猪链球菌2型次黄嘌呤核苷酸脱氢酶(IMPDH)重组蛋白的酶活性和免疫原性检测

通过PCR方法从猪链球菌2型(Streptococcus suis)05ZYH33分离株基因组中扩增出次黄嘌呤核苷酸脱氢酶基因(inosine 5-monophosphate dehydrogenase,impdh),长度1 064 bp.PCR产物和pET28a载体分别经过EcoR Ⅰ和Xho Ⅰ双酶切,连接,成功地构建了重组表达质粒pET28a-impah,并转入大肠杆菌(Escherichia coli) BL21(DE3)中,经过1 mmol/L IPTG诱导获得表达,蛋白大小35kD.表达蛋白具有良好的抗原性和酶活性.蛋白经过亲和层析纯化后,在37℃,pH7.0～9.0表现出较强酶活性,NADH A_(340)介于0.662～0.816之间.     

乙醛脱氢酶基因（ALDH）转化番茄的研究

乙醛脱氢酶基因（ALDH）为目的基因,构建了pBI-ALDH 植物表达载体,番茄品系03HN31子叶为外植体,经发根农杆菌（Agrobacterium rhizogens）介导采用共培养法转化番茄。通过分子生物学技术（PCR、Southern杂交、RT-PCR）和抗逆相关生理指标测定（相对电导率及丙二醛含量）相结合的方法对转化番茄植株进行检测,研究结果表明：乙醛脱氢酶基因（ ALDH ）导入并整合到番茄的基因组中,在转录水平上可以稳定表达;在干旱、高盐和低温胁迫条件下,转基因番茄植株和对照株的相对电导率和丙二醛含量有差异,证明转化番茄植株的质膜受损程度有所降低,抗氧化胁迫性能有所提高,转化率为10.78%。     

Ascorbate levels and the activity of key enzymes in ascorbate biosynthesis and recycling in the leaves of 22 Chinese persimmon cultivars

The objective of this study was to determine diversities of ascorbic acid (AsA) and activities of enzymes involved in AsA metabolism, and to determine their relationships to AsA content in the leaves of Chinese persimmon (Diospyros kaki) cultivars. Results showed that there were huge differences not only in the contents of AsA and glutathione (GSH) but also in the enzyme activities of l-galactono-1,4-lactone dehydrogenase (GLDH, EC 1.3.2.3), dehydroascorbate reductase (DHAR, EC 1.8.5.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) in the leaves of 22 Chinese persimmon cultivars. Control of AsA levels mainly depended on the capacity for biosynthesis and GLDH activity played a main role in determining AsA levels in the leaves of persimmon. Both MDHAR and DHAR, MDHAR in particular, also played an important role in maintaining AsA redox state by reducing oxidized AsA back to AsA. But contents of GSH and hydrogen peroide (H₂O₂) and activity of APX were not significantly correlated with AsA content in the leaves of persimmon.     

马铃薯抗坏血酸含量及其代谢相关酶活性关系的研究

 为探讨马铃薯不同器官中抗坏血酸(AsA) 含量及其代谢相关酶活性关系, 研究了马铃薯幼叶、功能叶、老叶、茎和块茎中AsA和其氧化态脱氢抗坏血酸(DHA) 的含量与L - 半乳糖- 1, 4 - 内酯脱氢酶( GalLDH) 、脱氢抗坏血酸还原酶(DHAR) 、谷胱甘肽还原酶( GR ) 、抗坏血酸过氧化物酶(APX) 、抗坏血酸氧化酶(AO) 和单脱氢抗坏血酸还原酶(MDHAR) 等6种酶活性之间的相关性。结果表明, 马铃薯AsA在幼叶和块茎中含量很高。叶片和茎的抗坏血酸库(AsA与DHA之和) 水平与GalLDH活性显著相关, 而AsA含量与DHAR活性显著相关, DHA含量与APX活性显著相关。说明在马铃薯幼叶中高含量的AsA可能由于GalLDH和DHAR的高活性; 而块茎中AsA的积累, 主要来自于叶片的运输和DHAR催化的DHA再生。     

阔叶猕猴桃果实GalDH cDNA 克隆及其在大肠杆菌中的表达

 以猕猴桃属植物中果实维生素C含量最高的阔叶猕猴桃(Actinidia latifolia Merr. ) 果实为试 材, 经RT2PCR扩增获得约110 kb的L - 半乳糖脱氢酶cDNA片段。生物信息学分析表明, 该cDNA片段长为997 bp, 最大开放阅读框为960 bp, 可编码319 个氨基酸残基, 命名为A lGalDH, GenBank登录号为EU525846, 核苷酸序列及其推导的氨基酸序列与已知其它植物GalDH核苷酸、氨基酸序列间的同源性分别在76%和70%以上, 且具有醛酮还原酶的保守结构域。构建了其原核表达载体pET-A lGalDH并转化大肠杆菌BL21, 经0.1 mmol·L ^{- 1} IPTG诱导, 获得具有较高活性的表达融合蛋白6 ×His-AlGalDH。经Ni-His亲和磁珠分离纯化, 获得单一的融合目的蛋白条带, 测定其酶活为120 pmol·min^{- 1} ·mg^{- 1}。     

