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 solu...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.展开更多
Ethylene plays an important role in the regulation of many growth and developmental processes of higher plants. In tomato,Le-ACS6,a member of the ACC synthase multigene family involved in system 1 ethylene biosynthesi...Ethylene plays an important role in the regulation of many growth and developmental processes of higher plants. In tomato,Le-ACS6,a member of the ACC synthase multigene family involved in system 1 ethylene biosynthesis during fruit ripening,is subject to negative feedback regulation by ethylene. To identify the cis-elements that are responsible for the negative feedback control,we established an in vitro transient assay system employing particle bombardment on mature-green tomato fruit pericarp to examine the expression of a luciferase(LUC) reporter gene driven by a 5′-serially deleted Le-ACS6 promoter. The results localized putative cis-elements required for negative ethylene-response between -347 and -266 upstream from the translational start site ATG. Several lines of stable transformation of the Le-ACS6 promoter and GUS reporter fusion gene containing internal deletion from -347 to -266 were generated. The expression pattern of the GUS reporter showed that removal of the nucleotides from -347 to -266 completely eliminated the response of the Le-ACS6 promoter to exogenous ethylene.展开更多
以微型番茄M icro-T om核基因组DNA为模板,利用PCR方法扩增编码有色体特异性的番茄红素β-环化酶的B基因编码区,并将B基因与在高等植物中组成型表达的C aM V 35S启动子融合,构建成双元载体pL e-BZN,导入农杆菌,利用农杆菌介导的遗传转...以微型番茄M icro-T om核基因组DNA为模板,利用PCR方法扩增编码有色体特异性的番茄红素β-环化酶的B基因编码区,并将B基因与在高等植物中组成型表达的C aM V 35S启动子融合,构建成双元载体pL e-BZN,导入农杆菌,利用农杆菌介导的遗传转化方法转化到微型番茄M icro-T om中,通过抗性筛选和PCR鉴定,成功获得了多株转基因微型番茄,为获得富含β胡萝卜素的转基因微型番茄奠定了基础.展开更多
本文报告利用EST筛选结合RT-PCR的方法从番茄中克隆了LeCOP1LIKE基因的1 060 bp cDNA片段,并利用其非保守域构建反义RNA表达载体。利用农杆菌介导法,将LeCOP1LIKE基因的反义RNA表达载体转入微型番茄Micro-Tom,获得了10株反义LeCOP1LIKE...本文报告利用EST筛选结合RT-PCR的方法从番茄中克隆了LeCOP1LIKE基因的1 060 bp cDNA片段,并利用其非保守域构建反义RNA表达载体。利用农杆菌介导法,将LeCOP1LIKE基因的反义RNA表达载体转入微型番茄Micro-Tom,获得了10株反义LeCOP1LIKE转基因微型番茄。RT-PCR分析表明其中4个转基因株系中的LeCOP1LIKE表达被显著抑制,并发现抑制LeCOP1LIKE基因的表达导致转基因番茄的株高下降、叶片的叶绿素含量提高、果实中番茄红素含量增加。并且明显抑制转基因种子的发育。这些实验结果证明了LeCOP1LIKE基因为番茄发育过程中光形态建成的抑制因子。展开更多
从‘Micro-Tom’番茄(Solanum lycopersicum)中分离克隆了一个与叶片衰老相关、编码LRR型类受体蛋白激酶(receptorlike protein kinase,RLK)的基因。序列分析显示,该基因的编码产物与大豆和拟南芥中的SARK(SENESCENCE-ASSOCIATED RECEPT...从‘Micro-Tom’番茄(Solanum lycopersicum)中分离克隆了一个与叶片衰老相关、编码LRR型类受体蛋白激酶(receptorlike protein kinase,RLK)的基因。序列分析显示,该基因的编码产物与大豆和拟南芥中的SARK(SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE)类似,都具有典型的LRR-RLK结构,且可能具有丝/苏氨酸和酪氨酸双底物特异性激酶活性,因此命名为SlSARK-like。半定量RT-PCR分析表明,无论是自然衰老还是AtSARK过表达诱导衰老的‘Micro-Tom’叶片中,SlSARK-like基因的表达水平都明显提高。进一步利用农杆菌介导的番茄转化技术获得了6个GVG:SlSARK-like转基因‘Micro-Tom’株系,发现DEX诱导SlSARK-like基因过表达导致转基因植株早衰,说明SlSARK-like基因对‘Micro-Tom’叶片衰老起正向调控作用。上述研究结果进一步支持了编码LRR-RLK的SARK基因参与叶片衰老调控的机制普遍存在于高等植物中。展开更多
基金supported by the Key Technologies R&D Program of China during the 12th Five-Year Plan period(2011BAD12B03)
文摘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.
基金Supported by the National Natural Science Foundation of China (Grant Nos. 30470174 and 30070075) Natural Science Foundation of Tianjin City (Grant No. 05YFJZJC00800)
文摘Ethylene plays an important role in the regulation of many growth and developmental processes of higher plants. In tomato,Le-ACS6,a member of the ACC synthase multigene family involved in system 1 ethylene biosynthesis during fruit ripening,is subject to negative feedback regulation by ethylene. To identify the cis-elements that are responsible for the negative feedback control,we established an in vitro transient assay system employing particle bombardment on mature-green tomato fruit pericarp to examine the expression of a luciferase(LUC) reporter gene driven by a 5′-serially deleted Le-ACS6 promoter. The results localized putative cis-elements required for negative ethylene-response between -347 and -266 upstream from the translational start site ATG. Several lines of stable transformation of the Le-ACS6 promoter and GUS reporter fusion gene containing internal deletion from -347 to -266 were generated. The expression pattern of the GUS reporter showed that removal of the nucleotides from -347 to -266 completely eliminated the response of the Le-ACS6 promoter to exogenous ethylene.
文摘以微型番茄M icro-T om核基因组DNA为模板,利用PCR方法扩增编码有色体特异性的番茄红素β-环化酶的B基因编码区,并将B基因与在高等植物中组成型表达的C aM V 35S启动子融合,构建成双元载体pL e-BZN,导入农杆菌,利用农杆菌介导的遗传转化方法转化到微型番茄M icro-T om中,通过抗性筛选和PCR鉴定,成功获得了多株转基因微型番茄,为获得富含β胡萝卜素的转基因微型番茄奠定了基础.
文摘本文报告利用EST筛选结合RT-PCR的方法从番茄中克隆了LeCOP1LIKE基因的1 060 bp cDNA片段,并利用其非保守域构建反义RNA表达载体。利用农杆菌介导法,将LeCOP1LIKE基因的反义RNA表达载体转入微型番茄Micro-Tom,获得了10株反义LeCOP1LIKE转基因微型番茄。RT-PCR分析表明其中4个转基因株系中的LeCOP1LIKE表达被显著抑制,并发现抑制LeCOP1LIKE基因的表达导致转基因番茄的株高下降、叶片的叶绿素含量提高、果实中番茄红素含量增加。并且明显抑制转基因种子的发育。这些实验结果证明了LeCOP1LIKE基因为番茄发育过程中光形态建成的抑制因子。
