玉米C_4光合酶基因导入对拟南芥光合特性及抗旱性的影响

为了解玉米C_4型光合酶基因对C3植物拟南芥光合特性的影响及其对干旱胁迫的响应,分别以过表达ZmPEPC(磷酸烯醇式丙酮酸羧化酶)、ZmPPDK(丙酮酸磷酸二激酶)和ZmNADP-ME(依赖于NADP的苹果酸酶)单个酶基因的拟南芥株系(分别简写为PC、PK、ME),以及过表达PEPC+PPDK和PPDK+NADP-ME两个酶基因的拟南芥株系(分别简写为PCK、PKM)为供试材料,在开花期停止浇水,并分别于干旱胁迫处理前1天、第5天、第10天和结束干旱胁迫复水处理5d时测定转基因拟南芥中目标基因的表达量、光合速率、水分利用效率和酶活性。结果表明,在正常生长条件下,PCK类型株系的PEPC酶活性、PPDK酶活性、净光合速率(Pn)以及水分利用效率(WUE)较野生型拟南芥分别高52%、20%、24%和55%,除PPDK酶活性外,PCK类型株系各指标测定值的增幅均高于其他转基因类型株系,综合表现最优。不同类型株系的上述测定指标总体表现为PCKPCPK、PKMME。干旱胁迫处理5d时,各类型株系中目标基因的表达量、光合酶活性、Pn和WUE均有所上升。干旱胁迫处理10d时,拟南芥受到严重损伤,上述指标的测定值均下降。复水5d时上述指标得到不同程度的恢复,不同类型转基因株系的各项测定指标在不同干旱胁迫处理中总体仍表现为PCK最优,PC次之。各转基因株系均优于受体非转基因拟南芥。     

小麦和谷子C_4光合途径关键酶活性及其与光合和蒸腾的关系

为了解小麦和谷子叶片中C4光合途径关键酶磷酸烯醇式丙酮酸羧化酶(PEPC)、NADP-苹果酸酶(NADP-ME)、磷酸丙酮酸二激酶(PPDK)和苹果酸脱氢酶(MDH)的活性差异及其与光合速率和蒸腾速率的关系,以4个小麦品种(中国春、晋麦47、绵阳11、小偃6号)和4个谷子品种(豫谷1号、吨谷1号、冀谷19、冀谷20)为材料,在苗期、拔节期、抽穗期、开花期和灌浆期,分别测定了小麦和谷子叶片中四种光合酶的活性、净光合速率和蒸腾速率。结果表明,在小麦生育期,叶片中均可检测到这四种光合酶的活性,且酶活性与净光合速率变化趋势一致;谷子叶片中四种光合酶活性均远高于小麦;不同小麦和谷子品种间四种光合酶活性存在显著差异。小麦的PPDK和NADP-ME活性均与净光合速率呈显著正相关,与蒸腾速率相关不显著;谷子的MDH、PEPC和PPDK活性均与净光合速率呈显著正相关,仅豫谷1号的PEPC活性、冀谷20和吨谷1号的MDH活性与蒸腾速率呈显著正相关。     

转C43基因水稻能量亏缺及其补偿技术探讨

转C43基因(PEPC+ PPDK+ ME)水稻光合速率与转PEPC单基因水稻相近,存在能量亏缺现象.分析了转C43基因水稻能量亏缺的原因,探讨了生产上可用的能量亏缺补偿技术,为转C4基因水稻的推广提供技术途径.     

玉米C_4型全长pepc基因导入普通小麦的研究

为了获得具有类似C4光合途径的转pepc(磷酸烯醇式丙酮酸羧化酶)基因小麦材料,采用基因枪法将玉米C4型全长pepc基因导入小麦材料01H186-20-24,经在含4 mg.L-1L-PPT(L-phosphinothricin,草丁膦)的培养基上筛选,获得抗性再生植株,经PCR检测获得118株T0代阳性植株;进一步利用PCR对T1代植株进行筛选,T2代在隔离条件下种植于大田,Southern blot、SDS-PAGE检测表明,外源pepc基因在小麦基因组中得以整合和表达。测定了40个转基因T2代植株和受体对照的净光合速率(Pn)、PEPC活性,结果表明,在大田自然条件下,82.5%的转基因小麦的Pn增加,最大提高18.57%,达到了28.1μmol CO2.m-2.s-1,转基因植株中PEPC活性为对照的1.5~1.98倍。     