中国野生葡萄醛脱氢酶基因在拟南芥中的过量表达

将质粒pWR306中包含ED35s启动子、Omega元件及TNOS终止子的一段核苷酸序列定向克隆到质粒pCAMBIA1300,构建了植物中间表达载体pWR-II;将重组质粒pGEM-T-ALDH的醛脱氢酶(ALDH)基因片段定向克隆到pWR-II,构建了葡萄ALDH基因的植物过量表达载体pWR-II-ALDH。采用改进冻融法将其导入农杆菌EHA105,采用花序浸蘸法转化拟南芥,获得了潮霉素抗性的转化拟南芥植株。PCR检测证明外源基因已整合进拟南芥基因组,Real-timePCR结果表明ALDH基因在转化植株的表达量远高于野生种。转化植株和野生植株在形态上有一定的差异,说明葡萄ALDH基因在拟南芥中表达对其生长发育有一定影响。     

Uneven ripening in ‘Bangalore blue’ grape (Vitis vinifera × Vitis labrusca) berries in India is linked to seed viability

Uneven ripening (UR) is a physiological disorder of unknown origin in ‘Bangalore blue’ grape (Vitis vinifera × Vitis labrusca) leading to wine of inferior quality. A preliminary study found wide variations in total dehydrogenase activity (TDH) of seeds from unevenly ripe berries. In our experiments, gibberellin (GA₃) applied to young grapes increased seed TDH activity and reduced the incidence of uneven ripening to 2% compared with 35% in the control. In contrast, paclobutrazol (PBZ) decreased TDH activity and increased the incidence of the disorder to 58%. GA₃-treated berries had higher concentrations of sucrose and TDH activity in seed representing mature seeds with high viability. In contrast, PBZ-treated and control berries had higher concentrations of glucose and lower TDH activity, indicating immature seeds with low viability. These results suggested that competition among developing berries can lead to differences in seed gibberellin content, seed viability and the rate of berry growth resulting in green, purple, and black berries at harvest. The study established the role of seed viability in uneven ripening and demonstrated that the incidence of the disorder is reduced by the application of GA₃ to immature berries.     

Generation and analysis of expressed sequence tag sequences from a soft‐seeded pomegranate cDNA library

The cultivation of soft‐seeded pomegranate is an important direction in pomegranate breeding. To comprehensively understand the molecular mechanisms involved in the formation of soft‐seeded pomegranate (Punica granatum. var. Hongmanaozi), we established an expressed sequence tag (EST) resource. Two thousand valid sequences were generated, from which 907 unigenes were obtained after initial assembly using the clustalx program. Among these unigenes, 51 showed no similarity to any protein in the public databases, 433 matched with proteins of unknown function, and 423 matched with proteins of known or putative functions. The 423 unigenes were further classified into 13 categories. Among these categories, protein synthesis, cell structure, protein destination and storage, secondary metabolism, signal transduction and transporters accounted for 8%, 8%, 4%, 7%, 6% and 17%, respectively. We also successfully developed 10 highly polymorphic expressed sequence tag‐simple sequence repeat (EST‐SSR) markers for pomegranate. The results provide a new tool for future activities in pomegranate breeding.     

海鲈游泳续航时间和运动代谢规律研究

为研究海鲈(Lateolabrax japonicus)的游泳续航能力和游泳过程中的物质代谢规律,以深水网箱养殖的3月龄海鲈为研究对象,采用游泳续航时间和生化指标作为评测海鲈游泳能力和代谢规律的方法进行了试验。结果显示,海鲈的游泳续航时长为200 min,其续航能力强于大黄鱼而弱于美国红鱼。在游泳过程中,海鲈血糖浓度升高,但差异性不显著(P>0.05),肌糖原随着时间的增加显著减少(P<0.05),且肌糖原浓度在海鲈疲劳时接近于0。当鱼接近100%疲劳时,其肝糖原浓度也接近于0;当海鲈以不同疲劳程度游泳时,其肌肉乳酸脱氢酶浓度会显著升高(P<0.05)。表明海鲈的续航游泳过程伴随着无氧呼吸和有氧呼吸。     

乙醇脱氢酶应用研究现状

乙醇脱氢酶(ADH)是广泛分布于人和动物肝脏、植物及微生物细胞之中的含锌金属酶,具有广泛的底物特异性.ADH作为生物体内主要短链醇代谢的关键酶,近年来已经受到了国内外研究者的普遍关注,并对其同功酶的结构、理化性质、生物学功能、遗传学特性、分离提取技术等进行了较多的基础性研究工作,为其在科学研究和工农业生产中的应用奠定了良好基础.文章主要对ADH在工业分析、化工生产、食品和医药研究以及生物科学中的应用进行了概述,以便为其进一步研究开发提供参考.