甜蛋白基因MBLⅡ对番茄的遗传转化

以5～7d龄的“丽春”番茄无菌苗子叶作为外植体,研究了子叶外植体对抗生素卡那霉素的敏感性,抗生素卡那霉素对番茄筛选的适宜浓度为70mg／L。通过根癌农杆菌(Agrobacterium tumefaciens)介导,成功地进行了马槟榔甜蛋白基因MBLⅡ对番茄的遗传转化,获得转化番茄抗性植株,组织化学法有阳性表现、PCR特异扩增及Southern杂交检测出现特异条带,表明MBLⅡ基因已顺利整合到转基因番茄植株的基因组。     

转基因表达OPH提高番茄果实降解有机磷农药能力的研究

拟通过基因工程提高番茄果实降解有机磷农药残留的能力。构建了E8启动子基因驱动有机磷降解基因(OPD)的植物表达载体pSE8OP,经农杆菌介导遗传转化番茄子叶后,进行GUS染色、PCR和Southern blotting分析。证明OPD基因已整合进转基因植株基因组中,为1个拷贝。HPLC比较分析发现,转基因番茄果实能显著提高降解毒死蜱和对硫磷的能力,大大减少了番茄中的农药残留。     

番茄果实乙烯释放量的遗传模型分析

本试验选择2个番茄果实乙烯释放量显著不同的番茄品系,通过P_1、P_2、F_1、F_2、B_1和B_2六世代的分析方法,研究了番茄果实乙烯释放量的遗传规律.结果表明:番茄果实乙烯释放量遗传符合1对负向完全显性主基因+加性-显性多基因模型(D-4),主基因效应在B_1、B_2和F_(23)个世代的遗传率分别为36.33%、44.09%和35.14%,多基因效应在B_1、B_2和F_(23)个世代的遗传率分别为54.73%、40.50%和54.88%.     

番茄果实特异性启动子的克隆与遗传转化研究

为了实现外源基因在番茄果实中的高效和特异表达,克隆了番茄果实特异基因多聚半乳糖醛酸酶基因( Polygalacturonase,PG)的启动子.以中蔬四号番茄为材料,建立并优化了以子叶为外植体的番茄高效再生和遗传转化体系;以GUS为报告基因,构建PG:GUS植物表达载体,转化番茄.结果表明,在1.0 mg/L ZT的MS分化培养中,番茄子叶的发芽率最高,芽的诱导率高达91％,且发生畸态芽和褐化的外植体最少;通过抗生素浓度对农杆菌的抑制效果试验发现,当头孢霉素的浓度为200 mg/L时,抑制农杆菌的效果最好;成功克隆了番茄PG启动子,将PG启动子驱动的GUS基因转入番茄,对转基因后代果实的GUS染色表明,PG启动子驱动的外源基因在果实中特异表达.     

液泡转化酶反义基因对番茄的遗传转化

以'中蔬4号'番茄的子叶为试材,通过农杆菌介导法遗传转化,将液泡转化酶反义基因导入再生植株,经PCR和Southern斑点杂交检测证明,5株转化植株基因组中整合有目的基因.遗传转化最佳条件为:在附加1.5 mg/L 6-BA和0.1 mg/L IAA的MS培养基上再生培养,外植体预培养2 d,菌液浓度OD600=0.5,侵染时间5 min,共培养2 d.遗传转化后,对整合有目的基因的再生番茄叶片液泡转化酶活性测定,表明液泡转化酶活性明显受到抑制.获得的转基因植株为进一步研究液泡转化酶基因的功能奠定了基础.     

甜蛋白基因MBLII对番茄的遗传转化

以5～7d龄的 丽春 番茄无菌苗子叶作为外植体,研究了子叶外植体对抗生素卡那霉素的敏感性,抗生素卡那霉素对番茄筛选的适宜浓度为70mg/L.通过根癌农杆菌 Agrobacteriumtumefaciens 介导,成功地进行了马槟榔甜蛋白基因MBLII对番茄的遗传转化,获得转化番茄抗性植株,组织化学法有阳性表现、PCR特异扩增及Southern杂交检测出现特异条带,表明MBLII基因已顺利整合到转基因番茄植株的基因组.     

