转基因(SAG12-IPT)青菜的迟衰特性

利用根癌农杆菌感染方法将融合基因SAG12 IPT导入青菜 ,转基因植株明显表现出衰老延迟的生理现象。SAG12 IPT的抗衰老作用表现为 :在衰老过程中转基因青菜叶片中叶绿素含量高于未转基因的青菜 ,PCR分析结果表明该融合基因已经转入青菜中。激素检测结果表明转基因青菜叶片中细胞分裂素含量高于未转基因植株 ,说明抗衰老与叶片内细胞分裂素含量提高有关。另外 ,转基因植株不仅表现出活体植株衰老延迟 ,而且长在植株上的与离体的叶片滞绿时间延长。这些为蔬菜的耐储存育种提供了新的思路 ,同时为该融合基因在十字花科经济作物中的应用提供了理论依据。     

玉米不同叶位叶片叶绿体超微结构与光合性能的研究

对玉米植株基部（第3叶）,中部（果穗叶）和上部（倒2叶）叶位叶片,进行叶绿体超微结构的观察,并测定了叶绿素含量和光合强度,结果表明,不同叶位叶片叶肉细胞中叶绿体的超微结构,随叶位上升而渐趋复杂化,果穗叶最为显著,向上又趋简单,具体表现为基粒片层的数目随叶位上升而增多基质片层和基质也随之增加,果穗叶最多,向上又趋减少,不同叶位叶片叶绿素含量和光合强度,果穗叶高于其它叶位。     

玉米不同叶位叶片叶绿体超微结构与光合性能的研究

对玉米植株基部（第3叶）,中部（果穗叶）和上部（倒2叶）叶位叶片,进行叶绿体超微结构的观察,并测定了叶绿素含量和光合强度,结果表明,不同叶位叶片叶肉细胞中叶绿体的超微结构,随叶位上升而渐趋复杂化,果穗叶最为显著,向上又趋简单,具体表现为基粒片层的数目随叶位上升而增多基质片层和基质也随之增加,果穗叶最多,向上又趋减少,不同叶位叶片叶绿素含量和光合强度,果穗叶高于其它叶位。     

转基因抗早衰棉的获得

利用花粉管通道技术将含有抑制衰老嵌合基因PCSAG12-ipt的pBG121质粒转入早衰型陆地棉品种中棉所10号中,通过对T1代植株进行卡那霉素田间筛选、PCR检测及GUS组织化学染色,获得了12株转基因棉花。在棉花发育进入衰老时期,对转基因植株进行叶绿素和细胞分裂素含量的测定及形态观察,结果表明转基因植株的衰老得到延迟。     

小麦叶片细胞的研究——冬小麦个体发育过程中叶肉细胞及其它类型细胞结构变化的观察

冬小麦农大183植株的主茎在其个体发育过程中共生长13—14片叶片。随着叶位的上升,各叶片的叶面积,叶鞘面积及各叶片所有各类型细胞的形态和结构互有差异,并反映出逐步复杂化的规律,尤其是叶肉细胞;在返青以前的低叶位叶片中,它们的叶肉细胞,三环以下的占80%左右,在高叶位的叶片中,随着叶位上升,多环细胞的类型随着增多。在旗叶中,四环以上的细胞占77%,并常看到多环而又有分枝的细胞。高叶位的叶鞘、小穗的护颖和稃片等绿色组织中的绿色细胞都有着和叶肉细胞相同的峰谷腰环(图1)形态。高叶位的表皮细胞形态也比低叶位的表皮细胞复杂。经过低温春化处理的冬小麦春播植株的第七叶片(旗叶)叶肉细胞类型的组合与秋播植株旗叶(第十三叶)一样。未经春化处理的春播植株第七叶虽然生长时间及生态环境和上述二种植株的旗叶相同,但其叶片细胞的形态和组合却和年前秋播植株的越冬前低叶位叶片相同。这说明了叶片细胞的组成主要决定于内在的生长发育规律。     

水稻谷蛋白启动子驱动下的ipt基因在转基因烟草中的表达

利用农杆菌系统介导 ,采用叶盘转化法 ,将在水稻谷蛋白启动子驱动下的外源ipt基因导入烟草植株中 ,经过抗生素筛选、PCR与测序分析检测出转基因植株。成熟的转基因烟草种子经过ELISA细胞分裂素试剂盒检测 ,发现iPAs含量为对照的 2 .43倍 ,此外 ,种子的重量也增加了 7.8%。     

