华南地区3个棕榈藤种光合途径的判别

C3植物可以通过转入C4植物基因而具备C4植物光合特性,从而提高产量。有鉴于此,本文通过测定我国华南地区分布的黄藤(Daemonoropsmargaritae(Hance)Becc.)、单叶省藤(CalamussimplicifoliusC.F.Wei)和白藤(C.tetradactylusHance)等3个棕榈藤种苗木和成年植株叶片的叶绿素含量、气孔密度、磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvatecarboxylase,PEPC)、丙酮酸磷酸二激酶(pyruvatephosphatedikinase,PPDK)和稳定碳同位素比值等指标以判别3个藤种的光合途径,为棕榈藤转入C4植物基因工作提供理论依据。结果表明,3种藤种苗木和成年植株的叶绿素含量和气孔密度比常见C3植物和C4植物高,但叶绿素a/b值、叶片上下表面气孔比值、PEPC酶活性、PPDK酶活性和稳定碳同位素比值等指标均较低,与常见C3植物的对应指标相当或略低,而远小于常见C4植物,因此认为黄藤、单叶省藤和白藤等3个藤种是C3植物。     

转C4光合酶基因水稻的CO2交换和荧光特性

用转PEPC、PPDK、NADP_ME、PEPC PPDK酶基因水稻 (OryzasativaL .)及原种为材料 ,研究了光合作用对光照、温度、CO2 的响应和光抑制条件下的叶绿素荧光特性 ,结果如下 :1.转C4 光合酶基因水稻的饱和光合速率比原种高 ,其中转PEPC、PEPC PPDK双基因水稻的光饱和点比原种高 2 0 0 μmol·m-2 ·s-1,饱和光合速率比原种分别高5 1.6 %和 5 8.5 % ;转PEPC基因水稻的羧化效率比原种高 49.3% ,CO2 补偿点降低 2 6 .2 % ;在高温 (35℃ )下 ,转PEPC基因水稻的光合速率比原种高 17.5 %。 2 .经光抑制处理 8d后 ,转PEPC、PEPC PPDK酶基因水稻的PSⅡ光化学效率 (Fv/Fm)和光化学猝灭 (qP)下降 2 0 % - 30 % ,非光化学猝灭 (qN)增加了约 30 % ;但原种的Fv/Fm 和qP下降了 5 0 %多 ,qN变化不明显 ,表明转C4 光合基因水稻耐光抑制能力增强。这些结果为用生物技术提高水稻光合效率研究提供了新的依据和途径     

转C4光合酶基因水稻的CO2交换和荧光特性

用转PEPC、PPDK、NADP-ME、PEPC+PPDK酶基因水稻(Oryza sativa L.)及原种为材料 ,研究了光合作用对光照、温度、CO2的响应和光抑制条件下的叶绿素荧光特性,结果如下: 1.转C4光合酶基因水稻的饱和光合速率比原种高,其中转PEPC、PEPC+PPDK双基因水稻的光饱和点比原种高200 μmol*m-2*s-1,饱和光合速率比原种分别高51.6%和 58.5%;转PEPC基因水稻的羧化效率比原种高49.3%,CO2补偿点降低26.2%;在高温(35 ℃)下,转PEPC基因水稻的光合速率比原种高17.5%.2.经光抑制处理8 d后,转PEPC、PEPC +PPDK酶基因水稻的PSⅡ光化学效率(Fv/Fm)和光化学猝灭(qP)下降20%- 30%,非光化学猝灭(qN)增加了约30%;但原种的Fv/Fm和qP下降了5 0%多,qN变化不明显,表明转C4光合基因水稻耐光抑制能力增强.这些结果为用生物技术提高水稻光合效率研究提供了新的依据和途径.     

转PEPC基因水稻具有初级CO2浓缩机制的生理特点

以原种粳稻Kitaake为对照, 研究了转玉米PEPC基因水稻的PEPC的高表达和碳同化特性的关系. 结果显示: 与原种相比, 转PEPC基因水稻气孔导度和光合速率显著增加, 经统计分析, 气孔导度的增加与光合速率的增加并无相关性. 而在高光强下, 与CO2浓缩有关的PEPC, CA酶蛋白的表达显著增加. 因此在大气CO2浓度下可显著增加光合能力(50%); 无CO2条件下, 可减少叶内CO2释放量, 从而降低了CO2补偿点. 用专一的抑制剂DCDP处理, 证明转PEPC基因水稻叶内PEPC的高表达与碳同化能力的提高和Fv/Fm的稳定性有关. 用14C示踪20 s, 转PEPC基因水稻14C较多的分配在C4光合原初产物天冬氨酸中, 意味着叶内存在着一定的C4光合代谢途径. 上述结果说明, 用代谢工程可以在叶内构建初级的CO2浓缩机制, 为转基因的高光效育种技术提供了生理依据.     

