) of the Clean Water Act requires that owners of certain water cooled power plants evaluate technologies and operational measures that can reduce their impacts to aquatic organisms. The studies must discuss the social costs and benefits of alternative technologies including cooling towers (79 Fed. Reg. 158, 48300 - 48439). Cooling towers achieve their effect through evaporation. This manuscript estimates the property value, recreation, and hydroelectric generation impacts that could result from the evaporative water loss associated with installing cooling towers at the McGuire Nuclear Generating Station (McGuire) located on Lake Norman, North Carolina. Although this study specifically evaluates the effects of evaporative water loss from cooling towers, its methods are applicable to estimating the economic benefits and costs of a new water user or reduced water input in any complex reservoir system that supports steam electric generation, hydroelectric generation, residential properties, recreation, irrigation, and municipal water use.

牛种布鲁氏菌A19ΔBtpA缺失株生物学特性及其免疫原性研究

布鲁氏菌四型分泌系统(T4SS)效应物BtpA是布鲁氏菌的重要免疫抑制分子之一。为了进一步了解BtpA对牛种布鲁氏菌生物学特性的影响,并探究BtpA基因缺失是否会影响布鲁氏菌的免疫原性。在牛种布鲁氏菌疫苗株(A19株)上对BtpA基因进行了缺失,通过qRT-PCR检测布鲁氏菌A19ΔBtpA缺失株中的BtpA mRNA表达水平,并探究布鲁氏菌A19株和A19ΔBtpA缺失株在生长曲线、胞内生存、黏附侵袭和体外应激方面的差异。此外,将A19株和A19ΔBtpA缺失株免疫小鼠,在免疫后的第7、14、21、28和35天使用间接ELISA检测小鼠体内的布鲁氏菌特异性抗体水平;通过ELISpot检测免疫第21天时小鼠脾淋巴细胞中IFN-γ的表达水平,利用流式细胞术测定了小鼠脾淋巴细胞中IFN-γ阳性CD4^(+)、CD8^(+)T淋巴细胞以及CD4^(+)、CD8^(+)T淋巴细胞的分类情况。结果表明:成功构建了A19ΔBtpA缺失株,A19ΔBtpA缺失株中BtpA的转录水平显著低于A19株,A19ΔBtpA缺失株与亲本株在生长曲线、胞内生存和黏附侵袭方面呈现相似的趋势。而体外应激试验显示,高渗应激条件下A19ΔBtpA缺失株的存活菌量明显低于A19株(P<0.05)。免疫原性研究中,与PBS组相比,A19组和A19ΔBtpA组均可极显著诱导小鼠产生布鲁氏菌特异性抗体(P<0.01);与A19组相比,A19ΔBtpA组淋巴细胞IFN-γ表达水平显著升高(P<0.05),使小鼠产生的IFN-γ阳性CD4^(+)、CD8^(+)T淋巴细胞以及CD4^(+)/CD8^(+)T细胞比值显著增高(P<0.05)。BtpA基因缺失不会影响布鲁氏菌A19体外胞内增殖,但可能参与布鲁氏菌抗高渗环境能力相关。此外,A19ΔBtpA缺失株比A19株更好地诱导Th1型免疫应答,诱导宿主产生与A19株相当的IgG特异性抗体,具有布鲁氏菌基因缺失疫苗的潜力。该研究将为进一步探究布鲁氏菌的致病机制和开发基因缺失株疫苗提供理论基础和数据支持。

Glass-Ceramics with High Strength and High Transmittance Obtained by Multi-Factor Controlling

The presence of Li_(2)Si_(2)O_(5) and LiAlSi_(4)O_(10) could effectively improve the elastic modulus and transmittance of lithium disilicate(LD)glass-ceramics.Through synergistically modulation of the crystal content and grain size,we obtained high strength and high transmittance of LD glass-ceramics.The optimal sample had a high transmittance of 90.3%,the hardness was 7.72 GPa,the fracture toughness was 1.07 MPa·m^(1/2),and the elastic modulus was 103.1 GPa.

Animal Safety Test of Bacillus thuringiensis BT Protein

[Objectives]To determine the biological safety of BT protein from Bacillus thuringiensis(Bt)fermentation broth to mammals at high doses.[Methods]Healthy mice were randomly divided into 4 groups with 10 mice in each group.The experimental groups were fed with Bt fermentation supernatant at 0.2,0.6 and 1.0 mL/kg,respectively,once a day for 7 consecutive days.The blank control group was fed normally without intragastric administration.[Results]There was no significant difference in blood routine and blood biochemical analysis between the experimental group and the control group.After intragastric administration,the mice were dissected,and no obvious pathological changes were found;the heart,liver,spleen,lung and kidney were taken to make tissue sections,and no pathological changes were found by microscopic observation.[Conclusions]Mice can tolerate high doses of BT protein from B.thuringiensis fermentation broth.

Mechanical Property and Microstructure of Cement Mortar with Carbonated Recycled Powder

Carbonated recycled powder as cementitious auxiliary material can reduce carbon emissions and realize high-quality recycling of recycled concrete.In this paper,microscopic property of recycled powder with three carbonation methods was tested through XRD and SEM,the mechanical property and microstructure of recycled powder mortar with three replacement rates were studied by ISO method and SEM,and the strengthening mechanism was analyzed.The results showed that the mechanical property of recycled powder mortar decreased with the increasing of replacement rate.It is suggested that the replacement rate of recycled powder should not exceed 20%.The strength index and activity index of carbonated recycled powder mortar were improved,in which the flexural strength was increased by 27.85%and compressive strength was increased by 20%at the maximum.Recycled powder can be quickly and completely carbonated,and the improvement effect of CH pre-soaking carbonation was the best.The activity index of carbonated recycled powder can meet the requirements of Grade II technical standard for recycled powder.Microscopic results revealed the activation mechanism of carbonated recycled powder such as surplus calcium source effect,alkaline polycondensation effect and carbonation enhancement effect.

