通过表达ACC脱氨酶基因控制番茄果实的成熟

乙烯在跃变型果实的成熟过程中起着触发呼吸跃变和促进果实成熟的作用。细菌来源的1-氨基环丙烷-1-羧酸(ACC)脱氨酶能降解乙烯的直接前体ACC,从而抑制植物体内乙烯的合成。我们用PCR方法从假单孢杆菌中克隆到ACC脱氨酶基因并通过农杆菌介导的方法将其转入番茄(Lycopersicun esculentum)中。再生植株经Southern blot检测证明,ACC脱氨酶基因已整合到番茄基因组中并稳定表达。转基因番茄果实成熟期的推迟时间与体内乙烯的抑制程度有相关性。转基因番茄植株乙烯的合成降低80%左右,果实在离体条件下可保鲜75d左右。研究ACC脱氢酶基因在植物体内的作用可阐明高等植物体内乙烯的作用机理并为培育耐贮藏果蔬品种打下基础。     

含ACC脱氨酶的根际细菌提高植物抗盐性的研究进展

盐胁迫是抑制植物生长的主要非生物因素之一,高浓度的盐分不利于植物体的生长和发育,严重时会导致植物细胞及植物体死亡.已有大量实验结果显示含ACC脱氨酶的根际细菌可以缓解高盐对植物的危害.ACC脱氨酶可以降解乙烯的直接前体1-氨基环丙烷-1-羧酸(ACC),从而降低胁迫乙烯的合成量.胁迫乙烯是阻碍植物生长的主要原因.首先介...     

Combined Role of ACC Deaminase Producing Bacteria and Biochar on Cereals Productivity under Drought

Most of the cereal crops are widely cultivated to fulfil the humans food requirements. Under changing climate scenario, the intensity of drought stress is continuously increasing that is adversely affecting the growth and yield of cereal crops. Although the cereals can tolerate moderate drought to some extent, but mostly they are susceptible to severe drought stress. Higher biosynthesis of ethylene under drought stress has been reported. Many scientists observed that inoculation of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing plant growth promoting rhizobacteria (PGPR) is an efficacious tool to overcome this problem. These PGPR secrete ACC deaminase which cleavage the ACC into the compounds, other than ethylene. Furthermore, secretion of growth hormones also play imperative role in enhancing the growth of the cereals under limited availability of water. In addition, the use of biochar has also been recognized as another effective amendment to grant resistance against drought. Biochar application improves the soil physiochemical attributes i.e., porosity, nutrients retention and water holding capacity which decrease the loss of water and increase its bioavailability. In recent era, the idea of coapplication of ACC deaminase producing PGPR and biochar is becoming popular which might be more efficient to use water under drought stress. The aim of current review is to combine the facts and understanding of this novel idea to grant maximum resistance to crops against drought stress. Some scientists have observed significant improvement in yield of cereal crops by combined use of ACC deaminase producing PGPR and biochar. However, more research is suggested for deep understanding of complex synergistic mechanism of ACC deaminase activity in combination with biochar.     

番茄ACC合成酶cDNA克隆及其对果实成熟的反义抑制

利用RT—PcR技术克隆了ACC合成酶多基因家族成员之－LE-ACC2编码区约1．7kb的cDNA,经酶切图谱和序列分析鉴定无误后,反向插入到植物表达载体pBin437中,构建了表达Acc合成酶反望RNA的二元载体。经农杆菌途径转化番茄“丽春”品种后,通过PCR检测从抗卡那霉素再生植株中筛选到6株转基因植株,Southern杂交确证了外源基因是以单拷贝插入到番茄染色体中;对果实乙烯释放的测定结果表明转基因番茄果实的乙烯释放量仅为对照的30％左右,在室温下转基因番茄果实采后保存60 d以上仍然没有变红、软化。以上结果表明其反义RNA在转基因番茄中的表达能有效地抑制乙烯的生物合成从而延缓果实成熟,表现出良好的耐储保鲜特性。对转基因植株子一代(T1)的分析结果进一步表明反义ACC合成酶基因以典型的单基因方式传到子代。通过对子二代的分析已初步筛选到一 个耐储藏的转基因番茄纯合品系。     