大肠杆菌糖酵解途径和三羧酸循环启动子的表征及其应用

启动子是控制基因转录的重要元件,也是合成生物学研究和细胞工厂设计的关键环节。糖酵解途径和三羧酸循环是糖类分解代谢的中心代谢,受到包括启动子强度在内的严格调控。为了筛选一系列能满足合成生物学研究和细胞工厂设计需要的不同强度的内源性组成型启动子,利用报告基因——红色荧光蛋白m Cherry和在线分析软件,系统研究了大肠杆菌糖酵解和三羧酸循环中27个启动子的强度和核心结构元件。结果表明:这些启动子的强度范围变化很大,最强启动子Pgap A的强度是最弱启动子Pacn A强度的43. 6倍;启动子的-10序列和-35序列与它们的一致序列也不完全相同,两者之间的距离为17±3bp;但是,启动子的强度和启动子的结构特征基本一致。应用最强启动子Pgap A在重组大肠杆菌DH5αΔpck中分别表达磷酸烯醇式丙酮酸羧化酶基因和丙酮酸激酶基因,它们的酶活性分别提高了0. 32和1. 57倍,柠檬酸产量也提高了124. 7%和75. 5%。这些不同强度的启动子为大肠杆菌的合成生物学研究和细胞工厂设计奠定了一定的基础。     

磷酸烯醇式丙酮酸羧化酶和磷酸烯醇式丙酮酸羧激酶介导的草酰乙酸回补途径对谷氨酸棒杆菌V1氨基酸代谢的影响

【目的】谷氨酸棒杆菌是工业生产氨基酸的主要菌株,以缬氨酸高产菌株谷氨酸棒杆菌V1为研究对象,探讨磷酸烯醇式丙酮酸羧化酶(PEPC)和磷酸烯醇式丙酮酸羧激酶(PCK)介导的草酰乙酸回补途径对菌株生理特性以及主要氨基酸代谢流量的影响。【方法】通过基因工程手段,在谷氨酸棒杆菌V1中过表达pepc(编码PEPC)和pck(编码PCK),比较重组菌与出发菌关键酶活性、发酵特性以及主要氨基酸积累量变化。【结果】构建两株重组菌V1-pepc(强化草酰乙酸回补途径)和V1-pck(弱化草酰乙酸回补途径),重组菌生长均较出发菌延缓,总生物量、葡萄糖和硫酸铵消耗基本不变;过表达pck,PCK活性提高22.8%,丙氨酸、缬氨酸、谷氨酸、精氨酸积累量分别提高了11.8%、17.2%、27.8%和19.5%;过表达pepc,PEPC活性提高27.5%,同时PC活性降低12.9%,天冬氨酸族和谷氨酸族氨基酸的整体流量变化不大,丙氨酸族氨基酸的整体流量降低了14.7%。【结论】丙氨酸族氨基酸受此回补途径影响较大,天冬氨酸族氨基酸受此影响较小。     

蓝藻磷酸烯醇式丙酮酸羧化酶的生物信息学分析

应用NCBI、Expasy等在线生物信息学网站对蓝藻磷酸烯醇式丙酮酸羧化酶(PEPC)与其他物种进行序列同源比对,分析相同的保守序列及催化活性位点,构建分子进化树;预测跨膜结构、疏水性/亲水性、二级结构、功能域和模体等.结果显示,蓝藻PEPC与高等植物、细菌、真核藻PEPC同源性都比较低(约为33%),但是它们含有两个类似的活性部位和相同的催化活性位点;该蛋白质是非跨膜的亲水性不稳定蛋白,二级结构以a-螺旋和无规则卷曲为主,舍有一个功能结构域,主要的功能是参与氨基酸的合成.     

