利用免疫共沉淀技术研究RSV CP、SP和NSvc4三个蛋白的互作情况

为了检测水稻条纹病毒(Rice stripe virus, RSV)CP、SP和NSvc4蛋白自身和两两之间的互作关系,首先通过PCR扩增获得了分别融合有HA或c-Myc标签的RSV CP、SP和NSvc4基因,并将其插入到植物表达载体pEGAD或pKYLX71:35S2中。重组质粒转化农杆菌EHA105后,将含有不同重组质粒的农杆菌菌液混合后注射烟草(Nicotiana benthamiana)叶片。提取烟草中瞬时表达的蛋白,以HA抗体或c-Myc抗体进行免疫共沉淀实验,最后免疫混合物通过c-Myc抗体或HA抗体进行Western blot检测,确定蛋白的互作情况。结果表明,RSV CP蛋白自身能够发生互作,而CP与SP,CP与NSvc4,SP与SP,SP与NSvc4,及NSvc4与NSvc4蛋白之间均未有检测到互作现象。     

利用免疫共沉淀联合质谱技术筛选盐藻MAPK的互作蛋白

为进一步探究杜氏盐藻促有丝分裂原活化蛋白激酶(DsMAPK)的功能,采用免疫共沉淀联合质谱技术筛选盐藻MAPK的互作蛋白。将pGS21a-MAPK质粒转入大肠杆菌BL21中表达MAPK蛋白并制备多克隆抗体;将培养至对数生长期的盐藻细胞进行盐胁迫处理,然后提取盐藻总蛋白,并进行SDS-PAGE和Western blotting检测;以内源性靶蛋白为诱饵,将细胞总蛋白与MAPK抗体进行共孵育,将经蛋白A/G琼脂糖珠纯化的免疫共沉淀复合物进行质谱检测。结果表明,制备的多克隆抗体特异性良好;筛选出165种特有的差异蛋白。通过GO和KEGG分析发现,这些差异蛋白主要参与了新陈代谢、遗传信息的传递以及信号转导等生物学调控过程。蛋白质互作网络分析发现,直接与MAPK相互作用的蛋白有4种。本研究结果为深入研究杜氏盐藻响应盐胁迫的分子机制提供了参考。     

蛋白互作研究方法在水稻中的应用及主要互作数据库汇总

实验设计时选择合适的方法,有利提高科研效率。综述了多种研究蛋白质相互作用的方法,分析了各类方法的优缺点以及在水稻中的研究应用,归纳整理了现有的大型蛋白互作数据库。     

与南方水稻黑条矮缩病毒外壳蛋白P10互作的寄主因子筛选

为找到水稻中与南方水稻黑条矮缩病毒(SRBSDV)外壳蛋白P10(CP)相互作用的寄主蛋白,利用P10筛选水稻膜系统酵母双杂cDNA文库,寻找水稻中与P10互作的蛋白并结合生物信息学技术进行分析,回转验证.结果表明,筛选到10个可能与P10互作的水稻寄主蛋白,如胚发育晚期丰富蛋白Lea14-A、硫氧还原蛋白TRXh1、...     

烟草NtMYB4a基因酵母双杂交诱饵载体的构建及互作蛋白的筛选

为探究NtMYB4a是否与其他蛋白质发生互作,并共同影响烟草次生代谢产物的合成和响应非生物胁迫,通过构建NtMYB4a基因酵母双杂交诱饵载体,从烟草cDNA文库中筛选出与NtMYB4a互作的候选蛋白,并利用实时荧光定量PCR初步验证低温胁迫下候选蛋白和NtMYB4a的共表达关系。结果表明,从cDNA文库中初步筛选出39个与NtMYB4a互作的蛋白,经回交验证排除假阳性,最终得到6个与NtMYB4a具有互作关系的蛋白,分别是RALF-like 22蛋白、锌指A20/AN1结构域蛋白、液泡分选蛋白、天冬氨酸蛋白酶、B2蛋白和应激增强蛋白。在低温胁迫下,6种互作蛋白的基因表达量变化趋势与NtMYB4a相似,均呈升高—降低—升高的趋势。相关性分析发现,液泡分选蛋白、B2蛋白、RALF-like 22蛋白和应激增强蛋白基因的表达与NtMYB4a的表达呈正相关关系,锌指A20/AN1结构域蛋白和天冬氨酸蛋白酶基因的表达与NtMYB4a的表达呈较弱的负相关关系,推测NtMYB4a可能与这些蛋白互作参与烟草低温胁迫的防御调控。本研究结果为进一步探究NtMYB4a参与响应非生物胁迫的分子调控机制提供了依据。     