沙柳SpsLAS基因超表达对拟南芥营养生长的影响

用沙柳SpsLAS基因构建35S∷SpsLAS超表达载体并转化野生型拟南芥,对转基因拟南芥进行表型观察,利用荧光定量PCR,对分枝、生长素及细胞分裂素相关基因进行表达分析。结果显示:(1)成功构建35S∷SpsLAS超表达载体,并获得9株纯合转基因株系,且转基因株系的萌芽速率快于野生型(对照),生活周期也较长;其中7个株系表现为生长迅速、株高增加、莲座叶叶片增大、分枝增加,2个株系表现为矮化、分枝增加、育性降低等一系列变化。(2)荧光定量PCR显示,与对照相比24h时转基因株系幼苗生长素及细胞分裂素途径关键基因无明显变化,4d时各基因在各转基因株系呈上调趋势;30d时分枝相关基因RAX1、RAX3表达量均上调,而MAX1、MAX3、REV、AXR1无明显变化。研究表明,SpsLAS基因过表达对拟南芥株型、莲座叶有明显影响,该研究结果为进一步研究该基因对分枝调控机制奠定了基础。     

过量合成ALA转基因烟草叶片光合与叶绿素荧光特性的研究

以转酿酒酵母Hem1基因的转基因烟草和野生型植株为材料,用Li-6400光合测定仪和PAM-2100叶绿素荧光仪检测了2种烟草不同叶位叶片光合和荧光参数,并考察了它们的生长情况.结果表明:过量合成ALA的转基因烟草植株具有更强的光合能力,并伴随着气孔导度(Gs)和蒸腾速率(Tr)提高.暗适应下转基因植株不同叶位叶片的初始荧光(Fo)没有明显差异,而野生型植株下部叶片F0明显升高;转基因植株最大荧光(Fm)、可变荧光(Fv)、PSⅡ最大光化学效率(Fv/Fm)等参数均显著提高,特别是下部叶片表现得更为明显;在光照下,转基因植株PSⅡ有效光化学效率(Fv′/Fm′)和实际光化学效率(ΦPSⅡ)、荧光猝灭系数(qP)、电子传递速率(ETR)、光化学效率(Pc)以及进入PSⅡ反应中心的能量(Pc Ex)普遍高于野生型,而天线热耗散能量(Hd)以及非光化学荧光猝灭系数(NPQ)等明显低于野生型,且这些差异在基部叶片中表现得尤为突出.可见,过量合成ALA有利于延长烟草叶片光合寿命,提高光化学能量转换效应和光合产物积累,从而促进植株生长.     

转KN1毛果杨细胞分裂素、赤霉素含量及其相关形态结构

为了研究KN1基因超量表达对木本植物生长发育的影响,本研究测定了转KN1基因毛果杨细胞分裂素类激素及赤霉素的含量,并进行表型及解剖结构观察。结果表明,转基因毛果杨植株细胞分裂素类激素ZR、dhZR和iPA的含量分别为14.14ng/g、18.29ng/g和12.43ng/g,明显高于对照的9.44ng/g、12.47ng/g和7.60ng/g,而赤霉素GAs含量为9.61ng/g,低于对照的12.14ng/g,从而引起其植株矮化、分枝增加、叶片增厚、叶色变深、表皮细胞增大、栅栏和海绵组织发达等现象。本研究将为探索KN1基因对木本植物生长发育的影响提供依据。     

转大肠杆菌过氧化氢酶基因棉花的抗旱生理研究

以导入大肠杆菌过氧化氢酶基因KatE的T3代转基因棉花为供试材料,经卡那霉素检测和PCR鉴定,将筛选出的阳性转基因植株与对照棉花进行整个生育期的持续水分胁迫处理直至收获,比较材料间的生理生化指标的差异,鉴定转基因植株的耐旱能力。结果显示:(1)干旱胁迫持续至初蕾期时,转基因棉花与对照植株间各项抗旱生理指标差异均未达到显著水平。(2)水分胁迫持续至盛蕾和盛花期时,转基因棉花叶片相对含水量、光系统Ⅱ最大光化学效率(Fv/Fm)、CAT活性,以及叶片的净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)均显著或极显著高于对照植株,叶绿素含量也都明显高于对照植株。干旱胁迫持续至吐絮期时,转基因棉花的株高、果枝数和铃数均显著或极显著高于对照植株,且转基因棉花和对照的籽棉产量分别比正常灌溉处理降低57.5%和60.1%,全生育期的水分胁迫严重影响了棉花籽棉产量,但转基因棉花的籽棉产量仍显著高于对照。研究表明,在新疆石河子当地自然降水(干旱胁迫)条件下,转KatE基因棉花表现出了较好的生理和生长优势,KatE基因有助于提高棉花的抗旱性。     