Effect of Cryogenic Treatment on Microstructure and Tribological Property Evolution of Electron Beam Melted Ti6Al4V

Cryogenic treatment was used to improve the tribological properties of Ti6Al4V artificial hip joint implants.Cryogenic treatment at-196℃with different holding time were carried out on Ti6Al4V specimens fabricated using electron beam melting(EBM),and their microstructure and tribological properties evolution were systematically analyzed by scanning electron microscopy(SEM),vickers hardness,and wear tests.The experimental results show that the as-fabricated specimen consists of lamellarαphase andβcolumnar crystal.While,the thickness of lamellarαphase decreased after cryogenic treatment.In addition,it can be found that the fineαphase was precipitated and dispersed between the lamellarαphase with the holding time increase.Vickers hardness shows a trend of first increasing and then decreasing.The wear rate of the specimen cryogenic treated for 24 h is the minimum and the average friction coefficient is 0.50,which is reduced by 14.61%compared with the as-fabricated.The wear mechanism of the as-fabricated specimen is severe exfoliation,adhesive,abrasive,and slight fatigue wear.However,the specimen cryogenic treated for 24 h shows slight adhesive and abrasive wear.It can be concluded that it is feasibility of utilizing cryogenic treatment to reduce the wear of EBMed Ti6Al4V.

Capillary Property of Entangled Porous Metallic Wire materials and Its Application in Fluid Buffers:Theoretical Analysis and Experimental Study

Strong impact does serious harm to the military industries so it is necessary to choose reasonable cushioning material and design effective buffers to prevent the impact of equipment.Based on the capillary property entangled porous metallic wire materials(EPMWM),this paper designed a composite buffer which uses EPMWM and viscous fluid as cushioning materials under the low-speed impact of the recoil force device of weapon equipment(such as artillery,mortar,etc.).Combined with the capillary model,porosity,hydraulic diameter,maximum pore diameter and pore distribution were used to characterize the pore structure characteristics of EPMWM.The calculation model of the damping force of the composite buffer was established.The low-speed impact test of the composite buffer was conducted.The parameters of the buffer under low-speed impact were identified according to the model,and the nonlinear model of damping force was obtained.The test results show that the composite buffer with EPMWM and viscous fluid can absorb the impact energy from the recoil movement effectively,and provide a new method for the buffer design of weapon equipment(such as artillery,mortar,etc.).

Fluid property identification of the Lower Cretaceous reservoirs with complex oil-water contacts in Deseo Basin,Chad

Recently,exploration breakthroughs have been made in the Lower Cretaceous sandstone reservoirs in the Doseo Basin,but the identification of reservoir fluid property is difficult due to variable reservoir lithology,complex oil-water contact within and faint responses of the oil zone,which causes the lower accuracy of reservoir fluid property identification with conventional mudlogging and wirelogging techniques.Applying the geochemical logging,fluorescent logging,mud logging and cutting logging technology,in combination with formation test data,this paper distinguishes the crude oil types,analyzes the logging response characteristics of oil zone after water washing,and establishes the interpretation charts and parameter standards for reservoir fluid properties.The crude oil can be divided into two types,namely viscous-heavy and thin-light,based on total hydrocarbon content and component concentration tested by mud logging,features of pyrolysis gas chromatogram and fluorescence spectroscopy.The general characteristics of oil layers experienced water washing include the decrease of total hydrocarbon content and component concentration from mud logging,the decrease of S1 and PS values from geochemical logging,the decrease of hydrocarbon abundance and absence of some light components in pyrolysis gas chromatogram,and the decrease of fluorescence area and intensity from fluorescence logging.According to crude oil types,the cross plots of S1 versus peak-baseline ratio,and the cross plots of rock wettability versus fluorescence area ratio are drawn and used to interpret reservoir fluid property.Meanwhile,the standards of reservoir fluid parameter are established combining with the parameters of PS and the parameters in above charts,and comprehensive multiparameter correlation in both vertical and horizontal ways is also performed to interpret reservoir fluid property.The application in the Doseo Basin achieved great success,improving interpretation ability of fluid property in the reservoir with complex oil-water contact,and also provided technical reference for the efficient exploration and development of similar reservoirs.

Preparation and property of seal coating for gas turbine compressor

Double-layer structure of seal coating which consisted of a Ni5Al bond coating and a Ni25 graphite top coating were prepared on steel substrate of gas turbine compressor cylinder block.Bond coating was prepared by atmospheric plasma spraying and top coating was prepared by flame spraying.The microstructure,mechanical properties and abradability of the coating were characterized by scanning elec-tron microscope(SEM),hardness tester,universal testing machine,thermal shock testing machine and abradability testing machine.The res-ults show that the overall spraying structure of the seal coating is uniform,the nickel metal phase is the skeleton supporting the entire coat-ing,and the coating is well bonded without separation.The seal coating has a bonding strength of not less than 7.7 MPa,excellent thermal stability,and thermal shock resistance cycle numbers at 500℃more than 50;the scratch length,deepest invasion depth and wear amount of the coating increase with rise of test temperature,with almost no coating adhesion,indicating that the seal coating has excellent abradability.