二穗短柄草BdGI基因表达及其蛋白互作分析

光周期是调控植物开花的主要途径之一,GI在光周期途径中是控制生理节律和开花的关键因子。为探索二穗短柄草（Brachypodiam distachyon）BdGI基因的表达模式和蛋白互作关系,本研究通过实时荧光定量PCR(qRT-PCR)技术分析BdGI基因在不同光照条件下和长日照下不同生长发育阶段的表达情况;运用酵母双杂交初步筛选出互作蛋白BdZTL,并用双分子荧光互补法（BiFC）和免疫共沉淀法（CoImmunoprecipitation）进行验证。qRT-PCR分析结果表明,BdGI基因在不同光照下和长日照下不同生长发育阶段的表达情况不同,且都保持一定的昼夜节律,并且受到光周期的调控;利用酵母双杂交检测到GI与ZTL蛋白存在互作关系,双分子荧光互补与免疫共沉淀检测结果验证了两者互作关系的真实性。综上所述,BdGI的表达受光周期调控,具有昼夜节律性,在光周期诱导二穗短柄草开花的过程中也具备一定的调控作用。本研究结果为GI基因的功能研究奠定了理论基础。     

氮素吸收调控中铵转运蛋白与锚蛋白的互作研究

铵转运蛋白（AMT）介导的高亲和力铵跨膜运输是植物根系吸收铵态氮的主要途径。AMT蛋白水平上的调控能够快速有效地控制根系铵吸收能力,但参与调控的互作蛋白尚未见报道。本研究通过生物信息学手段预测得到铵转运蛋白AtAMT1;3可能和锚蛋白AtAnkTm8存在互作。基因表达分析实验发现AtAMT1;3和AtAnkTm8的根中组织特异性表达模式一致,并同时受到缺氮胁迫的上调表达,结果支持了它们互作的可能性。筛选鉴定出AtAnkTm8缺失突变体,分析在供铵条件下及对甲基铵敏感性的生长表型,结果发现AtAnkTm8的缺失没有影响拟南芥根的铵吸收能力,推测可能存在其他家族成员的功能冗余。AMT与AnkTm的互作为理解植物铵吸收调控过程提供了可能的新颖机制     

水稻条纹病毒与水稻互作中的生长素调控

利用real-time RT-PCR和高效液相色谱技术对水稻条纹病毒（Rice stripe virus, RSV）病株和RSV侵染的水稻悬浮细胞中生长素合成酶基因YUCAA1和内源生长素的相对表达量和含量进行分析结果表明,在细胞水平,RSV侵染能显著引起YUCAA1基因表达上调2.98倍和内源生长素含量升高2.66倍。而在植株水平,RSV侵染后却导致YUCAA1基因表达下调70%和内源生长素含量降低75%。这暗示RSV与寄主互作的不同阶段能够调控寄主植物生长素的信号传导。同时,利用KPSC缓冲液处理来消除病株内源生长素,能够引起RSV CP基因表达上调近3倍,30 μM IAA溶液处理可使病株内RSV CP基因表达下调45％。由此可见水稻体内生长素含量的变化能够影响RSV在寄主体内的复制。     

水稻赖氨酸和总黄酮含量的QTL定位及环境互作分析

为挖掘多环境下稳定存在的水稻赖氨酸和总黄酮含量相关QTL,以粳稻东农425和长白10号及其衍生的180个株系的F_(6:7)重组自交系(RIL)作为供试群体,采用完备区间作图法(ICIM)和基于混合线性模型的复合区间作图法(MCIM),对2014年和2015年水稻的赖氨酸含量和总黄酮含量进行加性QTL定位及环境互作分析。结果检测到10个影响赖氨酸含量的加性效应QTL和12个影响黄酮含量的加性效应QTL,分布在除第9、第10和第12染色体以外的9条染色体上,其中在第5染色体的RM538~RM1271标记区间内连续2年检测到总黄酮含量QTL。检测到6个存在环境互作效应的赖氨酸含量QTL、4个存在环境互作效应的总黄酮含量QTL,互作贡献率为0.15%~6.73%;一对影响总黄酮含量的上位互作效应的QTL,贡献率为0.99%。本研究结果为水稻赖氨酸和总黄酮含量QTL分子标记辅助育种提供了一定的理论依据。     

