The siderophore-interacting protein is involved in iron acquisition and virulence of Riemerella anatipestifer strain CH3

Riemerella anatipestifer causes epizootic infectious disease in poultry and serious economic losses especially to the duck industry. However, little is known regarding the molecular basis of its pathogenesis. The ability to acquire iron under low-iron conditions is related to the virulence of a variety of bacterial pathogens. In this study, a sip (Riean_1281) deletion mutant CH3Δsip was constructed and characterized for iron-limited growth, biofilm formation, and pathogenicity to ducklings. Results showed that siderophore-interacting protein (SIP) was involved in iron utilization and the sip deletion significantly reduced biofilm formation and adherence to and invasion of Vero cells. In addition, the sip gene was absent in 1 of 24 (4.17%) virulent strains and 2 of 3 (66.7%) avirulent strains of R. anatipestifer, and the sip gene from six R. anatipestifer strains, which belong to serotypes 1, 2, and 10, respectively, shared 100% amino acid identities to those of R. anatipestifer strains DSM15868 and RA-GD. These results suggested that siderophore-mediated iron acquisition may be an important iron-uptake pathway in R. anatipestifer. Animal experiments indicated that the median lethal dose of the CH3Δsip mutant in ducklings was about 35-fold higher than that of the wild-type CH3 strain. Thus, our results demonstrated that R. anatipestifer SIP was involved in iron acquisition and necessary for its optimal virulence.     

大豆脂肪酸脱氢酶GmFAD3C-1基因的克隆及功能分析

研究通过对大豆脂肪酸脱氢酶GmFAD3家族中4个关键酶基因序列进行比对,与对照JN18相比,大豆低亚麻酸突变体MT72中GmFAD3C-1基因在起始密码子后+966bp处存在一个碱基位点的缺失（G→?）,产生移码突变,导致氨基酸序列上产生较大变化（该突变基因命名为gmfad3c-1）。构建GmFAD3C-1基因超表达及CRISPR/Cas9编辑载体,利用花粉管通道法获得T₂转化植株。脂肪酸脱氢酶酶活测定结果显示,超表达植株T₂籽粒中,酶活与对照相比上升47.62%~78.85%,编辑载体T₂籽粒中,酶活与对照相比下降25.67%~47.11%。脂肪酸相对含量测定的结果进一步表明,GmFAD3C-1基因的表达与植株亚麻酸含量密切相关。     

人泛素结合酶UbcH5c活性突变体的构建·纯化以及催化活性研究

[目的]构建人泛素结合酶UbcH5c的活性位点突变体S22R和F62A,同时分析这2种突变体蛋白与野生型蛋白在泛素化反应中的活性差异。[方法]通过点突变(Site-Directed Mutagenesis)的方法构建2种突变体质粒,利用0.5 mmol/L IPTG分别诱导2种突变体蛋白在大肠杆菌中进行表达;将菌体超声破碎后,依次利用阳离子交换层析法对UbcH5c突变蛋白进行分离纯化,最后利用体外泛素化酶促反应结合蛋白免疫印迹杂交的方法分析野生型UbcH5c与其活性突变体UbcH5c S22R和UbcH5c F62A在泛素化修饰上的差异。[结果]人泛素结合酶UbcH5c的2个突变体蛋白S22R、F62A的泛素化修饰程度明显弱于野生型蛋白。[结论]突变体S22R和F62A突变均不同程度的改变了人泛素结合酶UbcH5c与泛素分子之间的结合活性,即2个突变的位点中第62位苯丙氨酸残基及第22位丝氨酸残基均是UbcH5c结合泛素的关键位点,而且后者对于UbcH5c结合泛素活性的影响更大。     

利用烟草表达一种新型鲑鱼降钙素的初步研究

利用烟草表达一种新型鲑鱼降钙素,以期为利用植物系统表达鲑鱼降钙素的研究提供理论参考。首先构建含外源基因“p il-m sCT”的植物双元表达载体p35S-2300-tw in T-DNA s∶p il-m sCT∶NO S-ter,然后通过农杆菌介导法转化烟草,获得卡那霉素抗性烟草128株,其中PCR阳性植株14株(阳性率10.94%);Sou thern b lotting分析表明外源基因已整合到烟草基因组中,拷贝数为2~8个;半定量RT-PCR结果显示,转基因烟草株间“p il-m sCT”转录水平存在较大差异。     

水稻极度分蘖突变体的分离和遗传初步研究

通过EMS诱变处理栽培稻粳灿89(Oryza sative L.indica JX89）种子，从M2代植株中筛选得到一株突变体，其植株形态表现为叶细、色淡、植株矮化和极度分蘖。在将近一年的营养生长过程中，分蘖总数达到2000多个，命名为极度分蘖突变体（Excessive tillering mutant）ext37.ext 37自交所得M3和M4代植株表现同一表型。突变体自交及其与JX89和丰矮占5号（FAZ-5）发杂交F1、F2代结果表明该突变为显性遗传方式，可能涉及两对等位基因。为了观察突变基因对下游基因表达影响的程度，取生长期44天的亲本JX89和突变体ext37的节间分生组织提纯细胞总RNA，进行基因表达谱差异显示分析。结果表明突变体与亲本的基因表达模式有明显的差异，说明可能是处在上游调控基因的突变影响了下游许多基因的表达。目前有关分子生物学的进一步研究正在进行中。     