光/暗对高粱叶片磷酸烯醇式丙酮酸羧化酶基因表达的调节

高粱幼苗黄化叶片经照光转绿后,其PEP-Case活性提高4～15倍,mRNA含量提高了1.03倍,并测定出PEPCase mRNA的分子量为3.4kb。以等量的总RNA及mRNA进行体外翻译,发现转绿后PEPCase专一性翻译活性提高了51％～53％。这表明光照可以在转录水平上调节PEP-Case的基因表达。     

氮素营养对丙酮酸磷酸二激酶的调节（简报）

成熟苋菜和玉米叶片以 KNO_3为主要氮源的完全营养液离体培养时,可溶性蛋白质和PPDK酶活性随氮浓度增加而增加,环己酰亚胺(20μmol/L)和水杨醛肟(0.5mmol/L )抑制可溶性蛋白质含量和PPDK酶活性。光下氮素促进PPDK酶活性的提高。     

菠萝叶片的稳定碳同位素比与PEP羧化酶及PEP羧激酶活性

专性景天酸代谢（ＣＡＭ）植物菠萝（Ａｎａｎａｓｃｏｍ ｏｓｕｓ （Ｌ．） Ｍｅｒｒ．）的下数第５ 到第３５ 位叶为材料,研究稳定性碳素同位素（δ１３Ｃ）值,磷酸稀醇式丙酮酸（ＰＥＰ）羧化酶和ＰＥＰ羧激酶活性的变化。１１ 个不同叶位叶片的δ１３Ｃ值平均为－ １２．９４‰,最大变幅相差－ ２．０６‰。两个酶的活性呈单峰形变化,第８—１１ 位叶的活性最高,低位叶的酶活下降。本试验条件下,ＰＥＰ羧激酶平均活性比ＰＥＰ羧化酶高３．４ 倍。结果表明,ＣＡＭ 植物的暗下羧化与光下脱羧之间具有一定的协调性。老叶虽然酶活较低,但总的看来,ＣＡＭ 水平变化不大     

Effects of growth light and nitrogen nutrition on the organization of the photosynthetic apparatus in leaves of a C4 plant, Amaranthus cruentus

Properties of C4 photosynthesis were examined in Amaranthus cruentus L. (NAD-malic enzyme (ME) subtype, dicot) grown under different light and nitrogen (N) conditions, from the viewpoint of N investment into their photosynthetic components. In low-light (LL) leaves, chlorophyll content per leaf area was greater and chlorophyll alb ratio was lower than in high-light (HL) leaves. These indicate that LL leaves invest more N into their light-harvesting systems. However, this N investment did not contribute to the increase in the quantum yield of photosynthesis on the incident photon flux density (PFD) basis (Qi) in LL leaves. N allocation to ribulose 1,5-bisphosphate carboxylasel oxygenase (Rubisco) was significantly higher in HL-high N (HN) leaves than in other leaves. On the other hand, N allocation to C4 enzymes [phosphoenolpyruvate carboxylase (PEPC) and pyruvate Pi dikinase (PPDK)] was unaffected by the growth conditions. Maximum photosynthetic rates (Pmax) per Rubisco content were similar irrespective of the growth light treatments. Carbon isotope ratios (delta13 C) in the leaf dry matter were more negative in LL leaves than in HL leaves (LL = -19.3% per hundred, HL = -16.0% per hundred) and independent of leaf N. Vein density was highest in HL-HN leaves, and leaf thickness was unaffected by the growth light treatments. From these results, we conclude that A. cruentus leaves would not acclimate efficiently to low growth light.     

转玉米PEPC基因水稻中有限的C4光合微循环及其生理作用

用转PEPC基因水稻(Oryza sativa L. subsp.japonica Kitaake)和原种水稻Kitaake为材料,研究了不同基因型水稻叶片中的C4光合微循环及其功能.通过测定与光合C4途径有关的关键酶,如磷酸烯醇式丙酮酸羧化酶(PEPC)、NADP -苹果酸酶(NADP -ME)、NADP -苹果酸脱氢酶(NADP -MDH)和丙酮酸磷酸双激酶(PPDK),说明原种水稻叶片中具有完整的C4光合酶体系;用外源OAA或MA饲喂叶切片或叶绿体后明显增加光合速率,证明原种水稻中具有一个有限的光合C4微循环.将玉米的PEPC基因导入原种水稻后,可大幅度提高光合C4微循环的速率.测定不同基因型的CO2交换速率,看出水稻中C4光合微循环的增强有提高净光合速率(Pn)和降低光呼吸速率/净光合速率(Pr/Pn)比值的作用.叶绿素荧光特性分析表明,C4光合微循环的增强伴随着PSⅡ电子传递效率(Fv/Fm)和光化学猝灭(qP)的增加以及非光化学猝灭(qN)的降低;这些结果为通过基因工程手段提高作物光合效率的遗传育种提供了科学根据.     

