两个褐飞虱海藻糖转运蛋白基因的结构及调控海藻糖代谢功能

【目的】海藻糖是昆虫中的主要血糖物质,在昆虫的发育及生理活动中发挥重要功能。其中,海藻糖转运蛋白（Tret）在将海藻糖从其生成组织（例如脂肪体）运输到其消耗组织的过程中起着重要作用。本研究通过分析褐飞虱（Nilaparvata lugens）两条Tret1的序列结构,进一步抑制NlTret1的表达,探讨这两个NlTret1在褐飞虱体内的生物学功能。【方法】以褐飞虱两条Tret1序列为研究对象,利用生物信息学技术分析其蛋白结构以及与其他昆虫之间的同源性。采用RNAi（RNA interference）技术将合成的外源dsRNA（double-stranded RNA）注射到实验室饲养褐飞虱种群体内,抑制其体内NlTret1的表达,分别在注射48 h后取材,抽取总RNA并用反转录试剂盒合成第一链cDNA,采用qRT-PCR（quantitative real-time PCR）技术检测dsNlTret1的干扰效果以及RNAi后褐飞虱体内海藻糖代谢通路中相关基因的表达,最后测定葡萄糖、海藻糖和糖原含量以及海藻糖酶活性。【结果】生物信息学分析表明,NlTret1-like X1和NlTret1-2 X1的开放阅读框长度分别为1 920和1 578 bp,分别编码639和525个氨基酸,预测蛋白分子量分别为69.29和58.71 kD,等电点分别为8.32和8.36;NlTret1-like X1和NlTret1-2 X1的二级结构主要包含螺旋和卷曲;保守结构域分析显示它们均属于MFS家族。进化分析结果显示,不同昆虫的Tret1蛋白具有较高的同源性,且褐飞虱与其他半翅目昆虫具有较近的亲缘关系;与注射dsGFP组相比,注射靶标基因的dsRNA后均能够显著沉默本基因的表达;褐飞虱体内糖原、葡萄糖在注射dsNlTret1-like X1和dsNlTret1-2 X1后,其含量均无显著变化,然而不同于注射dsNlTret1-2 X1组,在注射dsNlTret1-like X1后褐飞虱体内海藻糖含量极显著升高;干扰NlTret1-like X1 48 h后褐飞虱体内TPS1、TPS2、TRE1-1、TRE1-2和TRE2的表达均极显著下调;而干扰NlTret1-2 X1 48 h后褐飞虱体内TPS1、TPS2和TRE1-1的表达虽也极显著下降,但TRE1-2和TRE2极显著上调;在注射dsNlTret1-like X1后,试虫可溶性海藻糖酶和膜结合型海藻糖酶的活性均极显著降低,而在注射dsNlTret1-2 X1后无显著变化。【结论】褐飞虱的两个Tret1在不同组织间发挥着不同的功能,其中NlTret1-like X1在特异性转运海藻糖参与能量供应中起到更为显著的作用。研究结果有助于探索Tret1在昆虫或无脊椎动物中调控海藻糖代谢平衡的调节机制,可为将来通过调控血糖平衡来控制褐飞虱等害虫提供理论依据。     

Effect of trehalose supplementation in milk replacer on the incidence of diarrhea and fecal microbiota in preweaned calves

Trehalose, a nonreducing disaccharide consisting of d-glucose with α,α-1,1 linkage, was evaluated as a functional material to improve the gut environment in preweaned calves. In experiment 1, 173 calves were divided into two groups; the trehalose group was fed trehalose at 30 g/animal/d with milk replacer during the suckling period, and the control group was fed nonsupplemented milk replacer. Medication frequency was lower in the trehalose group (P < 0.05). In experiment 2, calves (n = 20) were divided into two groups (control group [n = 10] and trehalose group [n = 10]) based on their body weight and reared under the same feeding regimens as in experiment 1. Fresh feces were collected from individual animals at the beginning of the trial (average age 11 d), 3 wk after trehalose feeding (experimental day 22), and 1 d before weaning, and the fecal score was recorded daily. Fecal samples were analyzed for fermentation parameters and microbiota. The fecal score was significantly lower in the trehalose group than in the control group in the early stage (at an age of 14 to 18 d; P < 0.05) of the suckling period. Calves fed trehalose tended to have a higher proportion of fecal butyrate on day 22 than calves in the control group (P = 0.08). Population sizes of Clostridium spp. were significantly lower (P = 0.036), whereas those of Dialister spp. and Eubacterium spp. tended to be higher in the feces of calves in the trehalose group on day 22 (P = 0.060 and P = 0.083). These observations indicate that trehalose feeding modulated the gut environment and partially contributed to the reduction in medication frequency observed in experiment 1.     