水稻汕优63重组自交系重要农艺性状的QTLs和互作分析

利用水稻（Oryza sativaL.spp.indica)汕优63重组自交系群体241个株系，进行了株高，抽穗期，产量和产量构成因子等9个重要农艺性状分析，定位了这些数量性状的基因位点（QTLs）。结果表明，这些性状在重组自交系群体中均存在双向超亲分离，其分布接近正态分布，共检测到45个主效QTLs和47对互作QTL位点影响上述9个性状，贡献率为2．83％－28．46％，第7染色体C1023－R1440区间为最适跃区段，同时检测到5个性状的主效QTL与3个性状的互作有关，为典型的一因多效现象，为分析水稻杂种优势和分子标记辅助选择提供了理论基础。     

利用ISSR分子标记研究栽培稻和野生稻亲缘关系

本研究利用ISSR分子标记研究了37份栽培稻和野生稻的亲缘关系。结果表明，18个ISSR引物共扩增133个多态性条带，每个引物平均扩增多态性条带7.4个，扩增产物片段大小在0.1-3.0kb之间。而AA荆垢普通野生稻和栽培稻遗传关系密切，而与非AA型野生稻有一定亲缘关系。聚类分析将37份栽培稻和野生稻明显分为5组，第1组为AA类型的普通野生稻和栽培稻，第2组为疣粒野生稻，第3、4组分别为CC，CCDD染色体组野生稻，第5组为O.Rhychoryza。栽培稻与野生稻的亲缘关系体现了地妥由普通野生稻分化而来，而普通野生稻是从南向北逐渐分化的。本研究还讨论了栽培稻与普通野生稻的进货关系。     

利用膜分离技术和酶技术生产低乳糖牛奶的研究

提出了一种生产低乳糖牛奶的新工艺，即借助膜分离这一新型工程技术手段，对牛奶先进行超滤／稀释超滤处理，透过液经β－半乳糖苷酶水解，再与保留液混合、标准化、均质。从而解决了传统工艺中由于牛奶组分复杂，易引起β－半乳糖苷酶污染、失活的问题。     

我国大豆胞囊线虫主要生理小种的可溶性蛋白电泳比较初步研究

大豆胞囊线虫(Heteroderaglycines)是能造成大豆开花期死苗毁种的病害,按其危害性和土传难治的特点,最好的防治途径是选用抗病品种并施行交互轮作,但其易发生生理分化产生新小种,使抗病品种失抗,所以必须及时监测其生理小种动态,传统的方法是依据其在国际统一鉴别品种上的致病力差异来区分的.但其所需病土和胞囊量大,鉴定周期长,栽培管理费工,且易受环境条件影响,调查结果也难以掌握完全准确.     

Biochemical Studies of Proteins in Nondeveloped,Partially Developed,and Developed Doughs

Nondeveloped, partially developed with shear and extensional deformations, and developed doughs represent different stages of dough development. To understand the relationship between gluten proteins and dough rheology, this study used disulfide‐sulfhydryl analyses, gel filtration chromatography, SDS‐PAGE, acid polyacrylamide gel electrophoresis (A‐PAGE), and densitometry to examine proteins in the four types of doughs mentioned. Free sulfhydryl content was the lowest in native flour and nondeveloped dough, and the highest in partially developed doughs, while a reverse trend was observed for disulfide content. For each flour sample, the protein elution profile from gel filtration chromatography shifted with the level of dough development. With respect to the smallest sized molecules, native flour had the most, followed by nondeveloped, partially developed, and then developed doughs. SDS‐PAGE and A‐PAGE exhibited similar protein patterns among the same chromatographed protein fractions of each native flour and its different doughs. Densitometric data showed that the amount of high molecular weight (HMW) glutenins increased and the amounts of low molecular weight (LMW) glutenins, gliadins, and albumins/globulins decreased with progressive stages of dough development. In conjunction with previously published results, indications are that the increase in the size and the amount of HMW glutenins is related to the strength of dough and the amount of protein matrix present in the dough.     

