A Novel Alginate Lyase: Identification,Characterization, and Potential Application in Alginate Trisaccharide Preparation

Alginate oligosaccharides (AOS) have many biological activities and significant applications in prebiotics, nutritional supplements, and plant growth development. Alginate lyases have unique advantages in the preparation of AOS. However, only a limited number of alginate lyases have been so far reported to have potentials in the preparation of AOS with specific degrees of polymerization. Here, an alginate-degrading strain Pseudoalteromonas arctica M9 was isolated from Sargassum, and five alginate lyases were predicted in its genome. These putative alginate lyases were expressed and their degradation products towards sodium alginate were analyzed. Among them, AlyM2 mainly generated trisaccharides, which accounted for 79.9% in the products. AlyM2 is a PL6 lyase with low sequence identity (≤28.3%) to the characterized alginate lyases and may adopt a distinct catalytic mechanism from the other PL6 alginate lyases based on sequence alignment. AlyM2 is a bifunctional endotype lyase, exhibiting the highest activity at 30 °C, pH 8.0, and 0.5 M NaCl. AlyM2 predominantly produces trisaccharides from homopolymeric M block (PM), homopolymeric G block (PG), or sodium alginate, with a trisaccharide production of 588.4 mg/g from sodium alginate, indicating its promising potential in preparing trisaccharides from these polysaccharides.     

中国麇鹿朊蛋白核心片段的原核克隆表达与纯化

从采取的新鲜麋鹿血液中提取麋鹿基因组DNA,通过PCR的方法从麋鹿基因组中扩增麋鹿PrP蛋白核心片段的DNA,并分别在引物的5′和3′端引入EcoRⅠ和HindⅢ两个酶切位点,下游引物将原序列TATTAC突变为TAAT从变成两个终止密码子。通过两个酶切位点将麋鹿PrP蛋白核心片段DNA连接到pET-32a（＋）,转化DH5α,通过酶切鉴定和PCR鉴定,筛选出阳性克隆送菌液测序,从测序结果分析阳性克隆是否符合要求。测序正确后将核心片段基因连接到pET-32a（＋）,阳性质粒命名为MPrP-pET-32a（＋）,阳性质粒转化E.coli BL21（DE3）,进行诱导表达、蛋白质纯化鉴定。     

小菜蛾鱼尼丁受体基因克隆及序列分析

利用RT-PCR和RACE技术克隆了小菜蛾(Plutella xylostella)鱼尼丁受体(Px-RyR)基因,其核苷酸序列全长15 748bp,5′非编码区267bp,3′端非编码区109bp,开放阅读区全长为15 372bp(GenBank登录号:JF927788),编码5 123个氨基酸残基。估测其蛋白分子量为579.39ku,等电点为5.45。该基因编码氨基酸序列和其他鳞翅目昆虫RyR氨基酸序列比对相似性较高(92%),与哺乳动物3种亚型RyRs的相似性为45%～47%。此外,二级结构预测,其C-末端存在6个跨膜区域;且Px-RyR中存在一个出现4次重复,长度为89～95个氨基酸的RyR结构域,平均相似性为33%。     

苹果采前落果与内源激素的关系

为了探讨苹果采前落果与内源激素之间的关系,在采前8周间定期测定了不同苹果品种的果柄、果台和离层形成部位组织中IAA、ABA含量;在采收前20d内定期测定了离层部位组织中细胞壁分解酶(Cellulase)的活性;在收获期测定了果实乙烯的发生量。结果表明:不同品种果柄、果台和离层部位组织中IAA和ABA含量变化有差异,但它们变化的总趋势相似,都是随着果实成熟IAA含量下降,而ABA含量上升;不同品种的成熟果实中乙烯发生量有很大差异,以落果多的品种显著大于落果少的品种;采前落果重的品种离层部位组织中细胞壁分解酶的活性在果实成熟期急剧增加。由于果实进入成熟阶段后,IAA含量下降,ABA含量升高,ABA/IAA之间的相对平衡被打破,高ABA/IAA以及高乙烯会刺激离层组织中细胞壁分解酶的活性增高,进而促进离层形成,这可能是导致落果发生的原因。     

桑尺蠖越冬幼虫的耐寒性研究

研究了桑尺蠖越冬幼虫的耐寒能力 ,测定了虫体内抗寒物质的组成成分 ,并对其耐寒机理作了初步探讨。结果表明 :越冬幼虫从越冬初期至越冬滞育期 ,4龄幼虫的平均过冷却点从初期的-1 7 2℃下降至滞育期的 -2 4 4℃ ;5龄幼虫的平均过冷却点从 -1 3 4℃下降至 -2 4 8℃。越冬幼虫体内水分、糖元、脂肪含量下降 ,其中以糖元含量下降明显 ;越冬幼虫以“小分子糖—氨基酸—糖蛋白”物质系统形成抗寒基质 ,增强耐寒性。     

茶树病程相关蛋白基因CsPR5的克隆及表达分析

为探究茶树中病程相关蛋白5(pathogenesis-related protein 5,PR5)在茶树中的功能,采用cDNA末端快速扩增(rapid amplification of cDNA end,RACE)技术克隆了PR5基因的全长;通过荧光定量PCR方法检测了CsPR5在龙井43茶树不同组织部位、植物激素(茉莉酸、乙烯利和水杨酸)处理、茶炭疽病菌Colletotrichum camelliae侵染和小贯小绿叶蝉Empoasca onukii为害后的表达特征。结果表明,CsPR5基因开放阅读框为843 bp,编码281个氨基酸。CsPR5基因在茶树根、茎、嫩叶、花和种子5个组织中的相对表达量分别为0.187、3.093、0.928、0.045和0.012,且五者之间差异显著,主要在茎和叶中表达,其分别为根中相对表达量的16.54倍与4.96倍。水杨酸处理6 h及乙烯利处理6、12 h后,茶树CsPR5基因的相对表达量分别为1.622、2.344和1.845,分别比对照显著上调2.20倍、3.18倍和2.35倍;但是外施茉莉酸对茶树CsPR5基因的相对表达量无影响。茶炭疽病菌侵染显著诱导了茶树CsPR5基因的相对表达量,处理后的相对表达量为1.977,是对照的1.90倍;小贯小绿叶蝉取食显著抑制了其为害部位CsPR5的相对表达量,为7.273,仅为对照的38.80%,但该诱导不具系统性。     