水稻卫星搭载突变性状考察和品系选育

利用我国返回式卫星搭载水稻品种“特籼占13”种子，回收后经多代的种植选择，选育出性状稳定的13个突变品系进行主要经济性状考察，结果表明：空间诱变突变品系的多个性状都发生了不同程度的变异，其中谷粒长宽比、穗长、穗粒数、小区产量等性状，品系间变异达到显著和极显著水平。讨论了水稻空间诱变性状变异改良的有效性，并从参试空间诱变突变品系中选育出多个性状同时产生突变得到改良的优质高产新品系。     

假俭草体细胞抗寒突变体的获得及其SRAP分子鉴定

假俭草抗寒性差是限制其广泛应用的主要因素之一。本研究目标是以优良假俭草选系E-126为材料,经低温诱导和筛选获得体细胞抗寒突变体。结果表明,假俭草种子诱导的愈伤组织在继代培养的过程中,经0℃的低温条件下培养26 d,获得了2块存活的愈伤组织,该愈伤组织经过继代增殖后,进行分化、生根和移栽,获得株系1和株系2。苗期外部形态观察结果表明,假俭草低温诱导的株系1、株系2和对照在叶色、叶毛、叶长和叶宽上均没有显著性差异。半致死温度分析结果表明,株系1、株系2以及对照叶片半致死温度(LT₅₀)分别为-6.646,-6.546和-5.351℃,处理与对照之间差异显著,且都低于对照,但株系1和株系2之间无显著性差异。SRAP的结果表明,假俭草低温诱导的株系1和株系2在110,230以及240 bp处均存在相同特征带,表明假俭草体细胞突变体植株的变异稳定且在分子水平与对照存在差异,但株系1和株系2无差异。因此,株系1和株系2可作为同一体细胞抗寒突变体株系加以利用。     

苹果矮化砧木致矮机理研究进展

矮化密植栽培是现代苹果生产发展的方向,苹果矮化砧是实现苹果矮化密植栽培的主要途径,其致矮机理尚不清楚。揭示苹果矮化砧致矮机理,对加快苹果矮砧育种进程和改进苹果矮砧栽培技术有重要的意义。在总结前人对致矮基因、解剖结构、水分、物质运输、光合特性、酶与酚类物质、激素等7个方面研究进展的基础上,对致矮部位及致矮机理进行了探讨,提出苹果砧木致矮是矮化砧嫁接口、砧段的木质部及韧皮部综合作用的结果,嫁接的矮砧砧段是致矮的主要部位,苹果矮砧可能通过影响根系发育起到致矮作用,并指出今后应利用更为先进的显微仪器和测量软件对苹果矮砧和乔砧砧段解剖结构进行更微观精确地比较;在严格控制环境的条件下,尽量采取一致的试材,分析不同矮化性砧木在水分、矿质营养、同化物等物质代谢过程中的生理生化变化;借助分子生物学技术对植物激素物质合成、信号转导、降解及其互作在致矮中所起的作用进行更深入系统地研究,通过多方面研究对提出的问题进行研究验证,以求对苹果矮砧致矮机理解释更加透彻。     

保护地栽植矮甜李授粉方法选择试验

日光节能温室内种植的矮甜李选择授粉方法是获得高产的关键。通过人工点授、自然授粉、室内放蜂、液体授粉 4种方法的对比 ,总结出 4种授粉方法的坐果率为人工点授 (3 1 .0 7% ) >室内放蜂 (2 1 % ) >液体授粉 (9.5 3 % ) >自然授粉 (0 .1 6% ) ,从试验结果看出 ,保护地种植矮甜李必须进行辅助授粉 ,否则无产量。从经济、快捷、简便的角度出发 ,以室内放蜂为最佳授粉方法 ,放蜂量为 1 0 0 0头 /棚。     

The mutagenesis of a type IV secretion system locus of Piscirickettsia salmonis leads to the attenuation of the pathogen in Atlantic salmon,Salmo salar

Piscirickettsiosis is a threatening infectious disease for the salmon industry, due to it being responsible for significant economic losses. The control of outbreaks also poses considerable environmental challenges. Despite Piscirickettsia salmonis having been discovered as the aetiological agent of the disease more than 25 years ago, its pathogenicity remains poorly understood. Among virulence factors identified so far, type four secretion systems (T4SS) seem to play a key role during the infection caused by the bacterium. We report here the genetic manipulation of P. salmonis by means of the transference of plasmid DNA in mating assays. An insertion cassette was engineered for targeting the icmB gene, which encodes a putative T4SS‐ATPase and is carried by one of the chromosomal T4SS clusters found within the genome of P. salmonis PM15972A1, a virulent representative of the EM‐90‐like strain. The molecular characterization of the resulting mutant strain demonstrated that the insertion interrupted the target gene. Further in vitro testing of the icmB mutant showed a dramatic drop in infectivity as tested in CHSE‐214 cells, which is in agreement with its attenuated behaviour observed in vivo. Altogether, our results demonstrate that, similar to other facultative intracellular pathogens, P. salmonis’ virulence relies on an intact T4SS.