胡椒4-香豆酸：辅酶A连接酶Pn4CL基因的克隆及表达分析

根据胡椒4-香豆酸：辅酶A连接酶（4-coumarate:coenzyme A ligase, 4CL）基因的部分序列设计引物,运用RACE方法获得其家族成员的1个全长cDNA,命名为Pn4cl,长度2130 bp,开放阅读框1638 bp,编码545个氨基酸。预测Pn4CL分子量为59.57 kDa,理论等电点为5.70。该基因含有AMP-binding（AMP-binding enzyme）、CaiC[Acyl-CoA synthetase (AMP-forming) /AMP-acid ligaseⅡ]、PLN02246、AFD-class I等结合域,具有植物4CL所共有的保守结构域。系统进化分析表明,Pn4CL与北细辛的同源性最高,同时与木兰分支类植物的4CL聚类在一起,与菊分支的进化距离较近,与蔷薇分支的进化距离较远。亚细胞定位表明,该蛋白定位在细胞膜上。Real-time RT-PCR结果表明,该基因受外援激素SA和MeJA诱导表达,同时接种辣椒疫霉菌后,Pn4CL基因的表达量在抗/感2种胡椒中均出现先增加后减少的现象,并且在抗病种质中表达量较高。研究结果为Pn4CL的功能研究提供了理论依据。     

泛素/26S蛋白酶体途径调节作物种子大小的研究进展

泛素/26S蛋白酶体系统（ubiquitin/26S proteasome system, UPS）是蛋白质泛素化的重要途径,作为蛋白质翻译后重要的修饰系统,在作物生长发育的各个阶段发挥重要作用。大量研究表明,UPS中泛素受体蛋白、E3泛素连接酶、去泛素化酶等能相互协调,通过在目标蛋白上连接或去除不同数量的泛素来介导目标蛋白的降解、改变亚细胞定位、蛋白活性等,从而调控种子的大小。对不同作物中泛素/26S蛋白酶体系统调节种子大小方面的研究进展进行了综述,并对其未来的发展进行了展望,为研究种子大小的调控机制及育种改良提供参考。     

鱼类特有的Ring型E3泛素连接酶MARCH5A基因在刺激隐核虫感染下的表达分析

为探究大黄鱼MARCH5A的免疫作用,实验采用反转录PCR确认了该基因的c DNA序列。该序列全长1045 bp,包含1个长度为867 bp的开放阅读框,编码288个氨基酸;序列分析显示该蛋白含1个RINGv和4个跨膜结构域。进化树分析表明大黄鱼有11个MARCH家族蛋白(与哺乳动物相同),但鱼类存在多个亚型,MARCH5A为鱼类特有蛋白,其蛋白理化性质和三维结构均与MARCH5B存在一定的差异。荧光定量PCR分析显示,MARCH5A在健康大黄鱼的多个组织中均有表达,在血液和心脏中表达量最高,其次为鳃和脑,而在肾脏和皮肤中表达量最少。刺激隐核虫感染大黄鱼后,MARCH5A在皮肤中早期表达量显著上调,第2天为对照组的9.65倍,后期下调。在鳃、脾脏和头肾中的前期表达量也有所增加,后期下降。结果表明大黄鱼MARCH5A在抗刺激隐核虫免疫应答过程中可能发挥重要作用。     

Evaluation of the Abbott LCx Mycobacterium Tuberculosis Assay for Direct Detection of Mycobacterium Bovis in Bovine Tissue Samples

The commercial LCx amplification assay, usually employed to detect the Myocobacterium tuberculosis complex in respiratory specimens, was evaluated by comparing the results it gave with those obtained using Löwenstein-Jensen solid medium and pathological findings on 55 lymph nodes from cattle with positive and 10 lymph nodes from cattle with negative skin tests for tuberculosis. Fifty-three cultures (51 and 2, respectively) were positive for M. bovis, while the results for the LCx assay and the histological method were positive in 48 (45, 3) and 24 (20, 4) samples, respectively. None of the samples from cattle from certified tuberculosis-free herds were positive by any of the procedures. The results obtained with the LCx assay, compared with the culture procedure, regarded as the gold standard among the diagnostic techniques, gave a specificity of 91.6% and sensitivity of 90.5%. Although the sensitivity of LCx was suboptimal, DNA of M. bovis was detected in 81.8% of the skin test-positive animals. Amplification techniques could provide a rapid and reasonably reliable tool for detecting bovine tuberculosis.     

PCR法克隆紫穗槐4-香豆酸:CoA连接酶全长cDNA

4 香豆酸 :CoA连接酶 (4CL)是木质素生物合成过程中重要的酶 .该文利用简并寡核苷酸PCR法结合cDNA末端快速扩增PCR法直接获得了紫穗槐 4CLAcDNA全长序列 ,避免了复杂的构建、筛选cDNA文库的过程 .先用简并PCR得到了 4CLA1片段 ,又据此片段设计反向嵌套引物 ,用RACE方法获得未知的 5′和 3′端序列 .所获得的全长 4CLAcDNA ,编码 5 40个氨基酸 .氨基酸序列同源性分析表明4CLA是典型的 4CL蛋白 ,含有预计的AMP binding位点、催化反应区和保守的Cys .     

