水稻质体磷酸盐转运体OsPPT家族成员的功能分析

磷酸烯醇式丙酮酸/磷酸盐转运体(PPT)是植物质体磷酸盐转运蛋白家族(pPTs)成员之一,介导细胞质中的磷酸烯醇式丙酮酸(PEP)进入质体基质的同时,将磷交换到细胞质中。为对水稻OsPPT基因家族进行综合分析,探索其在水稻中的潜在功能。利用水稻原生质体瞬时转化分析OsPPT的亚细胞定位,通过酵母异源表达实验分析OsPPT的磷酸盐转运能力。设置正常供磷和缺磷等非生物胁迫水培实验处理,阐明OsPPT家族成员的组织特异性表达模式,以及对非生物胁迫逆境的响应。结果表明,OsPPT基因家族4个成员均定位于叶绿体膜,而且OsPPT可以在酵母中介导磷酸盐的跨膜转运。此外,通过实时荧光定量PCR(qRT-PCR)展示了OsPPT基因家族在应对环境胁迫时表达模式上的动态变化,比如磷饥饿,以及脱落酸(abscisic acid, ABA)、水杨酸(salicylic acid, SA)、氯化钠等非生物胁迫环境。OsPPT基因家族可能参与磷酸盐在细胞质和叶绿体之间的运输,同时也可能参与植物对逆境胁迫的响应。     

稗草(Echinochloa crusgalli)根型ppc基因对水稻的遗传转化及其对光合速率的调节效应

稗草(Echinochloa crusgalli)是稻田中的C4光合型杂草,为了探索稗草ppc基因(Eppc)对水稻遗传转化的可行性及其对光合速率的调节效应,首次将含有稗草根型磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase,PEPC)基因ppc c DNA的2个植物表达载体p Ubi-Eppc、p Rbc S-Eppc通过农杆菌介导法对水稻进行了遗传转化。对分化植株进行的PCR、RT-PCR、克隆测序和Western杂交等结果均表明稗草ppc基因已经整合到了水稻基因组中,并且在转录和翻译水平都得到了表达。转基因水稻PEPC活性和气体交换参数测定结果表明T0代多数植株的PEPC活性高于对照,最高达到了对照的5.85倍;T0代大多数转基因植株叶片的净光合速率(Pn)比对照提高了20.00%,最大地提高了47.16%,同时叶片水分利用效率(WUE)也得到增强;T6代大部分转化植株的PEPC活性及Pn仍保持高于对照,本研究表明C3根型ppc基因过量表达也可以提高水稻的Pn,且证明稗草PEPC对光合作用具有较强的调节作用。     

大豆磷酸烯醇式丙酮酸磷酸酯酶(PEPP)研究: Ⅱ.纯化与特性

从大豆叶片中分离磷酸烯醇式丙酮酸磷酸酯酶（phosphoenolpyruvate phosphatase，PEPP），通过硫酸胺分部（20％～50％饱和度）沉淀、DEAE-纤维素层析、羟基磷灰石层析将酶纯化了96．81倍，酶活性达17．91U／mg蛋白。该酶专一性较强，米氏常数（Km）为0．39mmol／L（PEP），最适pH6．8，在PH6．O～7．4范围内及50。C以下较稳定，被Mg^2＋、Mn^2＋激活，F^-、Cu^2＋、Zn^2＋、PO^3-、MoO4^2-抑制。     

Morphological and physiological studies on densely branched lateral roots triggered by localized phosphate in Sesbania cannabina

