叶绿体功能基因的工程改造

主要介绍叶绿体功能基因工程改造的目的、受体材料、主要方法、转入外源基因细胞的筛选和鉴定等.     

Chloroplast Integrity and ATP-Dependent CO(2) Fixation in Spinacia oleracea

Washed whole chloroplasts of Spinacia oleracea isolated and assayed in a tris (hydroxymethyl aminomethane)-HCl buffered sucrose solution exhibited low dark CO₂ fixing activity, whereas washed whole chloroplasts isolated in the same buffer but assayed in that buffer without sucrose exhibited much greater dark CO₂ fixing activity. The lowered activity could be attributed to the impermeability of the chloroplast membrane to ribose-5-phosphate or adenosine triphosphate. The preservation of the integrity of the chloroplast membrane, as reflected by its impermeability to either or both of the abovementioned compounds, was measured by the fixation of ¹⁴CO₂ into acid-stable products in the presence of ribose-5-phosphate and adenosine triphosphate by the whole chloroplast as compared with fixation by the chloroplast extract. An effect (i.e., apparent resistance to the passage of ribose-5-phosphate or adenosine-5-triphosphate into the chloroplast) similar to, but less pronounced than, that produced by the presence of sucrose in the isolation medium was observed upon the addition of MnCl₂ or CaCl₂ to the buffered sucrose isolation medium. The addition of KCl enhanced slightly the effect produced by addition of sucrose alone to the isolation medium. The presence of MgCl₂ in the isolation medium, however, either caused the chloroplasts to become leaky or more fragile since more of the activity of the carboxylative phase enzymes appeared in the cytoplasm. When a mixture of all of the metal ions was added to the buffered sucrose suspending medium, the chloroplasts exhibited the same response observed with MgCl₂ alone. The addition of ethylene diaminetetraacetate or dithiothreitol appeared to alter the permeability of the chloroplast membrane nonspecifically when the assay was conducted in the absence of sucrose. Specific activities (μmoles CO₂ fixed/mg chlorophyll × hr) as high as 329.6 have been observed for dark fixation by chloroplasts. The phosphoenolpyruvate carboxylase activity in the chloroplasts was only one-seventh that of ribulose diphosphate carboxylase. The phosphoenolpyruvate carboxylase activity in the cytoplasm was 5 times that of the chloroplasts.     

叶绿体分裂相关蛋白CrMinD的保守功能

细胞或质体中部正确分裂位点的选择是MinD蛋白与其他Min蛋白（MinC／E）相互作用的结果,MinD蛋白在原核细胞以及植物叶绿体的分裂过程中发挥着重要的作用。细胞中MinD蛋白浓度的明显升高可影响正常细胞的分裂过程而产生丝状体细胞。为了研究叶绿体分裂蛋白CrMinD的保守功能,构建了衣藻CrMinD-gfp的原核表达重组质粒进行了原核功能验证。试验结果表明,衣藻CrMinD蛋白的过量表达严重影响了大肠杆菌的分裂,其在原核细胞中运动和定位与用GFP标记的原核细胞MinD蛋白具有相似性。更进一步证明了叶绿体分裂同源物CrMinD蛋白与原核细胞MinD蛋白有着相似的功能,是一个进化上功能保守的蛋白。同时,这一结果也为研究植物细胞中质体的分裂机制奠定了一定的基础。     

Ontogenic Changes in Epicuticular Wax and Chloroplast Integrity of a Cotton (Gossypium hirsutum L.) Leaf

The progressive decline in cotton leaf photosynthesis with season could be accounted for by gaining an insight into ontogenic changes in chloroplast integrity and epicuticular wax ultrastructure. Therefore, the sequence of ultrastructural changes in chloroplast and epicuticular wax morphology were probed in 10-, 20-, 40-, and 60-d-old cotton (Gossypium hirsutum L.) leaves using electron microscopy. Scanning electron microscopy illustrated that the epicuticular wax on the periclinal walls of the convex epidermal cells occurred as striations and persisted as such during the course of leaf aging. The degree of wax spread, however, increased as the leaf progressed towards senescence. Transmission electron microscopy revealed that a 20-d-old photosynthetically active leaf possessed healthy chloroplasts (6.8 m long and an area of 9.7 m²) with absolute membrane integrity depicted by large appressed grana stacks of thylakoids interconnected by non-appressed stroma lamellae. The thylakoid membrane network was oriented parallel to the long axis of the chloroplast and a few small plastoglobuli (1.85 m²) scattered in the stroma. Conversely, membrane integrity was lost with leaf age after 20 d as evidenced by disruption of the grana and stroma lamellae. Concurrent with the membrane damage, extensive occlusion of chloroplast by several large spherical plastoglobuli (5.68 m²) occurred, the rate of occlusion increased with leafage distending the chloroplast as evidenced by proliferation of its cross-sectional area (12.8 m²). Of particular interest was the finding that the plastoglobuli ensued through the chloroplast envelope into the cytoplasm. The progressive loss of chloroplast membrane integrity coupled with increased leaf waxiness may have limited photosynthetic activities of cotton leaves during senescence.     

Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function

Pea (Pisum sativum L.) tissues with reduced chloroplast density (e.g. petals and stems) or function (i.e. senescent leaves and leaves darkened for prolonged periods) were surveyed to determine whether tissues with genetically or environmentally reduced chloroplast density and/or function also have significantly different amylolytic enzyme activities and/or isoform patterns than leaf tissues with totally competent chloroplasts. Native PAGE followed by electrophoretically blotting through a starch or β-limit dextrin containing gel and KI/I₂ staining revealed that the primary amylases in leaves, stems, petals, and roots were the primarily vacuolar β-amylase (EC 3.2.1.2) and the primarily apoplastic α-amylase (EC 3.2.1.1). Among tissues of light grown pea plants, petals contained the highest levels of total amylolytic (primarily β-amylase) activity and considerably higher ratios of β- to α-amylase. In aerial tissues there was an inverse relationship between chlorophyll and starch concentration, and β-amylase activity. In sections of petals and stems there was a pronounced inverse relationship between chlorophyll concentration and the activity of α-amylase. Senescing leaves of pea, as determined by age, and protein and chlorophyll content, contained 3.8-fold (fresh weight basis) and 32-fold (protein basis) higher α-amylase activity than fully mature leaves. Leaves maintained in darkness for 12 days displayed a 14-fold (fresh weight basis) increase in α-amylase activity over those grown under continuous light. In senescence and prolonged darkness studies, the α-amylase that was greatly increased in activity was the primarily apoplastic α-amylase. These studies indicate that there is a pronounced inverse relationship between chloroplast function and levels of apoplastic α-amylase activity and in some cases an inverse relationship between chloroplast density and/or function and vacuolar β-amylase activity.     

Chloroplast Structure and Function Is Altered in the NCS2 Maize Mitochondrial Mutant

The nonchromosomal stripe 2 (NCS2) mutant of maize (Zea mays L.) has a DNA rearrangement in the mitochondrial genome that segregates with the abnormal growth phenotype. Yet, the NCS2 characteristic phenotype includes striped sectors of pale-green tissue on the leaves. This suggests a chloroplast abnormality. To characterize the chloroplasts present in the mutant sectors, we examined the chloroplast structure by electron microscopy, chloroplast function by radiolabeled carbon dioxide fixation and fluorescence induction kinetics, and thylakoid protein composition by polyacrylamide gel electrophoresis. The data from these analyses suggest abnormal or prematurely arrested chloroplast development. Deleterious effects of the NCS2 mutant mitochondria upon the cells of the leaf include structural and functional alterations in the both the bundle sheath and mesophyll chloroplasts.     

Composition and Function of Thylakoid Membranes from Grana-rich and Grana-deficient Chloroplast Mutants of Barley

Chlorophyll-deficient barley (Hordeum vulgare) mutants were studied that had chlorophyll a/b ratios either higher or lower than the wild type. Mutants with high ratios (>5.2) had a reduced proportion of their photosynthetic lamellae appressed into grana (“grana-deficient” mutants) compared with wild type (chlorophyll a/b = 3.2), while the majority of lamellae in the chloroplasts with low chlorophyll a/b ratios (2.0-2.4) were organized into grana (“grana-rich” mutants).     