Propanil and swep inhibit 4-coumarate:CoA ligase activity in vitro

4-Coumarate:CoA ligase (4CL, EC 6.2.1.12) in the phenylpropanoid pathway in plants has attracted interest as a novel target for developing effective plant growth inhibitors (PGIs). In a previous study in which the 4CL inhibitory activity of 28 existing herbicides was investigated using an optimized in vitro screening assay, 4CL activity was found to be strongly inhibited by propanil and swep at 100 microM. Here, further experimental evidence is provided to substantiate the previous result. Using 4-coumaric acid as substrate, tobacco 4CL activity was inhibited by propanil or swep in a concentration-dependent manner, with 50% inhibition concentrations (I(50)) of 39.6 and 6 microM respectively. These herbicides also exhibited uncompetitive inhibition towards 4-coumaric acid. Furthermore, 4CLs from several plant species were inhibited by the herbicides within a range from 1 to 50 microM. It is proposed that these herbicides have another site of action as a result of the inhibition of 4CL in the phenylpropanoid pathway, and this enzyme represents a new target site for the development of PGI.     

水稻Os4CL5的Val337缺失突变激活其芥子酸催化活性

4-香豆酸辅酶A连接酶(4-coumarate:coenzyme A ligase,4CL)催化各种羟基肉桂酸生成相应的硫酯,从而调控木质素和黄酮类物质的生成.4CL的芥子酸催化活性一直是备受关注的一个问题,目前只有拟南芥(Arabidopsis thaliana)的At4CL4和大豆(Glycine max)的Gm4CL1具有芥子酸转化能力.本研究用水稻(Oryza sativa subsp.japonica)苗期的叶片为材料,提取总RNA并以此为模板,用反转录PCR扩增水稻的Os4CL5基因,并将其连接到原核表达载体pET22-b(+)上,构建了原核表达载体pET-4CL5(+Val).通过定点突变的方法删除了水稻Os4CL5的Val337,构建了原核表达载体pET-4CL5(-Val).重组质粒转化大肠杆菌(Escherichia coli)BL21(ED3)并经异丙基-β-d-硫代半乳糖苷(isopropyl-β-d-thiogalactoside,IPTG)诱导表达,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)检测表明,融合蛋白的分子量为57.0 kD,与预测值一致.His-SpinTrap蛋白纯化系统纯化回收重组蛋白,然后对重组蛋白的酶学性质进行表征.结果表明,野生型Os4CL5对不同底物表现出了不同的催化效率,香豆酸是最适底物,其次是阿魏酸,再次是咖啡酸,对芥子酸没有活性.与野生型Os4CL5相比,突变型Os4CL5对香豆酸、咖啡酸和阿魏酸的催化效率有所提高,并且其底物特异性发生了明显改变,获得了对芥子酸的催化活性.本研究为利用基因工程手段调控木质素的生物合成提供了理论依据.     

甜菜应答盐胁迫PUB基因的生物信息学分析

旨在为研究甜菜U-box型E3泛素连接酶提供理论基础。以甜菜T710-Mu品系为试验材料,前期通过转录组数据获得在盐胁迫下差异表达的甜菜PUB基因(plant U-box gene),利用生物信息学的方法对这些PUB基因进行分析,主要包括理化性质分析、染色体定位分析、基因序列分析、结构域分析、家族分类分析以及互作蛋白预测。结果显示甜菜PUB基因在甜菜9条染色体上均有定位,并且包含PUB蛋白具有的多个保守结构域,如U-box,Pkinase、Pkinase-Tyr、Usp、Arm以及KAP结构域,以拟南芥U-box家族分类为依据发现甜菜U-box多位于ClassⅡ、ClassⅢ、ClassⅣ中,甜菜PUB蛋白可作为E3泛素连接酶与多种功能蛋白相互作用,实现泛素化修饰。植物PUB蛋白具有E3泛素连接酶活性,在植物生长发育过程以及抵抗环境压力中起到重要作用。     

Effect of dietary fish oil intake on ubiquitin ligase expression during muscle atrophy induced by sciatic nerve denervation in mice

Dietary fish oil intake improves muscle atrophy in several atrophy models however the effect on denervation‐induced muscle atrophy is not clear. Thus, the aim of this study was to investigate the effects of dietary fish oil intake on muscle atrophy and the expression of muscle atrophy markers induced by sciatic nerve denervation in mice. We performed histological and quantitative mRNA expression analysis of muscle atrophy markers in mice fed with fish oil with sciatic nerve denervation. Histological analysis indicated that dietary fish oil intake slightly prevented the decrease of muscle fiber diameter induced by denervation treatment. In addition, dietary fish oil intake suppressed the MuRF1 (tripartite motif‐containing 63) expression up‐regulated by denervation treatment, and this was due to decreased tumor necrosis factor‐alpha (TNF‐α) production in skeletal muscle. We concluded that dietary fish oil intake suppressed MuRF1 expression by decreasing TNF‐α production during muscle atrophy induced by sciatic nerve denervation in mice.