Densely branched lateral roots (DBLRs) in Sesbania cannabina are formed in response to patchily distributed phosphorus (P) in volcanic soils. Little attention has been paid to morphological and physiological responses of DBLRs. Here, we investigated the relation between plant growth and DBLR development, enzymatic activities involved in P acquisition, and the influence of arbuscular mycorrhizal fungi (AMF), which contribute to P uptake, to clarify the function of DBLRs. We investigated DBLR development induced by localized application of P fertilizer and we compared the activities of phosphoenolpyruvate carboxylase (PEPCase) and acid phosphatase (APase) between DBLRs and non‐DBLRs. Additionally, plants were grown with or without AMF to investigate the effect of AMF colonization on the numbers of DBLRs and plant P uptake, and we compared AMF colonization between DBLRs and non‐DBLR roots. Secondary to quaternary lateral DBLRs were produced after the primary lateral roots passed near P fertilizer. P_i content per DBLR increased as DBLRs developed, promoting higher shoot growth. Under P deficiency, PEPCase and APase activities increased in non‐DBLR, but were significantly lower in DBLRs in the same plants. AMF inoculation changed the root system architecture by significantly decreasing the number of DBLRs, and AMF colonization was lower in DBLRs than in non‐DBLRs. Our results indicate that DBLR formation is a P‐coacquisition strategy of S. cannabina grown in P‐deficient andosolic soil. Roots that form DBLR are clearly different from non‐DBLR roots in morphological and biochemical response and AMF symbiosis.     

茶树磷酸烯醇式丙酮酸转运子基因CsPPT的克隆与表达分析

以白叶1号为试验材料,通过RT-PCR和RACE技术克隆获得茶树磷酸烯醇式丙酮酸转运子基因CsPPT（GenBank登录号：KJ652972）。CsPPT完整ORF长度为1β227βbp,编码408个氨基酸,蛋白分子量为44.7βkDa,理论等电点为10.16;无信号肽位点,属于非分泌型蛋白;建立了茶树CsPPT蛋白的系统发育树;磷酸化修饰预测该蛋白质多肽链中共有26个磷酸化位点;TMHMM预测表明CsPPT蛋白为跨膜蛋白;亚细胞定位发现,CsPPT蛋白定位于叶绿体上,推测CsPPT蛋白可能定位于叶绿体膜上。荧光定量PCR结果表明CsPPT基因在茶树花中表达量最高,其次为芽、叶和嫩茎,根中最低。     

盐胁迫对黄瓜幼苗光合作用及其关键酶基因表达特性的影响

以对盐胁迫敏感性不同的两个黄瓜品种中农大22号和戴多星为材料,研究了盐胁迫下黄瓜幼苗叶片光合特性、叶绿体超微结构以及光合作用关键酶1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)和磷酸烯醇式丙酮酸羧化酶(PEPC)活性及其编码基因表达的变化。结果表明:盐胁迫(150 mmol·L~(-1)NaCl处理)下,黄瓜幼苗光合色素(叶绿素和类胡萝卜素)含量、净光合速率、暗呼吸速率和蒸腾速率显著降低;叶绿体类囊体片层结构垛叠程度下降,淀粉粒变小且数量减少;Rubisco大亚基编码基因rbc L、PEPC编码基因Csppc2的表达水平先响应性上调,随后下降;Rubisco和PEPC活性呈降低趋势。相对中农大22号,戴多星具有较强的盐胁迫耐受性。     

Hypoglycemic effect of protopanaxadiol-type ginsenosides and compound K on Type 2 diabetes mice induced by high-fat diet combining with streptozotocin via suppression of hepatic gluconeogenesis