Expression of the <Emphasis Type="Italic">ipt</Emphasis> Gene with the AGPase S1 Promoter in Tomato Results in Unbranched Roots and Delayed Leaf Senescence

Transgenic tomato plants were produced with the isopentenyl transferase gene (ipt) ligated to a promoter that is active exclusively in sink tissue. Initially, transgenic plants had smaller, round-scale leaves, swollen stems, and exhibited early development of lateral shoots compared to wild type. Expression of the ipt gene resulted in the formation of unbranched roots on cuttings and delayed senescence in excised leaves. Callus and root formation occurred on excised leaves and leaf discs during dark incubation. The retention percentage of chlorophyll, as well as cytokinin in excised leaves or discs was significantly greater than wild type. Transgenic tomato fruit had elevated levels of cytokinins in the first days after fruit set and these levels were maintained longer during fruit development.     

Expression of isopentenyl transferase gene (<Emphasis Type="Italic">ipt</Emphasis>) in leaf and stem delayed leaf senescence without affecting root growth

A cytokinin biosynthetic gene encoding isopentenyl transferase (ipt) was cloned with its native promoter from Agrobacterium tumefaciens and introduced into tobacco plants. Indolebutyric acid was applied in rooting medium and morphologically normal transgenic tobacco plants were regenerated. Genetic analysis of self-fertilized progeny showed that a single copy of intact ipt gene had been integrated, and T₂ progeny had become homozygous for the transgene. Stable inheritance of the intact ipt gene in T₂ progeny was verified by Southern hybridization. Northern blot hybridization revealed that the expression of this ipt gene was confined in leaves and stems but undetectable in roots of the transgenic plants. Endogenous cytokinin levels in the leaves and stems of the transgenic tobaccos were two to threefold higher than that of control, but in roots, both the transgenic and control tobaccos had similar cytokinin levels. The elevated cytokinin levels in the transgenic tobacco leaves resulted in delayed leaf senescence in terms of chlorophyll content without affecting the net photosynthetic rate. The root growth and morphology of the plant were not affected in the transgenic tobacco.     

转ipt和反义ACO基因番茄的叶片衰老相关特性

以ipt和反义ACO转化的两类转基因番茄纯系为材料,研究在植株不同生长发育阶段,不同叶位中,与叶片衰老相关的生理生化指标.结果表明:两类基因导入番茄后,均可增强内源iPA和IAA表达水平,增加或保持番茄叶片的叶绿素含量、提高光合效率,进而明显地延缓植株的叶片衰老,提高单株果实产量.但它们调控叶片衰老的途径不同,ipt主要通过提高CTK的水平延缓叶片衰老,而反义ACO则主要是通过抑制乙烯生成,间接提高IAA的水平来实现.     

Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis

To determine the role of ethylene during tomato (Lycopersicon esculentum Mill. cv. Alisa Craig) leaf senescence, transgenic ACC oxidase antisense plants were analysed. Northern analysis of wild-type plants indicated that ACC oxidase mRNA accumulation normally begins in pre-senescent green leaves but was severely reduced in the antisense plants. Although the levels of ethylene evolved by wild-type and transgenic leaves increased during the progression of senescence, levels were extremely low in transgenic leaves. Leaf senescence, as assessed by colour change from green to yellow, was clearly delayed by 10–14 days in the antisense plants when compared with wild-type plants. Northern analysis of the photosynthesis-associated genes, cab and rbcS, indicated that levels of the corresponding mRNAs were higher in transgenic leaves which were not yet senescing compared with senescing wild-type leaves of exactly the same age. Northern analysis using probes for tomato fruit ripening-related genes expressed during leaf senescence indicated that once senescence was initiated the expression pattern of these mRNAs was similar in transgenic and wild-type leaves. In the antisense plants chlorophyll levels, photosynthetic capacity and chlorophyll fluorescence were higher when compared with senescing wild-type plants of the same age. Photosynthetic capacity and the quantum efficiency of photosystem II were maintained for longer in the transformed plants at values close to those observed in wild-type leaves prior to the visible onset of senescence. These results indicate that inhibiting ACC oxidase expression and ethylene synthesis results in delayed leaf senescence, rather than inducing a stay-green phenotype. Once senescence begins, it progresses normally. Onset of senescence is not, therefore, related to a critical level of ethylene. The correlation between higher levels prior to senescence and early onset, however, suggests that ethylene experienced by the plant may be a significant contributing factor in the timing of senescence.     