甘蔗C4磷酸烯醇式丙酮酸羧化酶基因转化烟草研究初报

将全长6.8 kb含所有内含子及部分5’侧端和3’末端的甘蔗C4磷酸烯醇式丙酮酸羧化酶(PEPC)基因插入植物目的基因载体pBI121,经农杆菌介导转化获得转基因烟草,PCR检测和dot southern分析证实外源基因已整合进入烟草基因组中。甘蔗C4Ppc基因导入烟草后其叶片PEPC活性有所提高,但提高较小。因此,有必要继续进行克隆目的基因的5’侧端和3’末端获得其完整序列后用于C3作物的遗传转化研究。     

共转化法获得无筛选标记的转PEPC、PPDK基因水稻恢复系纯合体

用根癌农杆菌共转化的方法将pSB130/CK质粒[目的基因磷酸烯醇式丙酮酸羧化酶基因(PEPC)、磷酸丙酮酸二激酶基因(PPDK)与筛选标记潮霉素抗性基因HPT分别构建于2个T-DNA区]导入水稻恢复系R299,经PCR、Southern检测,筛选到T0代13个转基因植株。对T1～T3代转基因后代进行PCR跟踪鉴定,分离出无潮霉素抗性基因的转基因植株(PEPC PPDK HPT-),分离株系比率为15%～21%,分离株系中PEPC PPDK HPT-型单株分离频率为7.2%～10.0%。在T3代成功获得了无筛选标记的转基因纯合系3个(06D351,06D352和06D353),PEPC酶活性和光合速率分析结果表明,其PEPC活性比对照提高了1.5～4.9倍,光合速率提高了19%～40%,说明这些转基因纯系材料在水稻高光效育种上具有很大的应用价值。     

水稻光合对不同光强的响应及品种间差异

 报道了水稻光合对不同光强的响应能力及品种间差异。应用14C同位素示踪技术在连续强光和遮荫条件下，测定了籼粳杂交稻亚优2号及其亲本粳稻02428，籼稻3O37和籼型杂交稻汕优63的光合速率，双磷酸核酮糖羧化酶(RuBPC)和磷酸烯醇式丙酮酸羧化酶(PEPC)活性。结果表明：籼粳杂交稻亚优2号在强光和弱光下光合速率和C3光合酶RuBPC活性比籼型杂交稻汕优63抑制较少，表现有比较稳定的光合特性。值得注意的是在光抑制条件下，耐光抑制水稻品种的C4光合酶PEPC有诱导增加活性的现象。与其两个亲本相比较，籼粳杂交稻亚优2号的光合特性更相似于粳稻02428。因此，在配组具有优良光合性状的籼粳杂交稻品种时，广亲和高光效种质02428是一个值得利用的材料。     

外源玉米pepc基因对小麦C_3、C_4途径相关基因表达的效应

为进一步探讨外源玉米pepc基因改良小麦光合性能的机制,以基因枪介导T4代的转玉米pepc基因小麦为试验材料,研究了抽穗期和灌浆期外源pepc基因对小麦内源光合相关酶基因表达的影响,并分析了转基因小麦的光合生理特性及其产量性状表现。结果表明,小麦抽穗期,pepc基因的表达上调了小麦C4微循环ppdk基因(丙酮酸磷酸双激酶基因)、nadp-me基因(NADP-苹果酸酶基因)、ca基因(碳酸酐酶基因)和C3循环rbcL基因(Rubisco大亚基基因)的表达;小麦灌浆期,pepc基因的表达仍显著上调C4微循环ppdk基因和nadp-me基因的表达,而ca基因和C3循环rbcL基因、rbcS基因(Rubisco小亚基基因)的表达量与对照差异不大。相应的酶活性在转基因植株中比对照有所提高,灌浆期增幅最大。两个测定时期的转基因小麦旗叶净光合速率(Pn)均比对照显著提高,在灌浆期增幅最大,比对照提高10.35%~22.77%。产量性状方面,转基因小麦的单穗粒数和收获指数均有所提高。上述研究结果表明,玉米C4型pepc基因导入小麦后,促进了小麦原有的C4循环,从而提高了小麦的光合效率和籽粒产量。     