日粮中添加不同有机酸对肉鸡生产性能的影响

选择体重相近、健康的1日龄罗曼肉用雏120只,在基础日粮中分别添加0.2%柠檬酸、乳酸和延胡索酸,观察有机酸对肉鸡生产性能的影响。结果表明:在22～42日龄,添加柠檬酸组和延胡索酸组采食量均比对照组显著增加(P<0.05);在1～21日龄及全期,添加柠檬酸组日增重比对照组显著增加(P<0.05),在22～42日龄添加柠檬酸组日增重比对照组和添加乳酸组有显著增加(P<0.05);3种有机酸对料肉比指标影响有下降趋势,但均未达到显著水平(P>0.05)。     

Antiviral activity of Alpinia katsumadai extracts against rotaviruses

In vitro anti-rotavirus activity of Alpinia katsumadai (AK) extracts were evaluated against bovine G8P[7] and porcine G5P[7] rotaviruses in two different assay strategies, a mixed treatment assay and a post treatment assay. In the mixed treatment assay, six AK extracts [AK-1 (EtOH extract), AK-3 (H(2)O layer), AK-5 (40% methanol fraction), and AK-9-11 (H(2)O extract, polysaccharide fraction, supernatant fraction)] exhibited inhibitory activities against G5P[7] rotavirus with the EC(50) values ranging from 0.7±0.4 to 33.7±6.5 μg/mL. Extracts AK-1, AK-3, and AK-5 inhibited rotavirus infection against G8P[7] rotavirus, the with EC(50) values of 8.4±2.2 μg/mL, 6.5±0.8 μg/mL and 8.4±5.0 μg/mL, respectively. By hemagglutination inhibition (HI) assay, six AK extracts completely inhibited viral adsorption onto human RBCs in both strains of rotaviruses at less than 11 μg/mL. However, in the post treatment assay, there was no anti activity shown against both strains of rotaviruses. As a result, six AK extracts were attributed mainly to having a strong interaction with hemagglutinin protein on the outer surface of rotavirus, resulting to blockage of viral adsorption.     

Effects of dietary oregano essential oil supplementation on growth performance,intestinal antioxidative capacity,immunity, and intestinal microbiota in yellow-feathered chickens

Essential oils are plant-derived aromatic volatile oils, and they contain bioactive compounds that have been shown to improve poultry nutrition. In this study, we investigated the effects of oregano essential oil (OEO) on intestinal antioxidative capacity, immunity, and gut microbiota of young yellow-feathered chickens. A total of nine hundred and sixty 1-d-old female Qingyuan partridge chickens were randomly allocated to four treatment groups with six replicates of 40 birds each, and the feeding trial was lasted for 30 d. The controls were fed on a basal diet without in-feed antibiotics; the birds in the antibiotic group were fed the basal diet supplemented with 20 mg/kg virginiamycin; the remaining birds were fed the basal diet containing 150 or 300 mg/kg OEO, respectively. Dietary supplementation with 150 or 300 mg/kg OEO increased average daily feed intake (P = 0.057) and average daily gain (P < 0.05). The activities of glutathione peroxidase and total antioxidative capacity in plasma, jejuna, and ileal mucosa were increased by OEO supplementation (P < 0.05), with a trend of lower jejunal content of malonaldehyde (P = 0.062). Moreover, dietary OEO increased the content of secretory immunoglobulin A (P = 0.078) and the relative expression of Claudin 1, Mucin 2, and Avain beta-defensin 1 in ileum (P < 0.05). Sequencing data of 16S rRNA indicated that dietary OEO increased the relative abundance of Firmicutes phylum, and Clostridium and Lactobacillus genera, and decreasing that of Romboutsia. Functional analyses indicated that microbial amino sugar and nucleotide sugar metabolism, replication, and repair systems were higher in OEO groups than those of controls and antibiotic treatment. In conclusion, dietary supplementation with OEO enhanced growth performance, alleviated local oxidative stress in intestine, improved production of natural antibodies, and favorably modulated intestinal microbiota composition.     

Inhibition of porcine circovirus type 1 and type 2 production in PK-15 cells by small interfering RNAs targeting the Rep gene

Porcine circovirus type 1 (PCV1) and type 2 (PCV2) are two genotypes of porcine circovirus. Both of them are presumed to be widespread in the swine population. Currently, there is no specific treatment for their infections. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), which represents a possible therapeutic application for the treatment of viral infections. In this study, three siRNA expression plasmids (pS-RepA, pS-RepB and pS-RepC) were generated to target three different coding regions of the Rep protein (Rep) of PCV. These siRNAs were used to inhibit PCV production in a porcine kidney cell line, PK-15 cells. Our results revealed that Rep gene expression was inhibited by pS-RepA, pS-RepB and pS-RepC to different degrees. Moreover, our study also showed that the production of PCV1 and PCV2 was reduced by these siRNAs. pS-RepC, which targets the middle region of Rep gene, proved to be the most efficient siRNA for inhibition of Rep expression and viral production. Taken together, our data suggest that RNAi could be investigated as a potential treatment for PCV infection.