遗传转化KN1基因促进毛果杨外植体再生

已有研究表明玉米同源异形盒(homeobox)基因Knotted1(KN1)超量表达会导致转基因烟草、拟南芥和番茄等植物中细胞分裂素含量增加,提高转化效率。由于木本植物遗传转化困难,且毛果杨可作为木本植物研究的模式植物,因此,本研究利用农杆菌介导法,将35S启动子驱动的玉米KN1(35S::KN1)基因导入毛果杨,并分析KN1基因表达对毛果杨再生率的影响。研究结果表明,经GUS组织化学染色及PCR分子鉴定,KN1基因已经成功导入毛果杨幼苗;KN1基因对毛果杨外植体愈伤诱导率影响不明显,但单个外植体芽诱导率比对照高0.96倍,毛果杨的再生频率明显提高;与移栽成活的野生对照植株相比,转化KN1基因植株出现叶片变小,叶色变深,在叶片边缘产生裂片,分枝增加,无顶端优势等特殊表型。本研究中KN1基因引起转化植株表型变化,因此在毛果杨的遗传转化中,可作为一种有效的正向选择标记基因。     

甜蛋白Brazzein基因在番茄果实中的特异表达

西瓜(Citrullus vulgaris S.)来源的AGPL1启动子在番茄(Lycopersicon esculentum L.)果实中具有较强的特异性驱动功能。将该启动子与甜味蛋白基因Brazzein融合构建植物表达载体, 通过根癌农杆菌(Agrobacterium tumefaciens)介导法成功地进行了对番茄的遗传转化, 获得转化植株。组织化学法、PCR特异扩增、Southern杂交分析及RT-PCR检测, 表明Brazzein基因已整合到转基因番茄植株基因组中并且稳定表达。通过AGPL1果实特异启动子的调控, 在不改变果实其他性状的前提下提高了番茄果实甜味品质, 并为甜蛋白的生产提供经验。     

利用多组学手段解析番茄育种过程中代谢物变化的机制

番茄(Solanum lycopersicum)在育种过程中经历了驯化、改良、分化和渐渗等不同阶段, 在这一选择过程中番茄的果重和风味等均发生了显著改变, 但是目前对于番茄育种过程中代谢物的变化及其遗传基础却不是十分清楚。近期, 中国农业科学院深圳农业基因组研究所黄三文研究组与华中农业大学罗杰研究组利用多组学(变异组、转录组及代谢组)手段系统解析了番茄育种过程中代谢物的变化。结果表明, 在番茄驯化过程中有46个甾醇糖基生物碱类物质(SGAs)含量逐渐降低, 并获得了7个与其中44个物质显著相关的遗传位点。因此, 在番茄育种过程中通过优异等位位点的组合可以显著降低SGAs的含量; 同时发现在番茄以果重为目标的选择过程中, 控制果重基因周围其它基因的“搭车效应”是引起许多代谢物变化的重要遗传因素, 及在育种过程中对某一性状的选择会对其它性状产生重要影响。该研究首次利用多组学手段系统解析了选择对作物代谢物的影响, 为番茄品质改良奠定了良好的理论基础。     

农杆菌介导Ds转座因子转化番茄群体的构建

以番茄Micro-Tom子叶为试材,绿色荧光蛋白GFP基因为报告基因,利用农杆菌介导的遗传转化法将构建的激活表达标签pAcGFP导入番茄Micro-Tom,获得转基因番茄群体.对转化群体进行了GFP基因活性及分子检测,结果表明,GFP已经整合到番茄基因组中.以GFP基因设计引物进行PCR检测表明,阳性率为91.43%.通过Southern杂交分析,31.25%的个体为一个插入位点,56.25%为2个插入位点,T-DNA在基因组中的平均拷贝数为1.8个.     

Breaking-off tissue specific activity of the oil palm metallothionein-like gene promoter in T1 seedlings of tomato exposed to metal ions

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are involved in cell growth regulation, transportation of metal ions and detoxification of heavy metals. A mesocarp-specific metallothionein-like gene (MT3-A) promoter was isolated from the oil palm (Elaeis guineensis Jacq). A vector construct containing the MT3-A promoter fused to the β-glucuronidase (GUS) gene in the pCAMBIA 1304 vector was produced and used in Agrobacterium-mediated transformation of tomato. Histochemical GUS assay of different tissues of transgenic tomato showed that the MT3-A promoter only drove GUS expression in the reproductive tissues and organs, including the anther, fruit and seed coat. Competitive RT-PCR and GUS fluorometric assay showed changes in the level of GUS mRNA and enzyme activity in the transgenic tomato (T₀). No GUS mRNA was found in roots and leaves of transgenic tomato. In contrast, the leaves of transgenic tomato seedlings (T₁) produced the highest GUS activity when treated with 150 μM Cu²⁺ compared to the control (without Cu²⁺). However, Zn²⁺ and Fe²⁺ treatments did not show GUS expression in the leaves of the transgenic tomato seedlings. Interestingly, the results showed a breaking-off tissue-specific activity of the oil palm MT3-A promoter in T₁ seedlings of tomato when subjected to Cu²⁺ ions.     