Compound K (CK) is a final intestinal metabolite of protopanaxadiol-type ginsenosides (PDG) from Panax ginseng. Although anti-diabetic activity of CK have been reported with genetic mouse models (db/db mice) in recent years, the therapeutic usefulness of CK and PDG in type 2 diabetes, a more prevalent form of diabetes, remains unclear. In the present investigation, we developed a mouse of non-insulin-dependent diabetes mellitus that closely simulated the metabolic abnormalities of the human disease. For this purpose, type 2 diabetes was induced in male ICR mice by combining of streptozotocin. The male ICR mice fed with HFD for 4 weeks received 100 mg/kg of STZ injected intraperitoneally. After 4 weeks, mice with fasting (12 h) blood glucose levels (FBG) above 7.8 mmol/L were divided into 3 groups (n = 12) and treated with vehicle (diabetes model, DM), 300 mg/kg/day of PDG and 30 mg/kg/day of CK for 4 weeks while continuing on the high-fat diet. Hypoglycemic effects of CK and PDG were consistently demonstrated by FBG levels, and insulin-sensitizing effects were seen during oral glucose tolerance testing (OGTT). Moreover, the mechanism of hypoglycemic effect in type 2 diabetic mice was examined. Gluconeogenic genes, Phosphoenolpyruvate carboxykinase (PEPCK) and Glucose-6-phosphatase (G6Pase), were decreased in two treatment groups with CK showing greater effects. These findings demonstrated the hypoglycemic and insulin-sensitizing capabilities of CK on type 2 diabetes induced by HFD/STZ via down-regulation of PEPCK and G6Pase expression in liver.     

Nutritional regulation of gene expression and enzyme activity of phosphoenolpyruvate carboxykinase in the hepatic gluconeogenesis pathway in golden pompano (Trachinotus ovatus)

Phosphoenolpyruvate carboxykinase (PEPCK) is known to exist as mitochondrial (PEPCK‐M) and cytosolic isoforms (PEPCK‐C). They are involved in glucose production and play vital roles in regulating glucose homeostasis. This study aimed to (1) clone and characterize PEPCK‐C cDNA from golden pompano fish (Trachinotus ovatus), which are known to have poor utilization of dietary carbohydrates, and (2) analyze the regulation of its expression by varying dietary carbohydrate‐to‐lipid ratios and nutritional status. A full length golden pompano PEPCK‐C cDNA fragment of 2,652 bp was cloned, which contains an open reading frame of 1,875 bp encoding 624 amino acids and shows a high homology to cobia (Rachycentron canadum) PEPCK‐C sequence (92% similarity). The analysis of tissue distributions of PEPCK‐C mRNA showed that a high abundance of PEPCK‐C was expressed in golden pompano liver, followed by kidney; while lower expression levels were found in the heart and intestine. In liver, PEPCK is under the regulation of dietary composition and nutritional status at the enzymatic and molecular levels. Our results indicate that PEPCK‐C plays a modulating role in the adaptation of hepatic gluconeogenesis to different nutrient conditions. Thus, the absence of molecular inhibition of gluconeogenic PEPCK gene expression in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata), which can at least partially account for their poor utilization of dietary carbohydrate, is specific to these species and is not observed in golden pompano. Further studies that examine, at the enzymatic and molecular levels, other key enzymes involved in hepatic glucose metabolism are necessary to understand the poor utilization of carbohydrates in golden pompano.     

导入玉米pepc基因杂交稻的光合特性和杂种优势

用含pepc基因的改良蜀恢881与3个不育系冈46A、776A、2480A配组,同时以野生型亲本蜀恢881与上述3个不育系杂交得到的F1为对照,比较了3个含有pepc基因F1及其相应对照在分蘖初期、分蘖盛期、拔节期、始穗期、齐穗期、成熟期以及剑叶完全展开后不同时期的光合指标的动态变化。含有pepc基因的3个F1在不同生育期和剑叶展开后的磷酸烯醇式丙酮酸羧化酶（PEPCase）活力均显著高于各自对照,净光合速率也有所提高,分析表明PEPCase活力与净光合速率有极显著的相关性（0.6081**）。3个含有pepc基因F1的表观量子效率、光补偿点、羧化效率均高于对照,CO2补偿点较对照低,在光饱和点和CO2饱和点时的净光合速率也高于相应对照。在单株产量表现中,含pepc基因的F1较对照的平均增幅达37.10%。试验表明,杂交稻由于pepc基因的导入,光合特性得到了一定改善,奠定了水稻高产的生理基础。