芽孢杆菌对桉树幼苗的促生效果及其ACC脱氨酶活性的研究

【目的】筛选出能显著促进桉树幼苗生长的芽孢杆菌菌株,探究酶活性与桉树幼苗生长的相关性,初步揭示芽孢杆菌对桉树幼苗的促生机制。【方法】以分离自广东广州、阳江桉树林地土壤的32个芽孢杆菌菌株为研究对象,测定桉树幼苗接种盆栽试验以及菌株ACC脱氨酶活性与幼苗N、P养分。【结果】接种菌株2306、2403、2301能够显著促进桉树幼苗高生长和生物量积累,尤以菌株2306的促生效果最佳,其苗高、生物量分别比对照增加53.1%和190.2%。【结论】芽孢杆菌的ACC脱氨酶活性与桉树幼苗高生长相关极显著,与生物量相关显著;而且上述3个菌株均能提高桉树幼苗的N、P含量。研究结果将进一步丰富桉树促生菌资源,促进桉树微生物肥料的开发。     

Ca2+对离体培养的番茄花柄脱落的影响以及脱落过程中离区内钙形态的变化

在自然条件下,Ca~(2 )对离体培养的番茄花柄脱落有抑制作用,而对经乙烯处理的则有显著的促进。无论是在自然还是乙烯条件下,未经任何处理的番茄花柄脱落过程中总钙与果胶酸钙含量下降,而水溶性钙含量升高,Ca~(2 )在不同程度上提高脱落过程中离区总钙、果胶酸钙和水溶性钙含量,尤其是在乙烯下的效果更显著,而经EGTA处理的则相反。     

Changes in Structural Features of Free N-Glycan and Endoglycosidase Activity during Tomato Fruit Ripening

In developing plants, free N-glycans occur ubiquitously at micromolar concentrations. Such oligosaccharides have been proposed to be signaling molecules in plant development. As a part of a study to elucidate the physiological roles of de-N-glycosylation machinery involved in fruit ripening, we analyzed changes in the amounts and structural features of free N-glycans in tomato fruits at four ripening stages. The amount of high-mannose type free N-glycans increased significantly in accordance with fruit ripening, and the relative amounts of high-molecular size N-glycans, such as Man_8-9GlcNAc₁, became predominant. These observations suggest that the de-N-glycosylation machinery, including endo-β-N-acetylglucosaminidase (ENGase) activity, is stimulated in the later stages of fruit ripening. But contrary to expectation, we found that total ENGase activities in the tomato fruits did not vary significantly with the ripening process, suggesting that ENGase activity must be maintained at a certain level, and that the expression of α-mannosidase involved in the clearance of free N-glycans decreases during tomato fruit ripening.     

番茄LeNHX1基因植物过量表达载体的构建及表达分析

从番茄幼苗中提取RNA,根据NCBI中番茄LeNHX1基因序列设计引物,通过RT-PCR获得了番茄LeNHX1基因的cDNA序列,包含一个1 605 bp的开放阅读框,编码534个氨基酸。将cDNA序列连接到植物过量表达载体PBI121上,对所获得的重组质粒进行双酶切鉴定,结果表明,植物过量表达载体PBI121-LeNHX1已构建成功。半定量RT-PCR结果表明LeNHX1基因在根、茎和叶中均表达,盐、低温和脱落酸的诱导能提高LeNHX1基因的表达量,推测番茄LeNHX1基因在逆境应答中可能起着重要作用。     

Water relations and growth of tomato fruit pericarp tissue

The water relations of young tomato fruit pericarp tissue were examined and related to tissue expansion. The relationship between bulk turgor pressure and tissue expansion (as change in fresh mass or length of tissue) was determined in slices of pericarp cut from young, growing fruit by incubation in different osmotic concentrations of polyethylene glycol 6000 or mannitol. The bulk turgor of this tissue was low (about 0.2 MPa), even in fruit from plants that were otherwise fully turgid, whether measured psychrometrically or by length change in osmotic solutions. The rate of tissue growth at maximum turgor was less than that at moderate turgor unless calcium was added to the incubation medium. However, added calcium also decreased the rate of growth at lower turgor pressures. Yield turgor was < 0.1 MPa, but it was increased by the addition of calcium ions. Electrolyte leakage from tissue was greatest at maximum turgor pressure but was decreased by the addition of calcium ions or osmoticum. Tissue growth was unaffected by a range of plant growth regulators (IAA, abscisic acid, benzyladenine and GA₃) but was inhibited, particularly at high turgor, by low concentrations of malic or citric acid. The low turgor pressure of pericarp tissue could be due to the presence of apoplastic solutes within the pericarp, and evidence for this is discussed. Thus, fruit tissue may be able to maintain optimal expansion rates only at moderate turgor and low calcium concentration.     