The effect of host plants on Tetranychus evansi, Tetranychus urticae (Acari: Tetranychidae) and on their fungal pathogen Neozygites floridana (Entomophthorales: Neozygitaceae)

In a series of tritrophic-level interaction experiments, the effect of selected host plants of the spider mites, Tetranychus evansi and Tetranychus urticae, on Neozygites floridana was studied by evaluating the attachment of capilliconidia, presence of hyphal bodies in the infected mites, mortality from fungal infection, mummification and sporulation from fungus-killed mite cadavers. Host plants tested for T. evansi were tomato, cherry tomato, eggplant, nightshade, and pepper while host plants tested for T. urticae were strawberry, jack bean, cotton and Gerbera. Oviposition rate of the mites on each plant was determined to infer host plant suitability while host-switching determined antibiosis effect on fungal activity. T. evansi had a high oviposition on eggplant, tomato and nightshade but not on cherry tomato and pepper. T. urticae on jack bean resulted in a higher oviposition than on strawberry, cotton and Gerbera. Attachment of capilliconidia to the T. evansi body, presence of hyphal bodies in infected T. evansi and mortality from fungal infection were significantly higher on pepper, nightshade and tomato. The highest level of T. evansi mummification was observed on tomato. T. evansi cadavers from tomato and eggplant produced more primary conidia than those from cherry tomato, nightshade and pepper. Switching N. floridana infected T. evansi from one of five Solanaceous host plants to tomato had no prominent effect on N. floridana performance. For T. urticae, strawberry and jack bean provided the best N. floridana performance when considering all measured parameters. Strawberry also had the highest primary conidia production. This study shows that performance of N. floridana can vary with host plants and may be an important factor for the development of N. floridana epizootics.     

Effect of Soils from Six Management Systems on Root-knot Nematodes and Plant Growth in Greenhouse Assays

The effects of soil management systems on root-knot nematode (Meloidogyne incognita) eggs and gall incidence on tomato (Lycopersicon esculentum) and cucumber (Cucumis sativus) following tomato were evaluated. Soil was collected from a replicated field experiment in which six management systems were being assessed for vegetable production. Soil management systems were conventional production, organic production, bahiagrass (Paspalum notatum) pasture, bahiagrass: Stylosanthes (Stylosanthes guianensis) pasture, bare ground fallow, and weed fallow. Soil was collected from field plots and used in greenhouse experiments. Identification of egg-parasitic fungi and the incidence of root-knot nematode galling were assessed both on tomato and cucumber planted in the same pots following the removal of tomato plants. Organic, bare ground fallow and conventional production treatments reduced galling both on tomato and on cucumber following tomato. Although no treatment consistently enhanced egg-parasitic fungi, management system did affect egg viability and the types of fungi isolated from parasitized eggs.     

Managing Nematode Population Densities on Tomato Transplants Using Crop Rotation and a Nematicide

Millet, milo, soybean, crotalaria, and Norman pigeon pea were used in conjunction with clean fallow and a nematicide (fensulfothion) for managing nematode populations in the production of tomato transplants (Lycopersicon esculentum Mill.). Glean fallow was the most effective treatment in suppressing nematode numbers. After 2 years in tomato, root-knot nematodes increased in numbers to damaging levels, and fallow was no longer effective for complete control even in conjunction with fensulfothion. After 4 years in tomato, none of the crops used as summer cover crops alone or in conjunction with fensulfothion reduced numbers of root-knot nematodes in harvested tomato transplants sufficiently to meet Georgia certification regulations. Milo supported large numbers of Macroposthonia ornata and Pratylenchus spp. and crotalaria supported large numbers of Pratylenchus spp. Millet, milo, soybean, crotalaria, and pigeon pea are poor choices for summer cover crops in sites used to produce tomato transplants, because they support large populations of root-knot and other potentially destructive nematodes.     