小麦不同进化材料叶与非叶器官C4光合酶活性及δ13C值差异

为明确小麦进化过程中C_4代谢酶活性的变化趋势,以二倍体小麦种(野生一粒小麦、栽培一粒小麦、拟斯卑尔脱山羊草、粗山羊草)、四倍体小麦种(野生二粒小麦、栽培二粒小麦、阿拉拉特、提莫菲维小麦)及六倍体小麦种(斯卑尔脱小麦、普通小麦)为试验材料,对不同染色体倍数小麦种不同绿色器官光合碳同化酶(PEPC、RuBPC、NADP-MDH、NADP-ME、NAD-ME)活性及稳定碳同位素(δ~(13)C)值进行了比较分析。结果表明,在同一种内,非叶器官(叶鞘、穗下节间、芒、护颖、外颖)RuBPC活性显著低于旗叶,除六倍体小麦品种外,其余小麦材料的PEPC及其他C_4途径酶活性均高于叶片。在小麦进化过程中,随着染色体倍数的增加,旗叶片的C_3和C_4同化酶活性均上升,且PEPC/RuBPC活性比值显著增加;非叶器官的RuBPC活性增加,但PEP羧化酶及其他C_4途径酶活性下降,PEPC/RuBPC活性比值显著降低。旗叶和穗器官的δ~(13)C值均在C_3途径范围内,穗器官的δ~(13)C值高于叶片;随染色体倍数的增加,旗叶δ~(13)C值增高,而穗器官的δ~(13)C值下降。综合来看,小麦非叶器官具有较强的C_4光合酶活性,在进化过程中,其C_4酶活性趋向减弱,而旗叶的C_4酶活性则趋向增加。     

不同大豆品种幼苗叶片光合及叶绿素荧光 特性对套作遮荫的响应

选用4个耐荫性有差异的大豆品种为材料,通过与单作比较,研究了不同大豆品种幼苗叶片光合及叶绿素荧光特性对套作遮荫的响应差异。结果表明,套作遮荫提高了大豆叶片叶绿素含量、总叶绿素与类胡萝卜素比值（Chl(a+b)/Car）、胞间二氧化碳浓度（Ci）、水分利用效率（WUE）、叶片初始荧光（Fo）、PSⅡ有效光化学量子产量（Fv′/Fm′）和实际光化学效率（ΦPSⅡ）;使叶绿素a与叶绿素b比值（Chla/b）、类胡萝卜素(Car)含量、净光合速率（Pn）、气孔导度（Gs）、气孔限制值（Ls）、蒸腾速率（Tr）、PSⅡ最大光化学量子产量（Fv/Fm）、光化学荧光猝灭系数（qP）、非光化学荧光猝灭（NPQ）呈减小趋势。套作遮荫降低了大豆地上部分干物质积累量,其干物质积累量与Pn呈极显著正相关。耐荫性相对较好的材料,如贡选1号和南豆12在套作遮荫下Pn下降较少,且有着相对较高的Chlb、Car分子组成比例、Fo和Fv/Fm。     