早春短命植物鸢尾蒜和准噶尔鸢尾蒜的光合途径

该研究以鸢尾蒜属两种早春短命植物准噶尔鸢尾蒜(Ixiolirion songaricum)和鸢尾蒜(Ixiolirion tataricum)为对象,通过解剖结构、光合参数、稳定碳同位素比值(δ¹³C)和光合关键酶活性分析二者的光合途径。结果发现：(1)显微结构和超微结构显示,两种短命植物的叶脉维管束鞘细胞1层、排列较紧密,鞘细胞内含有较多叶绿体且多离心分布,类似C₄花环结构。(2)准噶尔鸢尾蒜和鸢尾蒜最大净光合速率分别为14.81和15.04 μmol·m^-2·s^-1;两者的CO₂补偿点较低,分别为3.57和2.54 μmol·mol^-1,δ¹³C分别为-25.36±0.55‰和-25.76±1.38‰,光合酶PEPC/Rubisco比值分为别0.244和0.322。(3)最大净光合速率、δ¹³C和PEPC/Rubisco比值均说明两种植物为C₃光合途径,但二者均具有类似C₄的维管束鞘结构、较低的CO₂补偿点和暗呼吸速率,并具有高于部分C₃和C₃ C₄中间型植物的PEPC/Rubisco比值,表明两种短命植物的光合途径并非典型的C₃途径,而是C₃ C₄中间型。     

Growth and Photosynthetic Characteristics of Ceriops Roxburghiana under NaCl Stress

The plant growth, net photosynthetic rate (PN), intercellular CO2 concentration (ci), and dry matter production of Ceriops roxburghiana Arn. were significantly increased with increasing salinity from 0 to 400 mM NaCl. At 600 mM NaCl, shoot and root lengths, and dry mass were significantly depressed with respect to control. Absence of diurnal fluctuation of concentrations of organic acids, and the low activity of phosphoenolpyruvate carboxylase and high activity of ribulose-1,5-bisphosphate carboxylase confirmed the operation of C3 pathway in Ceriops even at increasing salinity.     

外源海藻糖增强高表达转玉米C4型PEPC水稻耐旱性的机制

为揭示海藻糖(Tre)调控转玉米(Zea mays) C4型PEPC基因水稻(Oryza sativa) (PC)的耐旱性机制,以PC及其野生型Kitaake (WT)为材料,通过水培试验,研究了Tre和12% (m/v)聚乙二醇(PEG)单独或联合处理对水稻生理生化特性的影响。结果表明,Tre处理可促进PC和WT水稻...     

Relationships between quantum yield for CO2 assimilation, activity of key enzymes and CO2 leakiness in Amaranthus cruentus, a C4 dicot, grown in high or low light

In C(4) photosynthesis, a part of CO(2) fixed by phosphoenolpyruvate carboxylase (PEPC) leaks from the bundle-sheath cells. Because the CO(2) leak wastes ATP consumed in the C(4) cycle, the leak may decrease the efficiency of CO(2) assimilation. To examine this possibility, we studied the light dependence of CO(2) leakiness (phi), estimated by the concurrent measurements of gas exchange and carbon isotope discrimination, initial activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and pyruvate, orthophosphate dikinase (PPDK), the phosphorylation state of PEPC and the CO(2) assimilation rate using leaves of Amaranthus cruentus (NAD-malic enzyme subtype, dicot) plants grown in high light (HL) and low light (LL). phi was constant at photon flux densities (PFDs) >200 micromol m(-2) s(-1) and was around 0.3. At PFDs <150 micromol m(-2) s(-1), phi increased markedly as PFD decreased. At 40 micromol m(-2) s(-1), phi was 0.76 in HL and 0.55 in LL leaves, indicating that the efficiency of CO(2) assimilation at low PFD was greater in LL leaves. The activities of Rubisco and PPDK, and the phosphorylated state of PEPC all decreased as PFD decreased. Theoretical calculations with a mathematical model clearly showed that the increase in phi with decreasing PFD contributed to the decrease in the CO(2) assimilation rate. It was also shown that the 'conventional' quantum yield of photosynthesis obtained by fitting the straight line to the light response curve of the CO(2) assimilation rate at the low PFD region is seriously overestimated. Ecological implications of the increase in phi in LL are discussed.