Induction of Drought Tolerance in Maize (Zea mays L.) due to Exogenous Application of Trehalose: Growth,Photosynthesis, Water Relations and Oxidative Defence Mechanism

The present investigation was conducted to assess the ameliorative effects of foliar‐applied trehalose on growth, photosynthetic attributes, water relation parameters and oxidative defence mechanism in two maize cultivars under field water deficit conditions. Various components of the experiment comprised two maize cultivars (EV‐1098 and Agaiti‐2002), two water‐stress levels (irrigation after 2 weeks and irrigation after 3 weeks during the entire period of growth), and two levels of trehalose (0 and 30 mm ) and four replicates of each treatment. Water stress significantly reduced the plant biomass production, photosynthetic attributes and water relation parameters in both maize cultivars. In contrast, water stress considerably increased the leaf malondialdehyde (MDA) contents, the activities of antioxidant enzymes such as peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), and the levels of non‐enzymatic compounds such as ascorbic acid and tocopherols. In contrast, water stress caused a marked reduction in leaf phenolic contents. Foliar‐applied trehalose significantly increased plant biomass production, and improved some key photosynthetic attributes and plant–water relation parameters. The ameliorative effect of exogenously applied trehalose was also observed on the activities of some key antioxidant enzymes (POD and CAT) and non‐enzymatic compounds (tocopherols and phenolics). Overall, exogenously applied trehalose considerably improved drought tolerance of maize plants by up‐regulating photosynthetic and water relation attributes as well as antioxidant defence mechanism.     

Prd29A启动子在小麦中的诱导表达活性及大肠杆菌海藻糖合成酶基因的转化

以gus基因为报告基因，通过瞬时表达测定了Prd29A诱导型启动子在小麦愈伤组织中的诱导表达活性。结果表明，Prd29A-gus基因的表达水平可在不同浓度NaCl盐的胁迫条件下得到显著提高。由此证实，Prd29A启动子在小麦中具有较强的诱导表达活性。在此基础上，将Prd29A诱导型启动子驱动下的大肠杆菌海藻糖合成酶基因（otsA）采用花粉管途径导入目的小麦品系，经PCR筛选、Southern鉴定及转化后代海藻糖含量的测定，获得了一批otsA转基因植株及株系，为进一步选育耐盐抗旱的转基因小麦新品系奠定了基础．     

Potassium fertilization improves apple fruit (Malus domestica Borkh. Cv. Fuji) development by regulating trehalose metabolism

Potassium (K) fertilization and antioxidant enzymes both positively influence plant growth and development. However, it is not known whether K treatment improves fruit development via increasing soluble sugar. In this study, K-treated apple (Malus domestica Borkh.) fruit was harvested from 60 to 150 days after full bloom and was analyzed for ROS production and trehalose metabolism. The results show that K fertilization improved fruit firmness, increased growth according to several parameters, increased soluble sugar content, and decreased ROS production. The ascorbate metabolic pathway more effectively reduced ROS production than catalase and peroxidase (POD) did under K treatment. Trehalose-treated fruit also showed higher activity of ascorbate-related enzymes (DHAR, GR, and APX) compared with non-treated fruit. The changes of antioxidant enzyme activity in trehalose-treated fruit corresponded to those in K-treated fruit. Moreover, trehalase (TREH) activity in fruit was notably reduced by K treatment. This demonstrates that K influences ROS production via regulating trehalose content and TREH activity in fruit. This study provides new insight into the K mechanism which improves fruit development, including fruit firmness and size.     