水稻孕穗期不同叶位叶片的气体交换与叶绿素荧光特性

研究了6个水稻品种孕穗期不同叶位的气体交换和叶绿素荧光特性,结果表明净光合速率([i]P[/i][sub]n[/sub]）、气孔导度（[i]g[/i]）、蒸腾速率（[i]E[/i]）和水分利用效率（[i]WUE[/i]）均随叶位降低而明显下降;而胞间CO[sub]2[/sub]浓度（[i]C[/i][sub]i[/sub]）基本维持不变,说明叶片衰老时[i]P[/i][sub]n[/sub]的下降主要不是气孔因素引起的。叶绿素含量降低与[i]P[/i][sub]n[/sub]下降的表现一致,叶绿素荧光分析表明[i]F[/i][sub]v[/sub]/[i]F[/i][sub]m[/sub]和[i]F[/i]'[sub]v[/sub]/[i]F[/i]'[sub]m[/sub]没有随叶位发生变化,而PSⅡ的光化学效率[i]Ф[/i][sub]PSⅡ[/sub]和电子传递速率([i]ETR[/i]）随叶位降低明显下降,这表明可能是PSⅠ电子传递受阻导致CO[sub]2[/sub]同化下降。     

聚乙二醇诱导水分胁迫引起水稻光合下降的原因探讨

在水稻品种Azucena(粳稻)6叶期用不同浓度的聚乙二醇(PEG 6000)模拟干旱处理,结果表明,随着PEG处理浓度提高,净光合速率（Pn）呈下降趋势, 但各阶段起主导作用的因素不同。第一阶段, 在10%PEG处理下Pn显著下降,色素和叶绿素荧光保持不变,气孔导度(Gs)和胞间CO2浓度(Ci)反而上升,表明为非气孔因子限制光合速率;第二阶段,在15%PEG处理下色素和叶绿素荧光虽开始变化,Pn继续降低, 并伴随Gs和Ci的下降而下降,因此气孔导度成为光合速率的主要限制因子;第三阶段,在20%PEG处理下Pn继续降低,Gs虽下降,但Ci并没有进一步下降,而色素和荧光参数均进一步下降,此时光合膜的损伤成为限制光合的主导因子。上述结果表明,严重水分胁迫虽使光合机构吸收和传递光能效率下降,但过剩的激发能仍可能导致活性氧积累;尽管水稻叶内启动了超氧化物防御系统,但仍能导致光合器官受损及PSⅡ光化学活性降低。     

光抑制条件下不同水稻品种叶片的PSⅡ光化学效率和CO2交换特性的差异

 比较了光抑制条件下两个水稻品种叶片的PSⅡ电子传递活性、D1蛋白量、叶绿素荧光Fv／Fm、净光合（PN）和光呼吸（PR）速率、碳酸酐酶（CA）、PEPC和RuBP羧化/加氧酶活性的变化,并对RuBP羧化酶进行了动力学分析。经光抑制处理后,耐光抑制品种02428较对光抑制敏感品种3037中D1 蛋白净降解少、PSⅡ活性和PSⅡ原初光化学效率高,光抑制较轻;RuBP羧化/加氧酶活性和RuBP羧化酶的Km(CO₂)和Vmax(CO₂)值没有变化,且在品种间无差异。CA和PEPC活性出现诱导增高,尤以品种02428中活性诱导的幅度更高。CO₂交换特点发生了显著变化,在PN降低的同时,PR／PN比值明显增加,品种3037的PR／PN比值比02428的更大。在水稻耐光抑制特性上,D1蛋白、CA和PEPC可能分别对PSⅡ光化学效率和CO₂交换起着重要的调节作用。     

蜀恢881含玉米C4型pepc基因改良系的遗传背景及其光合特性

从通过分子标记辅助选择育成的含玉米C4型pepc基因蜀恢881中筛选10株,利用530对SSR引物进行遗传背景分析,发现与蜀恢881表现差异的位点仅有1～4个,相似度在95.15%以上,最高的达9903%。进一步对相似度为9903%的转育玉米C4型pepc基因蜀恢881的6个生长时期测定有关光合指标,表明6个时期的PEPC酶活性均比蜀恢881增强,PEPC酶活性值最大时期是拔节期,为1264.2 μmol/(mg·h）,分蘖初期增加的幅度最大达到23.3倍;其净光合速率与蜀恢881相比,在分蘖初期、分蘖盛期、拔节期和始穗期都得到不同程度的提高,其中拔节期最高,达到22.3%。研究表明通过杂交利用C4型pepc基因特征引物对C4型pepc基因进行分子标记辅助选择,能够获得携有C4型pepc基因且与受体亲本遗传背景十分接近的籼型水稻材料,而且C4型pepc基因在新的遗传背景下能够高水平表达和稳定地遗传。据此,建立了分子标记辅助选择、光合生理生化指标鉴定和田间综合农艺性状考查相结合的筛选玉米C4型pepc基因水稻的技术体系;同时认为在拔节期进行转育C4型pepc基因水稻光合生理指标的测定和筛选的效果可能较理想。     