Effects of P(SAG12)-IPT gene expression on development and senescence in transgenic lettuce

An ipt gene under control of the senescence-specific SAG12 promoter from Arabidopsis (P(SAG12)-IPT) significantly delayed developmental and postharvest leaf senescence in mature heads of transgenic lettuce (Lactuca sativa L. cv Evola) homozygous for the transgene. Apart from retardation of leaf senescence, mature, 60-d-old plants exhibited normal morphology with no significant differences in head diameter or fresh weight of leaves and roots. Induction of senescence by nitrogen starvation rapidly reduced total nitrogen, nitrate, and growth of transgenic and azygous (control) plants, but chlorophyll was retained in the lower (outer) leaves of transgenic plants. Harvested P(SAG12)-IPT heads also retained chlorophyll in their lower leaves. During later development (bolting and preflowering) of transgenic plants, the decrease in chlorophyll, total protein, and Rubisco content in leaves was abolished, resulting in a uniform distribution of these components throughout the plants. Homozygous P(SAG12)-IPT lettuce plants showed a slight delay in bolting (4-6 d), a severe delay in flowering (4-8 weeks), and premature senescence of their upper leaves. These changes correlated with significantly elevated concentrations of cytokinin and hexoses in the upper leaves of transgenic plants during later stages of development, implicating a relationship between cytokinin and hexose concentrations in senescence.     

The effect of cytokinin and thidiazuron on tomato inoculated with endomycorrhiza

Seeds of tomato (Lycopersicon lycopersicum var. Peto 86) were planted in soil inoculated with two vesicular-arbuscular mycorrhizal isolates (Glomus intraradices Schenck & Smith). After 7 weeks, plants were transplanted to the field and sprayed twice with cytokinin and thidiazuron, at 50% flowering and 3 weeks later. These treatments significantly increased the percentage of infected roots (30–43%) and chlorophyll content (8–13%). Mycorrhizal plants had less carbohydrates in the roots than in the leaves. Total protein and phenylalanine contents showed pronounced increases, while proline content decreased. Treatment of the mycorrhizal plants with thidiazuron or cytokinin significantly increased plant dry weight (51–54%), and the tomato yield significantly increasecd after inoculation with both isolates (18–35% and 14–32%).     

Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.     

Transgenic tomato plants expressing strawberry <Emphasis Type="ItalicSmallCaps">d</Emphasis>-<Emphasis Type="Italic">galacturonic acid reductase</Emphasis> gene display enhanced tolerance to abiotic stresses

After analyzing tomato plants transformed with GalUR gene for their ascorbic acid contents, it was found that some transgenic lines contained higher levels of ascorbic acid compared to control plants. In the present study, callus induction rate was 50.2 % in the explant and shoot regeneration rate was 51.5 % from the callus with transformation efficiency of 3.0 %. Based on PCR and Southern blot analysis, three independent transformants containing the insert gene were selected. Phenotypic traits of these transgenic progeny were similar to those of control tomatoes. Tomatoes (H15) with high fruit ascorbic acid contents were selected for next generation (GalUR T₃) analysis. Transgenic tomatoes with increased ascorbic acid contents were found to be more tolerant to abiotic stresses induced by viologen, NaCl, or mannitol than non-transformed plants. In leaf disc senescence assay, the tolerance of these transgenic plants was better than control plants because they could retain higher chlorophyll contents. Under salt stress of less than 200 mM NaCl, these transgenic plants survived. However, control plants were unable to survive such high salt stress. Ascorbic acid contents in the transgenic plants were inversely correlated with MDA contents, especially under salt stress conditions. The GalUR gene was expressed in H15 tomatoes, but not in control plants. Higher expression levels of antioxidant genes (APX and CAT) were also found in these transgenic plants compared to that in the control plants. However, no detectable difference in SOD expression was found between transgenic plants and control plants. Results from this study suggest that the increase in ascorbic acid contents in plants could up-regulate the antioxidant system to enhance the tolerance of transgenic tomato plants to various abiotic stresses.     

Effect of Disbudding on Root Cytokinin Export and Leaf Senescence in Tomato and Tobacco

Bud removal and decapitation (disbudding) of plants of tomato(Lycopersicon esculentum Miller) and tobacco (Nicotiana tabacumL.) resulted in an increase in both the concentration and thetotal flux of cytokinin in bleeding xylem sap. There was a retardationin the rate of chlorophyll loss from leaves of disbudded plantsof both species. Ultrastructural examination of the chloroplastsof disbudded tomato plants revealed a maintenance of chloroplastintegrity, compared with chloroplasts from control plants. Itis suggested that the delaying of leaf senescence observed indisbudded plants is due to an increased availability of cytokininto these leaves.