在体分离小鼠小肠隐窝、绒毛上皮细胞的生化完整性研究

目的:检测低温条件下用螯合剂沉淀法分离的小鼠小肠上皮隐窝和绒毛细胞是否具有生化完整性.方法:使用螯合剂在低温(冰浴)条件下分离和富集小肠上皮绒毛和隐窝细胞;抽提DNA、RNA和总蛋白,用电泳的方法检测完整性;用Real-time PCR检测溶菌酶Lysozyme的表达以判断隐窝、绒毛细胞富集程度.结果:低温条件下分离的肠上皮隐窝、绒毛细胞形态完整;基因组DNA完整,未出现明显的DNA ladder现象;富集细胞的RNA完整;富集隐窝、绒毛细胞的蛋白未降解,两组总蛋白具有表达谱差异性;隐窝细胞富集物溶菌酶mRNA表达水平较绒毛细胞富集物高30倍以上.结论:小肠隐窝绒毛的生物学性状可在低温螯合剂沉底法分离过程中得到保存,提示此方法可以用来分析生理和创伤痛理条件下小肠上皮基因和蛋白表达改变.     

Effect of {alpha}-Tomatine on the Integrity and Biochemical Activities of Isolated Plant Cell Organelles

Respiration in isolated mung bean shoot mitochondria was notaffected by either of the tomatine treatments (10^–3 M,pH 5, and 10^–5 M, pH 8) but was reduced in the excisedshoots by both treatments, although only at the higher of thetwo temperatures (5 °C and 25 °C). Inhibition was gradualand took at least 2 h. Tomatine treatment of excised shootsalso resulted in an increased leakage of K+. PS II activityin isolated spinach leaf chloroplasts was reduced only by thehigh pH tomatine treatment at 25 °C. Again, about 2 h treatmentwas required before significant effects were observed but thealkaloid did not cause disintegration of the chloroplast asmeasured by pigment release. Disruption of lysosomes isolatedfrom cauliflower inflorescence tissue and release of acid phosphatasewas enhanced by tomatine. Initially only 10^–5 M tomatineat pH 8 was effective but, later, effects could only be obtainedwith 10^–3 M alkaloid at pH 5. The differential susceptibilityof these organelles to tomatine is discussed in relation tomembrane structure and to the mode of toxicity of the alkaloid.     

稀释液中添加海藻糖对山羊精子功能和膜完整性的影响

选用5只年龄为3～4岁的波尔山羊公羊研究在稀释液中添加海藻糖对山羊精子功能和膜完整性的影响。山羊精子分别用含6.6 mmol/L、13.2 mmol/L、19.8 mmol/L、26.4 mmol/L、39.6 mmol/L、52.9 mmol/L、66.1mmol/L、79.3 mmol/L的不同海藻糖的Tris-柠檬酸-葡糖糖(TCG)稀释液(卵黄:18%;甘油:6%)稀释和冷冻。结果表明:39.6 mmol/L、52.9 mmol/L、66.1 mmol/L、79.3 mmol/L组降温后的精子活率显著(P<0.05)降低;52.9 mmol/L、66.1 mmol/L、79.3 mmol/L组降温后的精子畸形率和39.6 mmol/L组降温后的膨胀精子率显著(P<0.05)提高。26.4 mmol/L组和39.6 mmol/L组冻融后的精子活率显著(P<0.05)高于对照组;66.1mmol/L和79.3 mmol/L组冻融后的精子活率、畸形率分别显著(P<0.05)低于和高于对照组。19.8 mmol/L、26.4 mmol/L、39.6 mmol/L组冻融后精子获能率显著(P<0.05)低于对照组。39.6 mmol/L组冻融后顶体完整率和膨胀精子率显著(P<0.05)高于对照组,而66.1 mmol/L组和79.3 mmol/L组显著(P<0.05)低于对照组。39.6 mmol/L组的受胎率显著(P<0.05)高于对照组,而66.1mmol/L组和79.3 mmol/L组的受胎率显著(P<0.05)低于对照组。结果表明,在含18%的卵黄(v/v)、6%甘油(v/v)的TCG稀释液中,添加适宜浓度(26.4mmol/L和39.6 mmol/L)海藻糖,可显著提高山羊精子功能和膜的完整性。     

Chloroplast and cytoplasmic enzymes

Summary Chloroplastic and cytoplasmic forms of pea (Pisum sativum L.) leaf carbonic anhydrase were separated by isoelectric focusing. The two forms have identical pH optima, 7.0 for the hydration reaction and 7.5 for the dehydration reaction, and identical Michaelis constants for CO₂, 0.03 M. Neither isozyme is affected by any of several compounds involved in carbon metabolism in the green plant.IV=Anderson and Pacold (1972).