大别山野生油茶中茶氨酸检测与茶氨酸合成酶基因克隆

茶氨酸是茶树叶片中最丰富的游离氨基酸,具有重要的生理药理功能,但迄今仅在蘑菇、蕈和一些山茶科(属)植物中检测到茶氨酸。茶氨酸因有一种独特的风味特色"umami鲜爽味"而被人类营养学广泛研究,并发现合成茶氨酸的植物不仅在植物分类上具有积极意义,而且对于植物资源的有效发掘有巨大经济价值;同时还可以间接去研究茶树中茶氨酸的代谢机理以及茶氨酸合成酶的分离纯化和TS基因的克隆表达。该文运用HPLC、LC-TOF/MS对大别山地区野生幼年与成年油茶根、叶中茶氨酸进行检测,并结合分子生物学手段对油茶中茶氨酸合成酶(theanine synthetase,TS)基因进行克隆与生物信息学分析。结果表明:在幼年的油茶根中检测到茶氨酸,含量为0.08mg·g-1(鲜重),而在幼年的油茶叶片和成年油茶根、叶中均未检测到茶氨酸;在幼年油茶根中克隆出一条长为1 071bp油茶TS基因开放阅读框,其基因序列与茶树谷氨酰胺合成酶(glutamine synthetase,GS,AB117934)基因和TS(DD410896)序列的同源性达到98%,氨基酸序列与茶树中GS(AB117934)和TS(DD410896)的相似性高达99%。经生物信息学分析,该序列编码的TS蛋白具有20个磷酸化位点,不存在信号肽序列与跨膜结构,含有卷曲螺旋结构的亲水性细胞质蛋白。该研究将为油茶新经济价值的发掘,为茶氨酸在油茶中合成代谢途径的研究提供一定的理论基础,同时也为进一步研究茶氨酸在茶树中代谢机理提供了新的研究思路。     

A pathogenicity determinant maps to the N‐terminal coat protein region of the Pepino mosaic virus genome

Pepino mosaic virus (PepMV) poses a worldwide threat to the tomato industry. Considerable differences at the genetic level allow for the distinction of four main genotypic clusters; however, the basis of the phenotypic outcome is difficult to elucidate. This work reports the generation of wild‐type PepMV infectious clones of both EU (mild) and CH2 (aggressive) genotypes, from which chimeric infectious clones were created. Phenotypic analysis in three solanaceous hosts, Nicotiana benthamiana, Datura stramonium and Solanum lycopersicum, indicated that a PepMV pathogenicity determinant mapped to the 3′‐terminal region of the genome. Increased aggression was only observed in N. benthamiana, showing that this factor is host specific. The determinant was localized to amino acids 11–26 of the N‐terminal coat protein (CP) region; this is the first report of this region functioning as a virulence factor in PepMV.     

诱发血管瘤型J亚群禽白血病病毒gp85基因的克隆与表达

2007年7月至11月,中国开产前后的商品海兰褐蛋鸡群大面积暴发血管瘤,造成巨大经济损失。将病料接种DF1细胞,通过PCR和间接免疫荧光(IFA)确定此次暴发的血管瘤为J亚群白血病病毒(ALV-J)感染引起。从患病鸡群中分离到5株ALV-J(前4株已经报道),将第5株病毒命名为WS0705。为研究该毒株抗原性的特点,用PCR方法扩增出gp85基因,并克隆进pMD18-T载体进行测序。氨基酸系统进化树分析显示WS0705与英国ALV-J原型毒株HPRS-103同源性最高。从已构建的质粒pMD18-T-WS0705gp85中酶切回收gp85基因,构建重组转移载体pFastBacH Tb-WS0705gp85。利用Bac-to-Bac表达系统获得了重组杆状病毒rBac-WS0705gp85。间接免疫荧光和Western blot检测WS0705gp85基因的表达产物。间接免疫荧光显示,构建的重组杆状病毒感染的Sf9细胞呈现明显的强阳性反应;Western blot分析,重组病毒感染的Sf9细胞蛋白显示出约35kD的阳性条带。结果表明,WS0705gp85基因在Sf9细胞中得到良好的表达,并且其编码产物完全可以被外源性ALV-J的特异性单抗JE9识别,进一步证明本次暴发血管瘤的病原为ALV-J,并为进一步开发ALV-J相关诊断产品奠定了基础。     

孝顺竹肉桂醇脱氢酶基因的克隆及表达研究

肉桂醇脱氢酶(cinnamoyl alcohol dehydrogenas,CAD)是木质素生物合成过程中的一类关键酶.采用RT-PCR及RACE方法从孝顺竹笋中分离出CAD基因家族的一个基因,cDNA全长是1131 bp(GenBank注册号为GU985522),含有一个1 107 bp的读码框,编码一个368 aa...     