Variations in structural,biochemical, and physiological traits of photosynthesis and resource use efficiency in Amaranthus species (NAD-ME-type C4)

C₄ plants show higher photosynthetic capacity and productivity than C₃ plants owing to a CO₂-concentrating mechanism in leaves, which reduces photorespiration. However, which traits regulate the photosynthetic capacity of C₄ plants remains unclear. We investigated structural, biochemical, and physiological traits associated with photosynthesis and resource use efficiency in 20 accessions of 12 species of Amaranthus, NAD-malic enzyme-type C₄ dicots. Net photosynthetic rate (P_N) ranged from 19.7 to 40.5 μmol m^?2 s^?1. P_N was positively correlated with stomatal conductance and nitrogen and chlorophyll contents of leaves and was weakly positively correlated with specific leaf weight. P_N was also positively correlated with the activity of the C₃ enzyme ribulose-1,5-bisphoshate carboxylase/oxygenase, but not with the activities of the C₄ enzymes phosphoenolpyruvate carboxylase and NAD-malic enzyme. Structural traits of leaves (stomatal density, guard cell length, leaf thickness, interveinal distance, sizes of mesophyll and bundle sheath cells and the area ratio between these cells) were not significantly correlated with P_N. These data suggest that some of the biochemical and physiological traits are involved in interspecific P_N variation, whereas structural traits are not directly involved. Photosynthetic nitrogen use efficiency ranged between 260 and 458 μmol mol^?1 N s^?1. Photosynthetic water use efficiency ranged between 5.6 and 10.4 mmol mol^?1. When these data were compared with previously published data of C₄ grasses, it is suggested that common mechanisms may determine the variations in resource use efficiency in grasses and this dicot group.     

Effects of Nitrogen Fertilizer Level on Chlorophyll Fluorescence Characteristics in Flag Leaf of Super Hybrid Rice at Late Growth Stage

To compare the effects of slow-release nitrogen fertilizer at six different levels on the flag leaf chlorophyll fluorescence characteristics of super hybrid rice,a field fertilization experiment was conducted with super hybrid rice Y Liangyou 1 as a test material.The photosynthetic electron transport rate (ETR),effective quantum yield (EQY),photochemical quenching coefficient (q P),and non-photochemical quenching coefficient (NPQ) of flag leaves were measured at the initial heading,full heading,10 d after full heading and 20 d after full heading stages.Results showed that the values of ETR,EQY and q P increased with rice development from initial heading to 20 d after full heading,whereas the NPQ decreased.During the measured stages,ETR,EQY and q P increased initially and then decreased as nitrogen application amount increased,but they peaked at different nitrogen fertilizer levels.The maximum ETR and EQY values appeared at the treatment of 135 kg/hm 2 N.In conclusion,the optimum nitrogen amount for chlorophyll fluorescence characteristics of super hybrid rice was 135 180 kg/hm 2.     

Variations in physiological,biochemical, and structural traits of photosynthesis and resource use efficiency in maize and teosintes (NADP-ME-type C4)