具有ACC脱氨酶活性的绞股蓝内生细菌的分离鉴定及其抑菌促生作用

采用富集筛选法从绞股蓝根中筛选得到6株具有ACC脱氨活性的细菌,其中菌株JDG-6、JDG-7、JDG-14、JDG-16、JDG-23均具有较强的分泌铁载体能力,但菌株JDG-32没有产铁载体能力。抑菌试验结果显示,菌株JDG-6、JDG-7、JDG-14和JDG16对一种或多种供试菌有抑菌作用,其中菌株JDG-14能抑制大肠埃希菌、藤黄八叠球菌和白色念球菌的生长。促生试验表明,菌株JDG-6、JDG-7和JDG-14均能促进水稻幼苗根的伸长,其中菌株JDG-14的促生作用最为明显,与对照组相比水稻幼苗的根长和根鲜重分别增长了26%和21%。通过形态特征观察、生理生化试验以及16S rDNA序列分析,菌株JDG-6、JDG-7、JDG-16和JDG-23属于假单胞杆菌属（Pseudomonas）,菌株JDG-14为木糖葡萄球菌（Staphylococcus xylosus）,而菌株JDG-32为枯草芽胞杆菌（Bacillus subtilis）。菌株JDG-6、JDG-7和JDG-14均具有ACC脱氨酶活性和抑菌活性的促生菌,具有农业应用潜力。     

A model for an early stage of tomato fruit development: cell multiplication and cessation of the cell proliferative activity

Changes in cell number during the early period of tomato fruit development were analysed by means of a deterministic model of cell multiplication. The period commenced at the seed stage with one theoretical cell undergoing intensive cell division, and ended when the cell number became nearly constant. The model takes into consideration the proliferative activity of the fruit cell population which, a few days before flower anthesis, begins to decrease progressively after each mitotic cycle. Model parameters, namely the time at which proliferative activity diminishes, its rate of decrease and the length of the cell cycle, were estimated by fitting the model to observed cell population dynamics in tomato fruits growing in three different positions on the truss. It is hypothesized that the molecular mechanism responsible for the cessation of mitosis in growing fruits is associated with shortening of telomeric ends of nuclear DNA, as suggested previously for other growing cell populations.     

Identification and quantitation of gibberellins in fruits of Lycopersicon esculentum, and their relationship to fruit size in L. esculentum and L. pimpinellfolium

GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀ and GA₂₉ were identified by combined gas chromatography-mass spectrometgry (GC-MS) in immature seeds and pericarp of Lycopersicon esculentum Mill. (tomato). Higher levels of these GAs were present in the seeds than in the pericarp; seeds in addition contained GA₁₅, GA₂₄, GA₂₅, and GA₄₄. Fruits of the Lycopersicon pimpinellifolium Mill. mutant I were smaller and contained lower GA₁ concentrations, but higher GA₂₀ concentrations, than those of mutants III and IV. In contrast, differences in fruit size in L. esculentum due to position on the truss did not correlate with GA₁ concentration in either the pericarp or seeds.     

中华补血草内生与根际具ACC脱氨酶活性细菌的筛选及其生物多样性

【目的】获得江苏沿海滩涂盐生药用植物中华补血草内生及根际具有1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性的细菌,研究其遗传多样性和潜在促生活性。【方法】从中华补血草和根际土壤分离筛选具有ACC脱氨酶活性的菌株,对其ACC脱氨酶活性定量检测,通过16S r RNA基因序列分析确定菌株系统发育地位。同时研究其固氮、溶磷、产植物生长素吲哚乙酸(IAA)及耐盐能力。【结果】分离筛选获得18株具有ACC脱氨酶活性的内生与根际细菌,定量检测发现其中有13株菌的ACC脱氨酶含量在20 nmolα-KA/(mg Pr·h)以上,有11株菌可以固氮,7株菌能够解磷,9株菌产生IAA。菌株的Na Cl盐耐受范围多数在0–13%之间。16S r RNA基因测序表明,活性菌株分属于7个属,多样性丰富,节杆菌属(Arthrobacter)为优势类群。其中菌株KLBMP 5180为节杆菌属的潜在新种。【结论】江苏沿海滩涂盐生药用植物中华补血草共生环境中具有丰富多样的具ACC脱氨酶活性的菌株,并存在潜在新物种资源,具有进一步研究价值。     

番茄多聚半乳糖醛酸酶cDNA的克隆及其对番茄中PG表达的反义抑制

通过PCR扩增获得了包含多聚半乳糖醛酸酶(PG)全部阅读框架的1.5kb cDNA,经限制酶酶谱和部分序列分析鉴定无误后,将其以反方向插入含两个增强子的35s启动子和Nos3＇端之间,构建成表达PG反义RNA的双元载体,经农杆菌途径转化番茄品种“丽春”,获得了60株抗卡那霉素再生植株,经PCR检测,证明有2／3的再生植株有外源PG基因导入,成熟果实的PG粗提液的SDS—PAGE分析表明：若干株系中PG蛋白量较对照有不同程度的下降。PG活性亦同步下降,其中一个株系3^＃,PG酶活下降了93%。这些结果表明外源PG基因的反方向导入有效地抑制了内源PG基因的表达。     