Caveolin-1真核表达载体的构建及其Hepa1-6细胞中的表达

Caveolin-1在不同肿瘤中发挥作用不同,既发挥抑癌基因样作用又发挥癌基因样作用.旨在分析caveolin-1 在小鼠肝癌细胞系中的表达情况及建立稳定表达外源caveolin-1的Hepa1-6细胞.利用RT-PCR和Western-blot方法检测caveolin-1在小鼠肝癌H22、Hea-F和Hepa1-6细胞中的表达;通过分子克隆构建小鼠caveolin-1 cDNA真核表达栽体,利用脂质体转染等方法建立稳定表达外源caveolin-1的Hepa1-6细胞株;通过RT-PCR、Western-blot、免疫细胞化学等方法鉴定其稳定表达细胞株.结果显示,caveolin-1在Hepa1-6细胞中表达呈阴性,在H22和Hca-F 中高表达;成功获得小鼠caveolin-1 cDNA真核表达载体pEGFP-N2/Cav-1,筛选并鉴定出高表达外源caveolin-1的Hepa1-6稳定细胞株C1和C4,为进一步分析caveolin-1在肝癌中所发挥的作用奠定了一定的研究基础.     

Investigation of bacterial community diversity in water of Zoige Alpine Wetland

Spatial isolation is currently thought to represent one of the major factors resulting in bacteria genetic variation and population abundance. The bacterial diversity in a distinct environment Zoige Alpine Wetland located in the northeast of the Qinghai-Tibetan Plateau with the altitude 3400 m on average aroused our great attention. This area belongs to Qinghai-Tibetan cold climate zone with the mean annual temperature about 1 °C. Although several studies on bacterial diversity in Qinghai-Tibetan Plateau had been reported, there is no report on wetland water in this area. In this work, six water samples were collected and the water qualities including COD_Cr, NH₄⁺-N, NO₃^--N, NO₂^--N, TN, TP, TOC were investigated, of which results indicated that more than 80% samples sorted as II–V class of surface water sources according to the National Water Quality Standard of China (GB3838-2002). Comparison of bacterial communities among the six samples was analyzed by DGGE of PCR-amplified 16S rDNA with universal bacterial primer sets. The profiles demonstrated that samples from the Flower Lake had more DNA bands than the Conservatory Station inferring higher diversity. In addition, the samples from the same environment shared similar compositions of bacterial communities. Bacterial community composition and predominant bacteria were analyzed by 16S rDNA clone library. The dominant group was Proteobacteria (51.6% of the total clones, which contained 24.2% alpha proteobacteria, 14.5% beta proteobacteria and 12.9% gamma proteobacteria). And the Bacteroidetes added to 17.7%, Verrucomicrobia to 4.8%. More than 24.2% of the total clones showed high similarity to uncultured bacteria. The above work provides some information on bacterial diversity for special site of spatial isolation.     

钛合金和钴铬合金表面白色念珠菌粘附的研究

目的比较钛合金(Ti-6Al-4V)和钴铬合金(Chromium-Cobaltalloy)表面白色念珠菌粘附能力的大小,研究表面粗糙度与细菌粘附的关系。方法将不同表面粗糙度的钛合金和钴铬合金试件进行白色念珠菌体外粘附试验,采用菌落形成计数法测定试件表面的细菌粘附量。结果各钛合金试件组的细菌粘附量均少于相同表面粗糙度的钴铬合金试件组,两种金属试件表面的细菌粘附量均随表面粗糙度的增大而增加。结论钛合金较钴铬合金更能减少由白色念珠菌引起的义齿性口炎等并发症,同时修复体表面严格的研磨抛光也能有效减少这些并发症。     

酿酒酵母CICC1747醛糖还原酶基因的克隆及表达

采用PCR技术以酿酒酵母CICC1747基因组DNA为模板扩增得到醛糖还原酶基因GRE3,插入到pET-15b载体的NdeⅠ和BamHⅠ酶切位点之间,构建了酿酒酵母醛糖还原酶原核表达载体pET-15b-GRE3。将该载体转化到大肠杆菌菌株Rosetta(DE3)中,重组菌株用IPTG诱导表达,采用紫外分光光度法测定醛糖还原酶活力,并对其表达条件进行初步优化。SDS-PAGE电泳结果显示在分子量约37 kD处有明显的特异性蛋白质条带。发酵液的比酶活最高为54.94 mU/mg,与酿酒酵母野生菌株相比提高了近10倍。