Availability of Micro-Tom mutant library combined with TILLING in molecular breeding of tomato fruit shelf-life

Novel mutant alleles of an ethylene receptor Solanum lycopersicum ETHYLENE RESPONSE1 (SlETR1) gene, Sletr1-1 and Sletr1-2, were isolated from the Micro-Tom mutant library by TILLING in our previous study. They displayed different levels of impaired fruit ripening phenotype, suggesting that these alleles could be a valuable breeding material for improving shelf life of tomato fruit. To conduct practical use of the Sletr1 alleles in tomato breeding, genetic complementation analysis by transformation of genes carrying each allele is required. In this study, we generated and characterized transgenic lines over-expressing Sletr1-1 and Sletr1-2. All transgenic lines displayed ethylene insensitive phenotype and ripening inhibition, indicating that Sletr1-1 and Sletr1-2 associate with the ethylene insensitive phenotype. The level of ethylene sensitivity in the seedling was different between Sletr1-1 and Sletr1-2 transgenic lines, whereas no apparent difference was observed in fruit ripening phenotype. These results suggested that it is difficult to fine-tune the extent of ripening by transgenic approach even if the weaker allele (Sletr1-2) was used. Our present and previous studies indicate that the Micro-Tom mutant library combined with TILLING could be an efficient tool for exploring genetic variations of important agronomic traits in tomato breeding.     

农杆菌介导的番茄Micro-Tom遗传转化体系的建立

Micro-Tom番茄植株矮小,生长密度高,生命周期短,转化效率高,成为功能基因组学研究的新型模式植物。对影响Micro-Tom遗传转化频率的共培养时间、AS的添加、工程菌液浓度和抑制农杆菌所用抗生素种类进行了分析,建立了Micro-Tom稳定高效的遗传转化体系。以bar基因设计引物对转化群体进行PCR检测,阳性率为72.3%,平均插入位点数为1.8个。遗传体系的建立为Micro-Tom在植物生物学研究中提供理论基础。     

SlDREB1B过量表达可促进番茄果实发育及成熟

为探讨DREBs在果实发育及成熟调控中的作用及机理,以番茄‘Micro-Tom’为试材,克隆得到1个DREBs转录因子SlDREB1B。研究证实,在果实中SlDREB1B的表达可受脱水胁迫和温度胁迫诱导。将SlDREB1B构建到带有e-GFP报告基因的载体上,采用叶盘法将SlDREB1B基因转入番茄,经报告基因筛选和SlDREB1B表达检测,成功获得转基因阳性植株。和转空载基因的植株相比,转SlDREB1B植株的果实发育和成熟进程明显加速。进一步分析表明,SlDREB1B的过量表达,可调控一系列与果实发育和成熟相关的基因表达,其中,色素合成相关基因和果实软化相关基因的表达受到强烈诱导,说明SlDREB1B可通过调节果实成熟相关基因的表达影响番茄果实发育和成熟进程。     

Genome sequencing and functional genomics approaches in tomato

Tomato genome sequencing has been taking place through an international, 10-year initiative entitled the International Solanaceae Genome Project (SOL). The strategy proposed by the SOL consortium is to sequence the approximately 220Mb of euchromatin that contains the majority of genes, rather than the entire tomato genome. Tomato and other Solanaceae plants have unique developmental aspects, such as the formation of fleshy fruit, not afforded by Arabidopsis or rice. Divergent phenotypes and habitats of the Solanaceae also make the family an ideal model to explore the bases of diversification and adaptation. Current progress in genome sequencing, genetic and genomic resources, and functional genomics approaches for tomato is summarized. Given the foreseen wealth of information in the upcoming genome sequence, the role of the laboratory-grown miniature tomato cultivar Micro-Tom as a valuable functional genomics tool for plant pathology and emerging areas of biology, such as omics biology, is emphasized.     

Visualization of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> cells during biocontrol of bacterial wilt disease in tomato with <Emphasis Type="Italic">Pythium oligandrum</Emphasis>

The biocontrol agent Pythium oligandrum (PO) can suppress bacterial wilt caused by Ralstonia solanacearum (RS) in tomato. To understand the primary biocontrol mechanisms of bacterial wilt by PO, we pretreated tomato plants with sterile distilled water or preinoculated them with PO, followed by inoculation with RS, then observed PO and RS in fixed sections of tomato tissues using a confocal laser-scanning microscope and fluorescence labeling until 14 days after the inoculation with RS. Horizontal and vertical movement of RS bacteria was frequently observed in the xylem vessels of roots and stems of tomato plants (cv. Micro-Tom) that had not been inoculated with PO. In plants that were preinoculated with PO, the movement of RS was suppressed, and bacteria appeared to be restricted to the pit of vessels, a reaction similar to that observed in resistant rootstocks. PO colonization was mainly observed at the surfaces of taproots, the junctions between taproots and lateral roots, and the middle sections of the lateral roots. PO was not observed near wound sites or root tips where RS tended to colonize. However, RS colonization was significantly repressed at these sites in PO preinoculated plants. These observations suggest that the induction of plant defense reactions is the main mechanism for the control of tomato bacterial wilt by PO, not direct competition for infection sites.     

转基因番茄试管内开花结实体系的建立

本研究建立了一种农杆菌介导法转化矮化型番茄Micro-Tom的试管内开花结实体系。以MS为基本培养基,附加2.0mg/L6-BA时,卡那霉素抗性再生芽的花芽诱导率为100%,在附加2.0mg/L6-BA和1mg/LGA即可诱导开花结实。经GUS组织化学染色和基因组PCR检测证实得到了转基因植株,本实验证明了该转基因植株在试管内开花结实体系从子叶共培养至转基因果实的获得最快仅需90d,为外源基因功能研究提供了一种快速方法。     

Accumulation of Carbohydrate and Regulation of 14-3-3 Protein on Sucrose Phosphate Synthase （SPS）Activity in Two Tomato Species

To explore the differences of carbohydrate metabolism in two tomato species and discuss the possible regulation of 14-3-3 proteins on the sucrose phosphate synthase （SPS） activity, we determined the contents of soluble sugar and starch through high performance liquid chromatography （HPLC）. The activities of sugar-metabolizing enzymes were assayed in desalted extract, and the relative expression levels of related genes in sugar metabolism were determined though real-time RT-PCR. The results indicated that glucose and fructose were mainly accumulated during the maturation of the fruit because of the high acid invertase （AI） and neutral invertase （NI） in Micro-Tom （Solanum lycopersicum） fruit, while in Solanum chmielewskii fruit, SPS which went along with the change of sucrose content led to the rapid sucrose increase during the fruit ripening. TFT1 and TFT10, belonging to 14-3-3 protein in tomato, were likely to down-regulated SPS activity during young and intumescence period.