The improvement of resistance to bacterial speck in transgenic tomato plants by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> mediated transformation

The pto gene, responsible for resistance to Pseudomonas syringae pv. tomato, was transferred to tomato genotype Urfa-2 by the LBA4404 strain of A. tumefaciens harboring the plasmid pPTC8. The presence of nptII and pto genes in transgenic plants was proved by PCR analysis. Insertion of the pto gene into the genome of transgenic plants and expression of the gene were confirmed by southern and northern hybridizations, respectively. The pathogen P. syringae pv. tomato was applied to all leaves of transgenic and control plants. While typical bacterial speck symptoms developed on the leaves of control plants, the transgenic plants did not display any typical symptoms of bacterial speck upon inoculation with strains 1 and 0. Some of these transgenic plants had thicker leaves than the control plants and produced abnormal flowers. The pollen of transgenic plants was used for crossing with control plants to produce F1 transgenic lines. Fruits from crossed transgenic and control plants were obtained, and F1 seeds germinated on Murashige and Skoog medium in the presence of kanamycin have developed F1 seedlings. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 1, pp. 102–110. The text was submitted by the authors in English.     

Isolation and characterization of an unusual 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4

A genomic library of the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth-promoting bacterium Enterobacter cloacae UW4 in pUC19 in Escherichia coli was screened for the ability to utilize ACC as a sole source of nitrogen. One of the clones that was isolated contained a plasmid with an insert of approximately 0.8 kb that conferred ACC deaminase activity. Sequence analysis revealed that this DNA fragment contains an open-reading frame of 696 nucleotides predicted to encode a protein of 232 amino acids, a member of the amidohydrolase protein superfamily, i.e., a deaminase that contains a mononuclear or binuclear metal center as compared to the canonical ACC deaminase which contains pyridoxal phosphate as a co-factor.     

大肠杆菌腺苷脱氨酶基因的克隆、表达与缺失

利用途径工程的基本原理,拟在大肠杆菌核苷酸代谢途径中构建腺苷（AR）转化为腺苷三磷酸（ATP）的新途径,故需使细胞内的腺苷脱氨酶基因（add）缺失。通过构建大肠杆菌MC4100　DNA的基因文库,筛选得到含腺苷脱氨酶基因的DNA片段。构建表达质粒pBD1和pBD2并实现了表达。在此基础上构建了add基因缺失的带卡那霉素抗性基因的线性52kb DNA分子,同时转化JM83、MC4100、BL21（DE3）。经遗传稳定性实验和DNA分子杂交鉴定,确认得到了来自JM83的两株add基因缺陷株J1和J2。再对菌株J1、pUC18／JM83、pBD1／JM83的细胞粗提液做腺苷脱氨酶的酶活鉴定比较,结果表明则没有腺苷脱氨酶活性,pBD1／JM83有比pUC18／JM83强的腺苷脱氨酶活性。     

An analysis of the accumulation of water and dry matter in tomato fruit

Abstract Previously published data from tomato plants grown in nutrient solutions having one of three electrical conductivities (2, 12 and 17 mS cm^?1) were analysed. The rate of water import into the fruit, and the proportion of this conducted by the xylem stream were calculated from the daily rates of transpiration and the net accumulation of water and calcium. The rate of water import decreased as the conductivity of the nutrient solution rose, the maximum daily import rates in the third week after pollination being 3.2, 3.0 and 1.8 g fruit^?1 d^?1 for fruit grown at 2, 12 and 17 mS cm^?1, respectively. During fruit development, the proportion of water imported via the xylem fell from 8–15% to 1–2% at maturity. The principal source of water for tomato fruit growth was phloem sap. Based on the daily rates of net dry matter accumulation, respiration and phloem water import, the calculated dry matter concentration of the phloem sap declined from 7 to 3%, or from 12.5 to 7.8% during fruit development in low or high salinity, respectively. The similar dry matter accumulation of fruit grown at different salinities was due to changes in both volume and concentration of phloem sap. Potassium salts in tomato fruit were calculated lo have contributed –0.29, –0.48 and –0.58 MPa to total fruit osmotic potential in the 2, 12 and 17 mS cm^?1 treatments, respectively, which accounted for 38% or 49% of the measured total osmotic potential of the 2 mS cm^?1 or 17 mS cm^?1 treatments. The contribution of hexoses to total fruit osmotic potential in the young fruit was from about –0.1 to –0.2 MPa at all salinities. The osmotic potential of tomato fruit is regulated more by potassium salts than by hexoses.