Interactions between the fungicide benomyl and soil microorganisms

Decomposition of benomyl and carbendazim was studied in field experiments following repeated applications during autumn to winter cereals. Effects of the fungicides on straw decomposition, balance of straw fungal flora and mineralization of nitrogen in the soils were investigated in field and in laboratory experiments. Persistence in the field of the fungicides at doses of 0.1–0.2 kg ha^?1 was 9–12 months in clay soils and 12 months or longer in sand soil. Decomposition of straw in the field was not affected in clay soils by doses up to 2 kg ha^?1. In sand soil, doses up to 0.5 kg ha^?1 gave no effect but in one case at 2 kg ha^?1 the initial stages of straw decomposition were slightly inhibited. All doses tested in both clay and sand soils caused changes in composition of the straw fungal flora. In a laboratory experiment with benomyl in sand soil an increase in rate of nitrate accumulation was observed at a dose corresponding to 2 kg ha^?1.     

Interactions between flavonoids and proteins: effect on the total antioxidant capacity

Flavonoids are potent antioxidants. It is also known that flavonoids bind to proteins. The effect of the interaction between tea flavonoids and proteins on the antioxidant capacity was examined. Their separate and combined antioxidant capacities were measured with the Trolox equivalent antioxidant capacity (TEAC) assay. It was observed that the antioxidant capacity of several components of green and black tea with alpha-, beta-, and kappa-casein or albumin is not additive; that is, a part of the total antioxidant capacity is masked by the interaction. This masking depends on both the protein and the flavonoid used. Components in green and black tea, which show the highest masking in combination with beta-casein, are epigallocatechin gallate and gallic acid. The results demonstrate that the matrix influences the efficacy of an antioxidant.     

Interactions between microclimatic variables and the soil microbial biomass

Summary Soil moisture, temperature, microbial substrate-induced respiration and basal respiration were monitored in two plots in an agricultural field from April 30 to September 25, 1987, and in a further two plots from May 26 to August 27, 1988. An attempt to relate biological variables to microclimatic variables was made through the use of correlation analysis. The microbial substrate-induced and basal respiration were both strongly positively correlated with the soil moisture content, and to a lesser extent positively related to soil temperature, especially when partial correlation was used to control for variation in soil moisture. Short-term changes in substrate-induced and basal respiration were correlated with changes in soil moisture but were largely independent of soil temperature. The ratio of basal to substrate-induced respiration (indicating the respiration: biomass ratio and therefore ecosystem stability or persistence) was negatively associated with the soil moisture content and in some instances with soil temperature when partial correlation analysis (correcting for soil moisture variation) was used. This suggests that the climatic conditions which contributed to the lowest ecosystem stability were low temperature, low moisture conditions.     

Interactions between earthworms, litter and trees in an old-growth beech forest

 Forty plots were selected in an old-growth beech forest (Biological Reserve of La Tillaie, Fontainebleau State Forest, France), to embrace the whole range of site conditions and phases of vegetation dynamics. Soils are sandy, thus the nutrient status of the topsoil is very poor except when trees have access to an underlying limestone layer. The study was focused on the role of calcium in the sustainability of the beech ecosystem. Calcium is mostly redistributed through leaf litter accretion and the activity of litter-consuming organisms, but other sources are fallen wood and uprooted mounds. In each of the 40 plots, earthworm species were sampled, and measurements were taken in order to describe humus profiles, growth of adult trees, litter quantity and quality, and access to lime. Densities of soil-dwelling earthworms, calcium content of beech leaf litter, height of tallest trees and depth of the limestone layer were correlated, indicating a gradient of soil fertility which mainly results from long-term interactions between soil organisms and trees in varying geological conditions. Possible causal relationships and implications of calcium turnover for nature conservation were discussed in the light of existing knowledge. Received: 6 July 1998     

Interactions between microbial community structure and the soil environment found on golf courses

Approximately 0.6% of the total UK land surface is occupied by golf courses, but little investigation into the biological properties of the soil under this type of amenity turf has been reported. The soil microbiota has a significant role within the majority of nutrient cycles. In order to analyse how golf course management affects the soil microbial community, an investigation of the phenotypic microbial community structure using phospholipid fatty acid (PLFA) analysis was carried out. Principal component analysis of PLFA biomarkers indicated that there were consistent relationships between the tees, fairways and greens and the soil microbial community structure. No conclusive mechanism could be demonstrated in one-way analysis with corresponding physical parameters (P>0.05 in all cases). Cannonical correlation analysis (CCA) using 28 PLFA biomarkers concurrently with 9 physicochemical parameters showed a highly significant relationship on different playing surfaces at all of the golf courses surveyed (P<0.01). The construction and maintenance of specific areas of a golf course, irrespective of geographical location, closely reflect the physicochemical status of the soil microbial habitat.