Impact of Paecilomyces lilacinus Inoculum Level and Application Time on Control of Meloidogyne incognita on Tomato

Microplot experiments were conducted to evaluate the effects of inoculum level and time of application of Paecilomyces lilacinus on the protection of tomato against MeIoidogyne incognita. The best protection against M. incognita was attained with 10 and 20 g of fungus-infested wheat kernels per microplot which resulted in a threefold and fourfold increase in tomato yield, respectively, compared with tomato plants treated with this nematode alone. Greatest protection against this pathogen was attained when P. lilacinus was delivered into soil 10 days before planting and again at planting. Yield was increased twofold compared with yield in nematode-alone plots and plots with M. incognita plus the fungus. Percentages of P. lilacinus-infected egg masses were greatest in plots treated at midseason or at midseason plus an early application, compared with plots treated with the fungus 10 days before planting and (or) at planting time.     

Association of Verticillium chlamydosporium and Paecilomyces lilacinus with Root-knot Nematode Infested Soil

Population densities of Meloidogyne incognita and the nematophagous fungi, Paecilomyces lilacinus and Verticillium chlamydosporium, were determined in 20 northern California tomato fields over two growing seasons. Paecilomyces lilacinus was isolated from three fields, V. chlamydosporium was isolated from one field, and both fungi were isolated from 12 fields. Verticillium chlamydosporium numbers were positively correlated with numbers of M. incognita and P. lilacinus. Paecilomyces lilacinus numbers were positively correlated with V. chlamydosporium numbers, but they did not correlate with M. incognita numbers. The correlation coefficients were low (R < 0.5) but significant (P < 0.05). All P. lilacinus and V. chlamydosporium field isolates parasitized M. incognita eggs in vitro. In a greenhouse study, numbers of V. chlamydosporium and P. lilacinus increased more in soils with M. incognita-infected tomato plants than in soil with uninfected tomato plants. After 10 weeks, the Pf/ Pi of second-stage juveniles in soils infested with P. lilacinus, V. chlamydosporium, and M. incognita was 47.1 to 295.6. The results suggest V. chlamydosporium and P. lilacinus are not effectively suppressing populations of M. incognita in California tomato fields.     

Pathological Interaction of a Combination of Heterodera schachtii and Meloidogyne hapla on Tomato

Increased culturing of a tomato population of Heterodera schachtii (UT1C) on tomato for 480 days (eight inoculation periods of 60 days each) significantly increased virulence to ''Stone Improved'' tomato. A synergistic relationship existed between Meloidogyne hapla and H. schaehtii on tomato. A combination of H. schachtii (UTIC) and M. hapla significantly reduced tomato root weights by 65, 64, and 61% below root weights of untreated controls, and single inoculations of M. hapla and H. schachtii, respectively. This corresponded to root reductions of 42, 44, and 46% from a combination of H. schachtii (UT1B) and M. hapla. Antagonism existed between H. schachtii and M. hapla with regard to infection courts and feeding sites. The root-knot galling index dropped from 6.0 with a single inoculation of M. hapla to 4.3 and 3.3 with combined inoculations of M. hapla plus UT1B and M. hapla plus UTIC cyst nematode populations. The pathological virulence of H. schachtii to sugarbeet was not lost by extended culturing on tomato; there were no differences in penetration, maturation, and reproduction between sugarbeet populations continually cultured on sugarbeet and the population continually cultured on tomato.     

Rhizosphere Colonization and Control of Meloidogyne spp. by Nematode-trapping Fungi

The ability of nematode-trapping fungi to colonize the rhizosphere of crop plants has been suggested to be an important factor in biological control of root-infecting nematodes. In this study, rhizosphere colonization was evaluated for 38 isolates of nematode-trapping fungi representing 11 species. In an initial screen, Arthrobotrys dactyloides, A. superba, and Monacrosporium ellipsosporum were most frequently detected in the tomato rhizosphere. In subsequent pot experiments these fungi and the non-root colonizing M. geophyropagum were introduced to soil in a sodium alginate matrix, and further tested both for establishment in the tomato rhizosphere and suppression of root-knot nematodes. The knob-forming M. ellipsosporum showed a high capacity to colonize the rhizosphere both in the initial screen and the pot experiments, with more than twice as many fungal propagules in the rhizosphere as in the root-free soil. However, neither this fungus nor the other nematode-trapping fungi tested reduced nematode damage to tomato plants.