谷氨酸棒杆菌麦芽寡糖基海藻糖合成酶基因的克隆与表达

采用PCR方法从谷氨酸棒杆菌（Corynebacterium glutamicum）基因组中分离得到2.4 kb的麦芽寡糖基     

外源海藻糖调节西瓜细胞渗透胁迫抗性的研究

为探究外源海藻糖对渗透胁迫下西瓜细胞生长的影响,本研究以西瓜悬浮培养细胞为试材,测定外源海藻糖预处理后,渗透胁迫对西瓜悬浮培养细胞生长量、细胞外pH、细胞内ROS（活性氧簇）相对含量和微管骨架的影响。结果表明：渗透胁迫下西瓜细胞的生长量明显受到抑制,并且渗透胁迫可诱导西瓜悬浮培养细胞质外体碱化,ROS迸发,细胞微管骨架发生解聚;外源添加海藻糖可在一定程度上缓解渗透胁迫对西瓜悬浮细胞生长量的抑制作用,调节pH、ROS表达水平、并维持微管骨架结构的完整性。以上研究结果表明渗透胁迫下,海藻糖对西瓜细胞具有维持细胞生长、保护亚细胞结构并调节抗性反应的功能。     

海藻糖生物特性及其在ELISA技术研发中的应用

海藻糖属非还原性双糖,在生物体内作为一种贮藏性糖类,可提供能量来源,同时它也是一种重要的应激代谢产物,对应激状态具有高度抗性。外源性海藻糖对细胞、抗体等生物活性物质同样具有非特异性生物保护作用。文章综述了海藻糖的能量储备、抗应激、细胞稳定剂等生物学特性,以及对细胞、抗体和ELISA中包板抗原或抗体的保护作用,并对其在ELISA技术改善中的应用前景做了推测与展望。     

木薯种茎离体脱水胁迫下生理生化响应研究

木薯是热带和亚热带地区重要的粮食和经济作物。木薯种茎是目前唯一的繁殖方式,然而采收后木薯种茎如何应对脱水胁迫及糖代谢基因的表达尚不清楚。本研究通过检测对照组和保水处理组的木薯种茎在采后不同时间点水分和糖分的含量变化来探究离体条件下木薯种茎内糖类发挥的作用。通过检测糖类代谢相关基因的表达来解析不同糖分间的代谢关系及离体种茎的活性状态。结果表明,在保水处理组中随着采后时间的延长,种茎中的果糖、葡萄糖和蔗糖的含量先降低后升高,而海藻糖含量变化正好相反。同时,保水处理组的果糖、葡萄糖和蔗糖的含量显著高于对照组,而海藻糖含量显著低于对照组。qPCR分析发现,严重的脱水胁迫显著地提高了糖酵解基因的表达。这些结论表明,脱水胁迫显著影响木薯茎中糖类的相互转化。推测脱水胁迫前20 d通过合成海藻糖来响应脱水胁迫,在20~30 d则通过分解海藻糖为木薯种茎提供必要的能量物质。同时本研究通过甘油处理提高了木薯种茎的贮藏时间,为木薯种茎的贮藏提供新的方法。     

外源海藻糖对NaHCO3胁迫下甘草幼苗生长调节及总黄酮含量的影响

本研究以甘草无菌苗为试验材料,采用植物组织培养的方法,分析50 mmol·L^-1 NaHCO₃ 胁迫下外源海藻糖对甘草幼苗生长量、叶绿素含量、渗透调节物质含量、抗氧化保护酶活性和总黄酮含量的影响。结果表明： 50 mmol·L^-1 NaHCO₃ 胁迫显著降低了甘草幼苗的生长量、叶绿体色素含量、K⁺ 浓度、抗氧化保护酶（SOD、POD、CAT）活性和总黄酮含量,显著提高了丙二醛、脯氨酸和可溶性糖含量以及Na⁺浓度;施加15 mmol·L^-1 海藻糖可显著提高甘草幼苗的生长量,提高叶绿素含量、K⁺ 浓度和总黄酮含量,降低丙二酸含量、脯氨酸含量、可溶性糖含量和Na⁺ 浓度,并且提高抗氧化保护酶活性。因此,NaHCO₃胁迫下施加外源海藻糖对甘草幼苗生长具有良好的调节作用,可以增强甘草的抗碱能力,促进甘草幼苗生长。本研究为外源海藻糖提高甘草耐碱性和揭示其调控机制提供理论依据。