C₄ plants show higher photosynthetic capacity and resource use efficiency than C₃ plants. However, the genetic variations of these traits and their regulatory factors in C₄ plants still remain to be resolved. We investigated physiological, biochemical, and structural traits involved in photosynthesis and photosynthetic water and nitrogen use efficiencies (PWUE and PNUE) in 22 maize lines and four teosinte lines from various regions of the world. Net photosynthetic rate (P_N) ranged from 32.1 to 46.5 μmol m^?2 s^?1. P_N was positively correlated with stomatal conductance, transpiration rate, and chlorophyll, nitrogen and soluble protein contents of leaves, but not with specific leaf weight. P_N was positively correlated with the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase and the C₄-acid decarboxylases, NADP-malic enzyme and phosphoenolpyruvate carboxykinase, but not with the activity of phosphoenolpyruvate carboxylase. Leaf structural traits (stomatal parameters, leaf thickness, and interveinal distance) were not correlated with P_N. These data suggest that physiological and biochemical traits are involved in the genetic variation of P_N, but structural traits are not directly involved. PWUE is in the lower class of values reported for C₄ plants, whereas PNUE is in the highest class of values reported for C₄ plants. PNUE was negatively correlated with leaf nitrogen content but not significantly correlated with P_N. PWUE was not correlated with δ¹³C values of leaves, indicating difficulty in using δ¹³C values as an indicator of PWUE of maize. In general, teosinte lines showed lower P_N but higher PWUE than maize lines.     

Structural and physiological responses of the C4 grass Sorghum bicolor to nitrogen limitation

Nitrogen (N) is one of the major nutrients influencing photosynthesis and productivity of C₄ plants as well as C₃ plants. C₄ photosynthesis operates through close coordination between mesophyll (M) and bundle sheath (BS) cells. However, how the development of structural and physiological traits in leaves of C₄ plants is regulated under N limitation remains uncertain. We investigated structural and physiological responses of leaves of the NADP-ME-type C₄ grass Sorghum bicolor to N limitation. Plants were grown under four levels of N supply (.05 to .6 g N per 5-L pot). Decreasing N supply resulted in decreases in net photosynthetic rate, stomatal conductance, leaf N and chlorophyll contents, and the activity ratio of phosphoenolpyruvate carboxylase to ribulose 1,5-bisphosphate carboxylase/oxygenase and increases in δ¹³C values and photosynthetic N use efficiency. Low-N leaves were thinner and had smaller photosynthetic cells, especially in M, resulting in lower M/BS tissue area ratio, and contained smaller and fewer chloroplasts. The BS chloroplasts in the low-N leaves accumulated abundant starch grains. The number of thylakoids per granal stack was reduced in M chloroplasts but not in BS chloroplasts. The low-N leaves had thicker cell walls, especially in the BS cells, which might be associated with less negative δ¹³C values, and fewer plasmodesmata in the BS cells. These data reveal structural and physiological responses of C₄ plants to N limitation, most of which would be related to cellular N allocation, light use, CO₂ diffusion and leakiness, and metabolite transport under N limitation.     

Photosynthetic Characteristics of an Amphibious C4 Plant,Eleocharis retroflexa ssp. chaetaria

Abstract

Eleocharis retroflexa (Poir.) Urban ssp. chaetaria (Roem. & Schult.) T. Koyama, an amphibious leafless sedge, grows not only under terrestrial conditions but also under completely submerged aquatic conditions. We investigated the photosynthetic traits and structural features of the culms, which are the photosynthetic organs, in the terrestrial and submerged forms of this species and compared them with those of other amphibious species of Eleocharis which are known to change the photosynthetic modes. The culms of the terrestrial form had Kranz anatomy with well-developed Kranz (bundle sheath) cells and high levels of C₄ enzyme activity typical of the NAD-malic enzyme (NAD-ME) subtype of C₄ metabolism. They also had a δ ¹³C value typical of C₄ plants, indicating that the terrestrial form fixes carbon through the C₄ pathway. The culms of the submerged form had not only a Kranz-like anatomy but also revealed anatomical traits typical of leaves of submerged aquatic plants. The activities of the C₄ enzymes in the submerged form were lower than those in the terrestrial form, but were still in the range typical of G4 plants, ¹⁴C pulse-¹²C chase experiments with the submerged form indicated that almost all of the fixed ¹⁴C was incorporated into G4 compounds, and subsequently the raioactivity was transferred into C₃ compounds and sucrose. The submerged form showed no diurnal fluctuation in malate level. These data demonstrate that a C₄ metabolism is operative even in the submerged form. This unique amphibious C₄ plant provides an intriguing example of the physiological and ecological adaptability of C₄plants.